HK1142630A - Highly efficient methods for reprogramming differentiated cells and for generating animals and embryonic stem cells from reprogrammed cells - Google Patents

Description

Efficient methods for reprogramming differentiated cells and producing animal and embryonic stem cells from reprogrammed cells

Technical Field

The present invention relates generally to the field of Somatic Cell Nuclear Transfer (SCNT), and the production of animals and cells.

RELATED APPLICATIONS

The benefit of U.S. provisional application No. 60/902,970 filed on 23/2/2007, U.S. provisional application No. 60/918,543 filed on 16/3/2007, U.S. provisional application No. 60/993,772 filed on 14/9/2007, and U.S. provisional application No. 61/009,432 filed on 28/12/2007 as claimed in this application, are incorporated herein by reference in their entirety.

Background

Advances in stem cell technology, such as the isolation and in vitro proliferation of human embryonic stem cells ("hES" cells), constitute an important new area of medical research. hES cells have the demonstrated potential to proliferate in an undifferentiated state and then be induced to differentiate into any and all cell types of the human body, including complex tissues. This has led to the proposal that many diseases caused by cellular dysfunction can be treated by administering hES-derived cells of various differentiation types (Thomson et al, Science 282: 1145-1147 (1998)). Nuclear transfer studies have demonstrated that it is possible to transform differentiated somatic cells back into a totipotent state, such as that of embryonic stem cells ("ES") (Cibelli et al, Nature Biotech 16: 642-646(1998)) or embryonic derived ("ED") cells. The development of techniques for reprogramming somatic cells back to a pluripotent ES cell state, such as by transferring the genome of a somatic cell into an enucleated oocyte and subsequently culturing the reconstructed embryo to produce an ES cell, commonly referred to as somatic cell nuclear transfer ("SCNT"), provides a method for transferring ES-derived somatic cells having the nuclear genotype of a patient (Lanza et al, Nature Medicine 5: 975-977 (1999)). It is expected that such cells and tissues will not be rejected despite the presence of allogeneic mitochondria (Lanza et al, Nature Biotech 20: 689-696, (2002)). Nuclear transfer also allows the reconstruction of telomeric repeat lengths in cells by reactivating telomerase catalytic components in early embryos (Lanza et al Science 288: 665-. Nevertheless, there is a need for improved methods of reprogramming animal cells to increase the frequency of successful and complete reprogramming. There is also a need to reduce the dependence on the availability of human oocytes.

There has been a long felt need for animals with certain desirable traits or characteristics, such as increased weight, milk content (milk content), milk production volume, length of lactation interval, and disease resistance. Traditional breeding methods are capable of producing animals with some traits that are clearly desirable, but these traits are often accompanied by many undesirable traits and are often too time consuming, costly and unreliable to develop. Furthermore, these methods simply fail to allow specific animal strains to produce gene products such as desired protein therapeutics (i.e., human or humanized plasma proteins or other molecules in milk) that are not found at all in the genetic complement of the relevant species.

The development of technologies capable of producing transgenic animals provides a means to produce animals with exceptional precision, engineered to carry specific traits or designed to express certain proteins or other molecular compounds of therapeutic, scientific or commercial value. That is, a transgenic animal is one that carries a gene of interest that is intentionally introduced into existing somatic and/or germ line cells at an early stage of development. As animals develop and grow, protein products or specific developmental changes engineered into the animals become apparent and exist in their genetic complements and in the genetic complements of their offspring.

Another problem associated with existing stem cell technology is the ethical considerations for obtaining stem cells using advanced human embryos (advanced human embryos). Therefore, obtaining cloned embryos at an early stage would be highly beneficial to limit ethical concerns.

In summary, the present invention solves the long standing problem related to efficiency, ethical dilemma, and how to clone embryos without oocytes.

Summary of The Invention

The present invention relates generally to methods of using a fertilized embryo as a recipient clonal somatic cell. In certain embodiments, the oocyte is the primary recipient and the fertilized embryo is the secondary recipient. In certain embodiments, the disclosure relates to methods of cloning a mammal, obtaining a pluripotent cell, or reprogramming a mammalian cell.

In certain aspects, the present disclosure provides a method of reprogramming a nucleus of a differentiated cell, the method comprising the steps of: providing a differentiated cell, an enucleated MII stage egg of an animal, and an enucleated 2-cell stage embryo of an animal, wherein said MII stage egg and said embryo are synchronized; injecting nuclei of said differentiated cells into said enucleated egg; activating the egg comprising the nucleus; allowing the activated egg comprising the nucleus to develop to a 2-cell stage; removing at least one nucleus and at least a portion of the surrounding cytoplasm of said activated 2-cell stage egg of the previous step; fusing said at least one nucleus removed in the previous step into said enucleated 2-cell stage embryo (preferably by placing said nucleus between 2 cells of said 2-cell stage embryo) to produce a single cell containing a reprogrammed nucleus of said differentiated cell.

In certain aspects, the present disclosure provides methods of producing an animal, comprising providing a differentiated cell, an enucleated MI I stage egg of an animal, and providing an enucleated 2-cell stage embryo of an animal, wherein the MI I stage egg and the embryo are synchronized; injecting nuclei of said differentiated cells into said enucleated egg; activating the egg comprising the nucleus; allowing the activated egg comprising the nucleus to develop to a 2-cell stage; removing at least one nucleus and at least a portion of the surrounding cytoplasm of the 2-cell stage egg of the previous step; fusing said at least one nucleus removed in the previous step into said enucleated 2-cell stage embryo to produce a single cell; and culturing said single cells from the previous step to allow development into an animal. In certain embodiments, culturing comprises implanting the cultured cells into the uterus of an animal. In certain embodiments, the implanted cells and the animal in which they are implanted are of the same species.

In certain aspects, the present disclosure provides methods of producing embryonic stem cells, the methods comprising the steps of: providing a differentiated cell, an enucleated MII stage egg of an animal, and an enucleated 2-cell stage embryo of an animal, wherein said MII stage egg and said embryo are synchronized; injecting nuclei of said differentiated cells into said enucleated egg; activating the egg comprising the nucleus; allowing the activated egg comprising the nucleus to develop to a 2-cell stage; removing the nucleus and surrounding cytoplasm of the egg at the 2-cell stage of the previous step; fusing the nuclei removed in the previous step into the enucleated 2-cell stage embryo (preferably by placing the nuclei between 2 cells of the 2-cell stage embryo to produce a single cell; and culturing the single cell from the previous step to a developmental stage where embryonic stem cells can be derived.

In certain embodiments, the embryonic stem cell is hemizygous or homozygous for an MHC allele, wherein the differentiated cell is hemizygous or homozygous for an MHC allele, or the embryonic stem cell is engineered to be hemizygous or homozygous for an MHC allele by homologous recombination or by loss of heterozygosity, or both, and wherein the same species is human. In certain embodiments, the method is repeated a plurality of times to produce a pool of embryonic stem cells, each embryonic stem cell being hemizygous or homozygous for a different MHC allele than the other embryonic stem cells of the pool.

In certain aspects, the methods of the present disclosure further comprise the step of culturing the single cells obtained from the foregoing methods to the blastomere, morula, or blastocyst stage. In certain aspects, the methods of the present disclosure further comprise a continuous nuclear transfer to the oocyte. In certain aspects, the methods of the present disclosure further comprise continuous nuclear transfer to an embryo.

In certain embodiments, the egg is coated with actinone (cyclohexamide), CsCl₂Calcium ionophore (ionocore), ionomycin, sperm factors (sperm factors), sperm fraction or fraction, 6-DMAP, SrCl₂Cytochalasin B or a combination thereof. In certain embodiments, the egg is coated with these agentsThe combination of (1) and (b). In a preferred embodiment, the egg is activated by a combination of ionomycin and 6-DMAP. In another preferred embodiment, the egg is activated by a combination of calcium ionophore and 6-DMAP. In another preferred embodiment, the egg is coated with SrCl₂And cytochalasin B.

In certain embodiments, the fusing step is performed electrically. In certain embodiments, the electrofusion is performed in two steps: a first step in which the nucleus is aligned with the anode and electrically shocked, and a second step in which the embryo and the nucleus are rotated by about 90 degrees and electrically shocked. In certain embodiments, the embryo and the nucleus are not rotated prior to impact. In certain embodiments, the fusing step is performed using sendai virus.

In certain embodiments, the MII stage egg is a human egg. In certain embodiments, the enucleated 2-cell stage embryo is a human embryo. In certain embodiments, the differentiated cell is a human cell. In certain embodiments, the cells of the present disclosure may be from any mammal. In yet another embodiment, the mammal is selected from a mouse, rat, cat, dog, rabbit, goat, hamster, pig, sheep, non-human primate, or primate.

In certain embodiments, the MII stage egg and the enucleated 2-cell stage embryo are from any animal. In certain embodiments, the MII stage egg and the enucleated 2-cell stage embryo are from the same species. In certain embodiments, the differentiated cell and the MII stage egg are from the same species. In certain embodiments, the differentiated cell and the enucleated 2-cell stage embryo are from the same species. In certain embodiments, the differentiated cell, the MII-stage egg, and the enucleated 2-cell stage embryo are from the same species. In certain embodiments, the same species is human.

In certain aspects, the disclosure relates to methods of cloning a mammal, obtaining a pluripotent cell, or reprogramming a mammalian cell. In certain embodiments, the method consists of the steps of: (a) obtaining donor nuclei from mammalian cells; (b) obtaining a fertilized embryo from a mammal; (c) transferring said donor nucleus to a cell of said fertilized embryo; (d) enucleating the primary nucleus of the fertilized embryo to allow the donor nucleus to reside in the fertilized embryo; and (e) culturing the fertilized embryo.

In certain embodiments, the enucleation step of the methods of the present application is performed between 3 and 6 hours of the nuclear transfer step, between 4 and 6 hours of the nuclear transfer step, between 5 and 6 hours of the nuclear transfer step, between 3 and 4 hours of the nuclear transfer step, between 3 and 5 hours of the nuclear transfer step, or between 4 and 5 hours of the nuclear transfer step. In certain embodiments, the enucleation step is performed within 3 hours of the nuclear transfer step, within 2 hours of the nuclear transfer step, or within 1 hour of the nuclear transfer step. In certain embodiments, the enucleation step is performed between 1 and 2 hours of the nuclear transfer step, between 1 and 3 hours of the nuclear transfer step, between 1 and 4 hours of the nuclear transfer step, between 1 and 5 hours of the nuclear transfer step, between 1 and 6 hours of the nuclear transfer step, between 2 and 3 hours of the nuclear transfer step, between 2 and 4 hours of the nuclear transfer step, between 2 and 5 hours of the nuclear transfer step, or between 2 and 6 hours of the nuclear transfer step.

In certain embodiments of the present disclosure, culturing the cloned embryos results in the development of blastocysts or blastocyst-like cell clusters (collections). In certain embodiments, embryonic stem cells can be derived from these blastocyst or blastocyst-like cell populations. In certain other embodiments, culturing the cloned embryos results in the development of 4-8 cell stage embryos or morula stage embryos. In certain embodiments, embryonic stem cells can be derived from all or a portion of such early stage division or morula stage embryos. In certain other embodiments, culturing the cloned embryos results in the development of embryos that continue to divide after the 2-cell stage. In certain embodiments, embryonic stem cell lines are derived and established.

In certain aspects of the present disclosure, the fertilized embryo is from a mammal of the same species as the mammalian donor cell. In certain embodiments of the present disclosure, the fertilized embryo is from a mammal that is a closely related species to the mammalian donor cell. In certain embodiments of the present disclosure, the fertilized embryo is a pre-nuclear stage embryo. In certain embodiments, the fertilized embryo is a 2-cell stage embryo. In certain embodiments of the present disclosure, the mammalian donor cell is an ES cell. In certain embodiments, the mammalian cell is a differentiated cell. In certain embodiments, the differentiated mammalian cell is a cumulus cell. In certain embodiments, the mammalian cell is a murine cell. In certain embodiments, the mammalian cell is a bovine cell. In certain embodiments, the mammalian cell is a human cell. In certain embodiments, the cells may be from other mammalian species, including, but not limited to, equine, canine, porcine, ovine origin; or rodent species such as mice may be used. In certain embodiments, the fertilized embryo is subjected to cryopreservation and thawed prior to the nuclear transfer step.

In certain embodiments, the donor nucleus is labeled. In certain embodiments, the nucleus is labeled by expression of a fluorescent transgene.

In certain aspects, the present disclosure relates to a method of cloning a mammalian cell, the method comprising the steps of: (a) obtaining donor nuclei from mammalian cells; (b) obtaining a first fertilized embryo from a mammal; (c) transferring the donor nucleus to the first fertilized embryo; (d) enucleating the primary nucleus of the first fertilized embryo to allow the donor nucleus to reside in the fertilized embryo; (e) culturing the fertilized embryo; (f) enucleating the second fertilized mammalian embryo; (g) separating the cells of the first fertilized embryo of step (e) and transplanting at least one cell to an enucleated second fertilized embryo; (h) fusing the transplanted cells with the cells of the enucleated second fertilized embryo to form a single cell embryo; and (i) culturing said cloned single cell embryos.

In certain embodiments, steps (f) - (i) are cycled more than once, starting with step (i) of the previous cycle with the fertilized embryo obtained in step (g). In certain embodiments, step (h) (the fusing step) is achieved by electrofusion. In certain embodiments, step (g) comprises transferring at least one nucleus of the fertilized embryo of step (e) to an enucleated second fertilized embryo.

In certain embodiments, the second fertilized embryo of the present disclosure is at the same stage of development as the first fertilized embryo. In certain embodiments, the second fertilized embryo of the present disclosure is at a similar developmental stage as the first fertilized embryo. Similar stages may include embryos at the same overall stage, such as the blastocyst stage, or embryos with similar cell number stage development times. In certain embodiments, the second fertilized embryo and the first fertilized embryo are at the 2-cell stage and only one of the two cells is transplanted.

In certain aspects, the present disclosure relates to a method of cloning a mammal, obtaining a pluripotent cell, or reprogramming a mammalian cell, the method comprising the steps of: (a) obtaining desired donor nuclei from mammalian cells; (b) obtaining at least one fertilized embryo from a mammal at least at the 2-cell stage; (c) transferring donor nuclei to one or more but not all cells of the fertilized embryo, one donor nucleus per cell; (d) enucleating the primary nucleus of each cell of said embryo into which a donor nucleus has been transferred, leaving said donor nucleus in said cell; and (e) culturing the fertilized embryo.

In certain embodiments, the fertilized embryo of the present disclosure is a 2-cell stage embryo and the donor nucleus is transplanted to only one of the two cells of the embryo. In various embodiments, a fertilized embryo of the present disclosure may be an embryo at any stage. In certain embodiments, the activated oocyte of the present disclosure is the recipient of a 2-cell stage nuclear transfer. In certain embodiments, the oocyte is at any stage. In certain embodiments, the oocyte and the embryo are in synchrony.

In certain embodiments, the step of transplanting donor nuclei is performed just prior to the enucleation step.

In certain aspects, the present disclosure relates to blastocysts derived from fertilized embryos produced by any of the methods of the present disclosure. In certain embodiments, the present disclosure relates to a blastocyst produced by any of the methods of the present disclosure.

In certain embodiments of any of the foregoing, the embryonic stem cells or embryonic stem cell lines can be produced using all or part of a cloned embryo. For example, embryonic stem cells or cell lines can be produced using all or part of a blastocyst stage cloned embryo or using all or part of an early stage division or morula stage embryo.

In certain aspects, the present disclosure relates to methods of producing embryonic stem cells, the methods comprising the steps of: (a) providing a differentiated cell, an enucleated MII stage egg of an animal, and an enucleated 2-cell stage embryo of an animal, wherein said MII stage egg and said embryo are synchronized; (b) injecting nuclei of said differentiated cells into said enucleated egg; (c) activating the egg comprising the nucleus; (d) allowing the activated egg comprising the nucleus to develop to a 2-cell stage; (e) removing the nucleus and surrounding cytoplasm of the 2-cell stage egg of step (d); (f) fusing said nucleus removed in step (e) into said enucleated 2-cell stage embryo (preferably by placing said nucleus between 2 cells of said 2-cell stage embryo) to produce a single cell; and (g) culturing said individual cells from step (f) to a developmental stage from which embryonic stem cells can be derived, comprising: (i) culturing the single cell from (f) to the morula stage to produce morula; (ii) isolating blastomeres from the morula; (iii) culturing the blastomeres to produce clusters of two or more blastomeres; (iv) contacting the cluster of two or more blastomeres in culture with embryonic or fetal cells, directly or indirectly; and (v) culturing the cluster of two or more blastomeres of (iv) until ES cells are produced.

In certain aspects, the present disclosure relates to methods of producing Embryonic Stem (ES) cells, the methods comprising: (a) culturing blastomeres removed or dissected from a mammalian maternal embryo together with the mammalian maternal embryo for 12 to 18 hours; (b) transferring the blastomeres to blastocyst medium further comprising laminin and inoculated with Mouse Embryonic Fibroblasts (MEFs), and (c) culturing the blastomeres of (b) until ES cells are produced.

In certain aspects, the present disclosure relates to methods of producing Embryonic Stem (ES) cells, the methods comprising: (a) culturing blastomeres removed or dissected from a mammalian maternal embryo together with the mammalian maternal embryo; (b) transferring the blastomeres to blastocyst medium further comprising laminin or fibronectin, and (c) culturing the blastomeres of (b) until ES cells are produced.

In certain aspects, the present disclosure relates to methods of producing embryonic stem cells, the methods comprising the steps of: (a) providing an enucleated MII-stage egg of an animal, a differentiated cell of an animal, and an enucleated 2-cell stage embryo, wherein said MII-stage egg and said embryo are synchronized; (b) injecting nuclei of said differentiated cells into said enucleated egg; (c) activating the egg comprising the nucleus; (d) allowing the activated egg comprising the nucleus to develop to a 2-cell stage; (e) removing the nucleus and surrounding cytoplasm of the 2-cell stage egg of step (d); (f) fusing said nucleus removed in step (e) into said enucleated 2-cell stage embryo (preferably by placing said nucleus between 2 cells of said 2-cell stage embryo) to produce a single cell; (g) culturing the single cell from step (f) to produce morula; (h) isolating blastomeres from the morula (e.g., a morula that is a maternal embryo); (i) culturing the blastomeres and the maternal embryos together for 12 to 18 hours; (j) transferring the blastomeres to blastocyst medium further comprising laminin or fibronectin and seeded with Mouse Embryonic Fibroblasts (MEFs), and (k) culturing the blastomeres of (j) until ES cells are produced.

In certain embodiments, MEFs are meiotically inactivated. In certain embodiments, step (c) comprises culturing under conditions that reduce the formation of oocytes.

In certain embodiments, the blastocyst medium comprises 2.5 μ g/ml laminin. In certain embodiments, the blastocyst medium comprises 10 μ l/ml laminin. In certain embodiments, the blastocyst medium comprises about 2.5, 5, 7.5, 10, 15, or 20 μ g/ml laminin. In certain embodiments, the medium is supplemented with 1-5, 1-10, 5-10, 10-20, or 1-20 μ g/ml laminin. In certain embodiments, the medium is supplemented with at least 1, 2.5, 5, 7.5, 10, 15, or 20 μ g/ml laminin.

In certain embodiments, step (c) of the above method comprises culturing for 5 days in blastocyst medium seeded with MEF cells. In certain embodiments, culturing in blastocyst medium seeded with MEF cells is performed for 1, 2, 3,4, 5,6, 7, 8, 9, or 10 days. In certain embodiments, step (c) of the above method further comprises culturing to a cell pellet of about 20 cells in the shape of the blastomere and transferring the cell pellet to a medium seeded with ES cells. In certain embodiments, the cell mass is about 5, 10, 15, 20, 30, 40, or 50 cells. In certain embodiments, the ES cells express a marker or are labeled. In certain embodiments, the ES cells express GFP. In certain embodiments, the maternal embryo is transferred to blastocyst medium and allowed to develop into a blastocyst. In certain embodiments, the blastomere is isolated from an embryo, comprising: (a) fixing the embryo; and (b) flicking the fixed embryo until the blastomeres separate.

In certain embodiments, more than one blastomere is removed or biopsied from the maternal embryo. For example, two blastomeres may be biopsied from a maternal embryo and used to derive ES cells.

In certain embodiments, embryonic stem cells are produced using methods that do not require and/or cause embryo disruption. For example, when embryonic stem cells are produced from a single blastomere of a morula stage maternal embryo, the remainder of the maternal embryo can then be frozen for long term or permanent storage, or used to produce pregnancy.

In certain embodiments, blastomeres removed or biopsied from a mammalian maternal embryo and the mammalian maternal embryo are cultured together for about 6 to 12, 6 to 18, 6 to 24, 12 to 18, 12 to 24, or 18 to 24 hours.

The present application contemplates the use of any of these aspects alone or in combination with any of the foregoing or following aspects and embodiments of the invention.

Brief Description of Drawings

FIGS. 1A-1B show mouse embryos cloned by the serial cloning (serial cloning) procedure. GFP positive ES nuclei were injected into recipient embryos. Embryos are displayed under bright field (a) and fluorescence microscopy (B).

FIGS. 2A-2C show the development of embryos from 2 cell clones in the presence of helper cells. Injected GFP-positive ES nuclei form mosaic embryos that can develop to the 4-cell stage (a), 8-cell stage (B) and blastocyst stage (C).

FIG. 3 shows cloned F2GFP mice. F2GFP mice at 10 weeks of age emit green fluorescence under UV light (arrow).

FIG. 4 shows the confirmation of the genetic composition of GFP cloned mice.

Figure 5 shows DBA2 clone 1 fingerprinting.

FIG. 6 shows H19 gene expression in embryos of F2GFP cumulus cell clones.

FIG. 7 shows IGF-2 gene expression in embryos of F2GFP cumulus cell clones.

FIG. 8 shows Oct-4 gene expression in embryos of F2GFP cumulus cell clones.

FIGS. 9A-9C show ES cell markers and ES cell teratoma formation from serial cloned embryos. (A) An ES cell marker. (B) Teratoma. (C) Chimeric young animals.

FIG. 10 shows a schematic of the nuclear transfer and serial nuclear transfer method.

FIGS. 11A-C show derivation and characterization of hESC lines of individual blastomeres without destroying the embryo. Drawing board A: stage of hES cell derivation from single blastomeres. (a) -blastomere biopsy, (b) -biopsy of blastomeres (arrows) and maternal embryos developing next to each other, (c) -initial outgrowth of single blastomeres on MEF, 6 days, magnification x200, (d) colony of single blastomere-derived hES cells, magnification x 200. A drawing board B: a blastocyst formed from a biopsied maternal embryo (a) and a pluripotency marker in a single blastomere-derived hES cell line (b-i); (b) alkaline phosphatase, (c) -Oct-4, (d) DAPI, corresponding to Oct4 and Nanog, (e) Nanog, (f) -SSEA-3, (g) -SSEA-4, (h) -TRA-1-60, (i) -TRA-1-81; original amplification: panel A (a), 400x, panels A (B-d) and B, 200x, except NED5g & h, 100 x. A drawing board C: individual blastomere-derived hESCs differentiate into three germ layers in vivo (a-d) and in vitro (e-g). (a) Teratomas show derivatives of all three germ layers. Cre, ciliated airway epithelium, including insets showing cilia at higher magnification; int, intestinal epithelium; cart, cartilage; ne, columnar neuroepithelium that binds to retinal pigment epithelium (rpe). (b-d), examples from other teratomas. (b) Bronchial nests (bronchialor nests); c) smooth muscle actin-stained muscle; (d) cdx2 stained intestinal epithelium. (e-g) -examples of in vitro differentiation derivatives: (e) hemangioblast colonies with both hematopoietic and endothelial potential, (f) embryoid bodies with beating heart cells; (g) the retinal pigment epithelium. Amplification: a-f 200x, g 100 x.

FIGS. 12A-12I show examples of single blastomere-derived hES cells differentiating into three germ layers (a-c) and cell types of therapeutic value (d-I). Immunostaining with antibodies against the following markers for the three germ layers: tubulin beta iii (a), smooth muscle actin (b), alpha fetoprotein (c). Examples of differentiation derivatives: hemangioblast colonies with both hematopoietic and epithelial potential (d). Immunostaining of epithelial cells using antibodies against kdr (e) and CD31 (f); embryoid bodies with beating heart cells (g), retinal pigment epithelium (h, i). RT-PCR showed the RPE markers PEDF (lane 1,300bp) and RPE65 (lane 2,285bp), positive control GAPDH (lane 3,465 bp). Amplification: a-c, e, f-20 x; d, g-10x, h-40 x.

FIGS. 13A-13B show microsatellite and PCR analysis of single blastomere-derived hES cells. A-DNA PCR confirmed the absence of GFP in individual blastomere-derived hES cells. Lane 1, positive control WA01(H1) hES cell line, lane 2, negative control (no template), lane 3, NED1, lane 4, NED2, lane 5, NED3, lane 6, NED 4. B-microsatellite analysis of single blastomere-derived hES cell lines.

FIG. 14 shows karyotypes of a single blastomere-derived hES cell line.

FIGS. 15A-15H show the effect of laminin on single blastomere development and hES cells. (A-C) formation of trophectoderm-like (trophelomem-like) vesicles in the absence of laminin. (A) Hoffman modulation contrast (Hoffman modulation contrast), (B) cdx2 immunostaining, and (C) cytokeratin 8 immunostaining. (D-F) formation of an ICM-like growth halo in the presence of laminin. (D) Phase contrast, (E) Oct-4 immunostaining, (F) corresponding DAPI images. (G-I) the depolarizing effect of laminin on hESC. (G, H) control (G) and laminin (H) covered hESC (WA07) confocal microscopy co-stained with the tight junction marker ZO-1 (green) and the pluripotent marker Oct-4 (red). Ultrastructural analysis (half-thin sections) of cross sections of control (left) and laminin-covered (right) hESC colonies (WA 09). Control colonies were organized into semi-stratified epithelia. The presence of apical microvilli (mv) and tight junctions (data not shown) indicate a structural specialization typical of epithelioid polarization. Laminin coverage induces cell depolarization as indicated by the lack of microvilli on the cell surface and the accumulation of cells to form a multi-layered structure. Amplification: (A-F)200X, (G, H)630X, and (I) 400X.

Detailed Description

Definition of

The term "embryonic stem cells" (ES cells) refers to cells derived from the inner cell mass of blastocysts or morulae that have been serially passaged like cell lines. ES cells can be derived from fertilization of egg cells with sperm or DNA, nuclear transfer, parthenogenesis, or by means of generating hES cells that are homozygous in the MHC region. The term "human embryonic stem cells" (hES cells) refers to human ES cells.

The term "pluripotent stem cell" refers to an animal cell capable of differentiating into more than one differentiated cell type. Such cells include hES cells, human embryo-derived cells (hedcs) and adult-derived cells, including mesenchymal stem cells, neural stem cells and bone marrow-derived stem cells. The pluripotent stem cells may be genetically modified or non-genetically modified. Genetically modified cells may include markers such as fluorescent proteins to facilitate their identification in ovo

The term "differentiated cell" as used herein refers to any cell during or that has been differentiated into an adult cell lineage. For example, embryonic cells can differentiate into epithelial cells that line the intestine. Differentiated cells may be isolated from, for example, a fetus or a fetal animal.

The term "implantation" as used herein in reference to an embryo refers to the conception of a female animal with an embryo as described herein. This technique is well known to those of ordinary skill in the art. See, e.g., Seidel and Elsden, 1997, Embryo Transfer in Dairy caltle (cow Embryo Transfer), w.d. hoard & Sons, co., Hoards Dairyman. The embryo may be allowed to develop in the uterus, or alternatively, the fetus may be removed from the uterine environment prior to delivery.

The term "synchronized" or "synchronized" as used herein, when referring to an estrus cycle, refers to assisted reproduction techniques well known to those of ordinary skill in the art. These techniques are fully described in the references cited in the previous paragraph. Estrogen and progesterone hormones are commonly used to synchronize the estrous cycle and embryonic development cycle of female animals. The term "developmental cycle" as used herein refers to an embryo of the invention and the time period between each cell division within the embryo. This period of time is predictable to the embryo and can be synchronized with the estrus cycle of the recipient animal.

The term "culturing" as used herein with respect to an embryo refers to a laboratory procedure that includes placing the embryo in a culture medium. The embryo may be placed in a culture medium for an appropriate amount of time to allow the embryo to remain static but functional in the medium, or to allow the embryo to grow in the medium. Media suitable for culturing embryos are well known to those skilled in the art. See, e.g., U.S. Pat. No. 5,213,979, entitled "Invitro Culture of Bovine Embryos" (in vitro Culture of Bovine Embryos), First et al, awarded 5/25.1993, and U.S. Pat. No. 5,096,822, entitled "Bovine Embryo Culture Medium", Rosenkrans, Jr. et al, awarded 3/17.1992, which are incorporated herein by reference in their entirety (including all illustrations, tables and drawings).

The term "suitable medium" as used herein refers to any medium that allows the proliferation of cells. Suitable media need not promote maximal proliferation, but only measurable cell proliferation.

The term "cloned" as used herein refers to a cell, embryonic cell, fetal cell, and/or animal cell that has a nuclear DNA sequence that is substantially similar or identical to the nuclear DNA sequence of another cell, embryonic cell, fetal cell, and/or animal cell. The terms "substantially similar" and "identical" are described herein. The cloned embryos may be generated from a nuclear transfer or, alternatively, the cloned embryos may be generated from a cloning process that includes at least one recloning step. If a cloned embryo is generated from a cloning procedure comprising at least one recloning step, said cloned embryo may be indirectly generated from an immortalized totipotent cell, since said recloning step may utilize embryonic cells isolated from an embryo generated from an immortalized totipotent cell. The term "pluripotent" as used herein when referring to an embryo refers to an embryo that can develop into a fetal animal.

The term "substantially similar" as used herein in reference to a nuclear DNA sequence means that the two nuclear DNA sequences are nearly identical. The difference between the two sequences may be a difference in replication errors that typically occur during nuclear DNA replication. Substantially similar DNA sequences are preferably greater than 97% identical, more preferably greater than 98% identical, and most preferably greater than 99% identical. Identity is measured by dividing the number of identical residues in both sequences by the total number of residues and then multiplying the result by 100. Thus, two copies of a sequence that are identical have 100% identity, while sequences that are less highly conserved and have deletions, additions or substitutions have a lower degree of identity. One of ordinary skill in the art will appreciate that there are a number of computer programs available for performing sequence comparisons and determining sequence identity.

The term "maternal embryo" is used to refer to an embryo from which a single blastomere is removed or biopsied. After biopsy, the remaining maternal embryo (maternal embryo minus biopsy blastomere) can be cultured with the blastomere to help promote proliferation of the blastomere. The remaining, viable maternal embryos may then be frozen for long-term or permanent storage, or for future use. Alternatively, the viable maternal embryo may be used to generate pregnancy. Alternatively, the viable maternal embryo may be destroyed. In certain embodiments, the maternal embryo is a cloned embryo produced by the serial transfer method of the invention. In other embodiments, the maternal embryo is an embryo produced by fertilization.

Summary of the invention

Although mammalian cloning has great potential for both basic scientific and therapeutic uses, the efficiency of mammalian cloning by Somatic Cell Nuclear Transfer (SCNT) is still low. The birth rate of viable pups after SCNT is less than 10%, regardless of the species, donor cell type, protocol or technique used. Similarly, the rate of development of cloned embryos is also lower than that of normal fertilized embryos, resulting in poor blastocyst development and smaller numbers of blastocyst cells. These defects also result in a relatively low success rate of establishing ES cell lines from cloned mouse embryos, regardless of the mouse strain or donor cell type, of about 5%, compared to about 30% using normal embryos. The inability to clone embryos is largely due to incomplete nuclear programming, as evidenced by abnormal expression of several genes during early developmental stages.

To overcome the inefficiency of SCNT, several approaches have been tried. More recently, Kishigami et al (2006) reported an improved mouse cloning technique for treating reconstituted mouse eggs with histone deacetylase inhibitor trichostatin A, which reduces abnormal DNA hypermethylation. Another approach is to use Pronuclear (PN) stage zygotes or 2-cell stage in vivo fertilized embryos as serial clones of the second cytoplast recipient. When the embryos cloned from the PN-staged mouse somatic cells are re-cloned into the enucleated in vivo fertilized PN-staged zygotes, there is some improvement in the in vitro development and survival pup rate of the cloned embryos. In fact, a similar approach was also successfully used for the first porcine somatic cell clone. 2-cell stage in vivo fertilized embryos have also been used for successful serial cloning. When 2-cell stage SCNT embryos are re-cloned into 2-cell stage in vivo embryos, their in vitro development is improved and culmination is achieved in surviving pups. However, none of these studies have investigated the molecular basis of the improvement, and the nuclear donor cells are ES cells or pre-treated somatic cells. Furthermore, the improvement in clonal embryo development was not significant. The pretreatment of donor cells addresses chromatin remodelling and cell cycle synchronization between nuclear donor cells and recipient oocytes in various ways, again resulting in somewhat improved cloning efficiency. However, regardless of the method used, the cloning efficiency remains so low that it cannot be widely used in basic scientific research, practical propagation of certain strains of mice, therapeutic cloning or stem cell derivation. Furthermore, we are not aware of any group that successfully derived blastocyst or blastocyst-like clusters from which embryonic stem cells could be derived, or successfully derived embryonic stem cells or stem cell lines, using serial cloning. Similarly, we also do not know that any group successfully derived morula stage embryos (NT clusters that are approximately identical to and correspond to the morula stage of development) using serial cloning. Such morula stage embryos can be used as a maternal embryo from which one or more blastomeres can be removed or biopsied and used to generate ES cells.

Another aspect of the present application uses an embryo from which one or more blastomeres can be removed or biopsied and used to generate ES cells.

Nuclear transfer method

It is an object of the present invention to provide means for cloning somatic cells more efficiently and without causing ethical problems. The methods of the present disclosure may be used to clone a mammal, obtain pluripotent cells, or reprogram mammalian cells.

Human or animal cells, preferably mammalian cells, can be obtained and cultured by well-known methods. Human and animal cells useful in the present invention include, for example, epithelium, nerve cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), other immune cells, erythrocytes, macrophages, melanocytes, monocytes, mononuclear cells, fibroblasts, cardiomyocytes, cumulus cells, and other muscle cells, and the like. In addition, human cells for nuclear transplantation can be obtained from various organs, such as skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra, and other urinary organs. These are merely examples of suitable donor cells. Suitable donor cells, i.e., cells that can be used in the present invention, can be obtained from any cell or organ of the body. This includes all somatic or germ cells such as primordial germ cells, sperm cells. Preferably, the donor cell or nucleus is an actively dividing, i.e., non-quiescent, cell, as this has been reported to increase cloning efficiency. Such cells include those at the G1, G2S, or M cell stages. Alternatively, resting cells may be used. Also preferably, such donor cells will be in the G1 cell cycle. In certain embodiments, the donor and/or recipient cells of the present application do not undergo a 2-cell block. In certain embodiments, the donor cells or nuclei are not pretreated prior to nuclear transfer. In certain embodiments, the donor cells or nuclei are not pretreated with spermine, protamine, or putrescine prior to nuclear transplantation.

In certain embodiments, a recipient fertilized embryo of the present invention may be from any mammalian species. In certain embodiments, a cryopreserved fertilized embryo is used as a recipient cell. In certain embodiments, the embryos are human. Cryopreservation and thawing are known to those skilled in the art (see Tucker et al, Curr Opin Obstet Gynecol.1995, 6 months; 7 (3): 188-92).

In certain embodiments, the donor nucleus may be labeled. The cells may be genetically modified with a transgene encoding a readily visualized protein such as green fluorescent protein (Yang, M.et al, 2000, Proc. Natl. Acad. Sci. USA, 97: 1206-1211) or one of its derivatives, or with a transgene constructed from the firefly (Photinus pyralis) luciferase gene (Fluc) (Sweeney, TJ. et al 1999, Proc. Natl. Acad. Sci. USA, 96: 12044-12049), or with a transgene constructed from the soft coral Renilla (Rhuc) (Bhaunik, S. and Ghambhir, S.S. 2002, Proc. Natl. Acad. Sci. USA, 99: 377). The reporter transgene may be constitutively expressed using a "housekeeping gene" promoter, such that the reporter gene is expressed at high levels in many or all cells, or may be expressed using a tissue-specific or developmental stage-specific gene promoter, such that only cells that have been targeted to a particular niche (niches) and developed into a particular tissue or cell type may be visualized. Additional labeling reagents include, but are not limited to, fluorescently labeled macromolecules (including fluorescent protein analogs and biosensors), luminescent macromolecule chimeras (including those formed with green fluorescent protein and mutants thereof), luminescently labeled primary or secondary antibodies reactive with intracellular antigens involved in physiological responses, luminescent colorants, dyes, and other small molecules. Labeled cells from chimeric blastocysts can be sorted, for example, by flow cytometry, to isolate a clonal population.

The Nuclear transfer (Nuclear transfer) technique or the Nuclear transfer (Nuclear transfer) technique is known in the literature. See, inter alia, Campbell et al, Theriogenology, 43: 181 (1995); collas et al, mol. reportdev., 38: 264-; keefer et al, biol. reprod, 50: 935-939 (1994); sims et al, proc.natl.acad.sci., USA, 90: 6143-6147 (1993); WO 94/26884; WO 94/24274 and WO 90/03432, which are incorporated herein by reference in their entirety. In addition, U.S. Pat. nos. 4,944,384 and 5,057,420 describe procedures for nuclear transfer of cattle. See also Cibelli et al, Science, vol.280: 1256-1258(1998).

The transfer of the donor nucleus into the recipient fertilized embryo may be performed by microinjection equipment. In certain embodiments, there is minimal cytoplasmic transplantation with the nucleus. Minimal cytoplasmic transplantation is achievable when microinjection is used to transplant nuclei, but not by cell fusion methods. In one embodiment, the microinjection apparatus includes a piezoelectric unit (piezo unit). Typically, a piezoelectric unit is operatively connected to the needle to transmit vibrations to the needle. However, any configuration of piezoelectric unit that can transmit vibrations to the needle is included within the scope of the present invention. In some cases, the piezoelectric unit may assist the needle in entering the target. In certain embodiments, the piezoelectric unit may be used to minimize cytoplasm following nuclear transfer. Any piezoelectric element suitable for the purpose may be used. In certain embodiments, the piezoelectric unit is a Piezo micromanipulation controller PMMl 50(PrimeTech, Japan).

Coring can be accomplished by known methods, such as described in U.S. Pat. No. 4,994,384, which is incorporated herein by reference. For example, metaphase II oocytes may be placed in HECM (optionally containing 7.5. mu.g/ml cytochalasin B) for immediate enucleation, or metaphase II oocytes may be placed in a suitable medium (e.g., CR1aa plus 10% estrus bovine serum) and then enucleated later.

Enucleation may be accomplished microscopically using a micropipette to remove the polar body and adjacent cytoplasm. The cells can then be screened to identify those that have been successfully enucleated. This screening can be achieved by staining the cells with 1. mu.g/ml 33342 Herchester dye used in HECM, and then observing the cells under UV irradiation for less than 10 seconds. The successfully enucleated cells can then be placed in an appropriate medium.

Non-invasive methods of enucleation in mammals have been reported very rarely, whereas in amphibians ultraviolet irradiation is used as a routine procedure (Gurdon q.j.microsc.soc.101299-311 (I960)). There has been no detailed report of the use of this method in mammals, although it has been noted that exposure of mouse oocytes to ultraviolet light for more than 30 seconds reduces the developmental potential of the cells during the use of DNA-specific fluorescent dyes (Tsunoda et al, J.reprod.Fertil.82173 (1988)).

The present invention may utilize "induced enucleation," which refers to the enucleation of oocytes by disrupting the meiotic spindle apparatus by destabilizing (e.g., multimerizing) the microtubules of the meiotic spindle (see U.S. patent application No. 20060015950). Destabilization of microtubules prevents segregation of chromatids (e.g., prevents successful nuclear division) and induces unequal segregation (e.g., skewing) of oocyte genomes (e.g., chromatin) during meiotic maturation, whereby nearly all endogenous chromatin of the oocyte is pooled in the second polar body.

In certain embodiments, the blastomeres may be dissociated using a glass pipette. In some embodiments, dissociation may occur in the presence of 0.25% trypsin (Coalls and Rob1, 43 BIOL. REPROD.877-84, 1992; Stice and Rob1, 39 BIOL. REPROD.657-664, 1988; Kanka et al, 43MOL. REPROD.DEV.135-44, 1996).

The NT unit (NT unit) can be activated by known methods. Such methods include, for example, incubating the NT unit at a sub-physiological temperature, essentially by applying a cold or substantially cool temperature to impinge the NT unit. Most conveniently, this is done by culturing the NT unit at room temperature, which is cold relative to the physiological temperature conditions to which the embryo is normally exposed.

Alternatively, activation may be achieved by the application of known activating agents. For example, sperm penetration of oocytes during fertilization has been shown to activate prefusion oocytes to produce a greater number of viable pregnancies and multiple genetically identical calves following nuclear transfer. In addition, treatments such as electrical and chemical shock or actinone treatments can also be used to activate NT embryos after fusion. Suitable oocyte activation methods are the subject of U.S. Pat. No. 5,496,720 to Susko-Parrish et al, which is incorporated herein by reference.

For example, oocyte activation may be achieved by performing the following operations, either simultaneously or sequentially:

(i) increasing the level of divalent cations in the oocyte, and

(ii) reducing phosphorylation of cellular proteins in the oocyte.

This is generally achieved by introducing a divalent cation, such as magnesium, strontium, barium or calcium, into the oocyte cytoplasm, e.g. in the form of an ionophore. Other methods of increasing the divalent cation level include the use of electric shock, treatment with ethanol, and treatment with caged chelators (caged chelators).

Phosphorylation can be reduced by known methods, such as by the addition of kinase inhibitors, such as serine-threonine kinase inhibitors, such as 6-dimethylamino-purine, staurosporine, 2-aminopurine, and sphingosine.

Alternatively, phosphorylation of cellular proteins can be inhibited by introducing phosphatases, such as phosphatase 2A and phosphatase 2B, into the oocyte.

Specific examples of the activation method are listed below.

1. Activation by lonomycin and DMAP

1- -place oocytes in lonomycin (5uM) and 2mM DMAP for 4 minutes;

2- -transfer the oocyte to a medium containing 2mM DMAP for 4 hours;

3- -rinsing four times and culturing.

2. Activation by Rochomycin, DMAP and Roscovitine

1- -place oocytes in lonomycin (5uM) and 2mM DMAP for 4 minutes;

2- -transfer the oocytes to medium containing 2mM DMAP and 200. mu.M Roscovitine for 3 hours;

3- -rinsing four times and culturing.

3. Activation by exposure to lonomycin, followed by cytochalasin and actinone.

1- -place oocyte in lonomycin (5. mu.M) for 4 minutes;

2- -transferring the oocytes to a medium containing 5ug/ml cytochalasin B and 5. mu.g/ml actinone for 5 hours;

3- -rinsing four times and culturing.

4. Activation by electric pulses

1- -Place the eggs in a medium containing 100uM CaCl₂The mannitol medium of (1);

2- -three administrations of 1.0kVcm^-120us ec pulses, each pulse spaced 22 minutes apart;

3- -transfer the oocytes to medium containing 5ug/ml cytochalasin B for 3 hours.

5. Activation by exposure to ethanol, followed by cytochalasin and actinone

1- -place the oocyte in 7% ethanol for 1 minute;

2- -transferring the oocytes to a medium containing 5ug/ml cytochalasin B and 5ug/ml actinone for 5 hours;

3- -rinsing four times and culturing.

6. Activation by microinjection of adenophorstine

1- -injection of oocytes with 10 to 12pl of a solution containing 10uM adenophorstine;

2- -culturing the oocyte.

7. Activation of sperm factor by microinjection

1- -injection of oocytes with sperm factor isolated from, e.g., primates, pigs, cows, sheep, goats, horses, mice, rats, rabbits or hamsters, using 10 to 12 pl;

2- -culturing the egg.

8. Activation of recombinant sperm factor by microinjection.

9. Activation by exposure to DMAP, followed by actinone and cytochalasin B

10. By exposure to SrCl₂And activation of cytochalasin B.

In certain embodiments, the oocyte or NT unit (typically about 22 to 28 hours after maturation) is placed in about 2mM DMAP for about 1 hour, followed by incubation in 5pg/ml cytochalasin B and 20ug/ml actinone for about 2-12 hours, preferably about 8 hours.

In certain embodiments, the activation of the reconstructed oocyte is in the absence of Ca⁺⁺Containing 10mM SrCl₂And 5. mu.g/ml cytochalasin B in CZB, 6 hours, 5.5% CO at high humidity₂An incubator.

As noted, activation can be achieved before, simultaneously with, or after nuclear transfer. In general, activation will be achieved from about 40 hours prior to nuclear transfer and fusion to about 40 hours after nuclear transfer and fusion, more preferably from about 24 hours prior to nuclear transfer and fusion to about 24 hours after nuclear transfer and fusion, and most preferably from about 4 to 9 hours prior to nuclear transfer and fusion to about 4 to 9 hours after nuclear transfer and fusion. Activation is preferably achieved near or after maturation of the oocyte in vitro or in vivo, such as at about the same time or within about 40 hours of maturation, more preferably within about 24 hours of maturation.

In certain embodiments, one step of the invention is fusion of the cloned nuclei with enucleated cytoplasts of germline Cells such as blastomeres, morula Cells, inner cell mass Cells, ES Cells, including hES Cells, EG Cells, EC Cells, as known in the art (Do & Scholer, Stem Cells 22: 941-. Fusion of cytoplasts with nuclei is performed using a number of techniques known in the art, including Polyethylene Glycol (see Pontervo "Polyethylene Glycol (PEG) in the Production of mammalian Somatic Cell Hybrids" Polyethylene Glycol "cytogene Cell Genet.16 (1-5): 399-400(1976)), direct injection of nuclei, Sendai virus mediated fusion (see U.S. Pat. No. 4,664,097 and Graham Wistar Inst.Symp. monogr.919(1969)), or other techniques known in the art such as electrofusion. electrofusion of cells includes bringing cells together in close proximity and exposing them to an alternating electric field. under appropriate conditions, the cells are pushed together and fused, then fused cells or hybrid cells are formed. electrofusion of cells and apparatus for performing electrofusion of cells are described in, for example, U.S. Pat. Nos. 4,441,972, 4,578,168 and PCT application No. 5,283,194/PCT for International application No. PCT/92/93/05166, pohl, "Dielectrophoresis", Cambridge university Press, 1978 and Zimmerman et al, Biochimica et Bioplzysia Acta 641: 160-165, 1981.

Fusion of the cloned nuclei with anucleated cytoplasmic bubbles of germline cells (such as hES cells) attached to a physical matrix as is well known in the art (Wright & Hayflick, exp. cell Res.96: 113-121, (1975); Wright & Hayflick, Proc. Natl. Acad. Sci., USA, 72: 1812-1816, (1975) can be combined with the present disclosure briefly, the cytoplasmic volume of the germline cells is increased by the addition of 10 μ M cytochalasin B for 20 hours, trypsinized and replated onto sterile 18mm coverslips, cylinders (cyclinders) or other physical matrices coated with a material facilitating attachment prior to the procedure, the cells are plated at a density such that after overnight incubation at 37 ℃ and gentle rinsing with medium once, the cells cover a certain portion of the surface area of the coverslips or other matrices, preferably about 90%, and then placed in a centrifuge tube in position, so that centrifugation will result in the removal of nuclei from the cytoplasts, said centrifugation containing 8mL of 10% Ficoll-400 solution and centrifugation at 20,000g for 60 minutes at 36 ℃. The cloned nuclei are then plated on coverslips or substrates at a density of at least cytoplasts, preferably at least five times the density of cytoplasts. Fusion of cytoplasts with nuclei is performed using Polyethylene Glycol (see Pontervo "Polyethylene Glycol (PEG) in the production of Mammalian Somatic Cell Hybrids" Polyethylene Glycol (PEG) "cytogene Cell Gene.16 (1-5): 399-.

Embryos derived by nuclear transfer have been suggested to be different from normal embryos and sometimes benefit from or even require in vivo culture conditions (at least in vivo) that are different from those under which embryos are normally cultured. The reason for this is not clear. In the routine manipulation of bovine embryos, reconstructed embryos (many of which are immediately) have been cultured in ovine oviducts for 5 to 6 days (as described by Willadsen in Mammalian Egg Transfer (Adams, E.E. eds.) 185CRCPress, Boca Raton, Fla. (1982)). In certain embodiments, embryos may be embedded in a protective medium, such as agar, prior to transplantation, and then excised from the agar after recovery from the temporary recipient. The function of the protective agar or other medium is twofold: first, it serves as a structural aid to the embryo by holding the zona pellucida together; and second, it acts as a barrier to cells of the recipient animal's immune system. Although this method increases the proportion of blastocyst-forming embryos, it has the disadvantage that many embryos may be lost.

The activated NT units can be cultured in a suitable in vitro medium until embryonic or stem cell-like cells and cell colonies are produced. Media suitable for the culture and maturation of embryos are well known in the art. Examples of known media that can be used for bovine embryo culture and maintenance include Ham 'sF-10 + 10% Fetal Calf Serum (FCS), tissue culture medium-199 (TCM-199) + 10% fetal calf serum, Tyrodes-albumin-lactate-pyruvate (TALP), Dulbecco's Phosphate Buffered Saline (PBS), Eagle's medium, and Whitten's medium. One of the most commonly used media for collection and maturation of oocytes is TCM-99 and 1 to 20% serum supplements, including fetal bovine serum, neonatal serum, estrus bovine serum, lamb serum or steer serum (steer serum). A preferred maintenance medium comprises TCM-199 and Earl salts, 10% fetal bovine serum, 0.2Ma pyruvate and 50ug/ml gentamicin sulfate. Any of the above may also include co-culture with various cell types, such as follicular cells, oviduct cells, BRL cells, and uterine cells, and STO cells.

In particular, epithelial cells of the human endometrium secrete Leukemia Inhibitory Factor (LIF) between the pre-implantation and implantation stages. Thus, addition of LIF to the culture medium may have an important role in enhancing the in vitro development of the reconstructed embryos. The use of LIF for embryonic or stem cell-like cell culture has been described in U.S. patent No. 5,712,156 (which is incorporated herein by reference).

Another maintenance medium is described in U.S. patent No. 5,096,822 to Rosenkrans, jr. This embryo culture medium (designated CR1) contains the nutrients necessary to support the embryo. cR1 contains calcium hemi-L-lactate in an amount in the range of 1.0mM to 10mM, preferably 1.0mM to 5.0 mM. The hemi-calcium L-lactate is an L-lactate salt having a hemi-calcium salt incorporated thereon.

In addition, suitable media for maintaining cultured human embryonic cells are exemplified by Thomson et al, Science, 282: 1145-: 7844-7848 (1995).

Thereafter, the cultured NT units are preferably washed and then placed in a suitable medium, such as CR1aa medium, Ham's F-10, tissue culture medium-199 (TCM-199), Tyrodes-albumin-lactate-pyruvate (TALP), Dulbecco's Phosphate Buffered Saline (PBS), Eagle medium or Whitten medium, preferably containing about 10% FCS. Such culturing will preferably be effected in a well plate containing a suitable confluent feeder layer. Suitable feeder layers include, for example, fibroblasts and epithelial cells, such as fibroblasts and uterine epithelial cells derived from ungulates, chicken fibroblasts, mouse (e.g., mouse or rat) fibroblasts, STO and SI-m220 feeder cell lines, and BRL cells.

In a preferred embodiment, the feeder cells will comprise mouse embryonic fibroblasts. Methods for preparing suitable fibroblast feeder layers are described in the examples below and are well within the ability of the ordinarily skilled artisan.

Methods for deriving ES cells from blastocyst-stage embryos (or equivalents thereof) are well known in the art. Such techniques may be used to derive ES cells from cloned embryos. Additionally or alternatively, ES cells may be derived from cloned embryos at an earlier developmental stage.

Applications of

In certain embodiments, the blastocyst or blastocyst-like cluster obtained by the present disclosure may be used to obtain an embryonic stem cell line. Such lines can be obtained, for example, according to Thoms on et al, Science, 282: 1145-: 7544-7848(1995) (incorporated herein by reference in their entirety).

Pluripotent embryonic stem cells can also be produced from a single blastomere that is removed from the embryo without interfering with the normal development of the embryo to birth. See the following U.S. applications: 60/624,827 filed on 11/4/2004; 60/662,489 filed on 3/14/2005; 60/687,158 filed on 3/6/2005; 60/723,066 filed on 3/10/2005; 60/726,775 filed on 14/10/2005; 11/267,555 filed on 4/11/2005; PCT application No. PCT/US05/39776, filed on 4.11.2005, the disclosure of which is incorporated by reference in its entirety; see also Chung et al, Nature, 2005, 10, 16 (electronically published before press) and Chung et al, Nature, volume 439, pages 216 and 219 (2006), the entire disclosures of each of which are incorporated by reference in their entirety.

In one aspect of the invention, the method comprises using cells derived from the reprogrammed cells of the invention in research and in therapy. Such reprogrammed pluripotent or totipotent cells can differentiate into any cell in the body, including without limitation skin, cartilage, bone, skeletal muscle, cardiac muscle, kidney, liver, blood and blood formation, vascular precursors (vasular precusor) and vascular epithelium, pancreatic beta, neurons, glia, retina, inner ear follicles (inner ear folliclele), gut, lung cells.

In particular, reprogrammed cells can differentiate into cells with a dermologically prenatal gene expression pattern that are highly elastic (angiogenic) or capable of regeneration without causing scarring. Dermal fibroblasts of mammalian fetal skin, especially in the areas corresponding to the coverings benefiting from high levels of elasticity, such as in the area around the joints, are responsible for the de novo synthesis of a complex structure of elastic fibers that function for many years without turnover. In addition, early embryonic skin can be regenerated without scarring. Cells at this point in time of embryonic development prepared from the reprogrammed cells of the present invention can be used to promote scar-free regeneration of skin, including the formation of normal elastin constructs. This is particularly useful in treating symptoms of the normal human aging process or actin-related skin damage, where there is profound elastic lysis of the skin, resulting in an aged appearance, including sagging and wrinkling of the skin.

In another embodiment of the invention, the reprogrammed cell is exposed to one or more differentiation inducers to produce other cells that are therapeutically useful, such as retinal pigment epithelium, hematopoietic and angioblast progenitors and many other useful cell types of ectoderm, mesoderm and endoderm. Such inducers include, but are not limited to: cytokines such as interleukin-alpha A, interferon-alpha A/D, interferon-beta, interferon-gamma-inducible protein-10, interleukin-1-17, keratinocyte growth factor, leptin, leukemia inhibitory factor, macrophage colony stimulating factor and macrophage inflammatory protein-1 alpha, 1-beta, 2, 3 alpha, 3 beta and monocyte chemotactic protein 1-3, 6kine, activin A, amphiregulin, angiogenin, B-epithelial cell growth factor, betacellulin, brain-derived neurotrophic factor, C10, cardiac neurotrophin-1, ciliary neurotrophic factor, cytokine-induced neutrophil chemoattractant-1, eotaxin (eotaxin)'s, Epidermal growth factor, epithelial neutrophil activating peptide-78, erythropoietin, estrogen receptor-alpha, estrogen receptor-beta, fibroblast growth factor (acidic and basic), heparin, FLT-3/FLK-2 ligand, glial cell line-derived neurotrophic factor, Gly-His-Lys, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, GRO-alpha/MGSA, GRO-beta, GRO-gamma, HCC-1, heparin-binding epidermal growth factor, hepatocyte growth factor, heregulin-alpha, insulin-growth factor-binding protein-1, insulin-like growth factor II, nerve growth factor, neurotrophin-3, 4. oncostatin M, placental growth factor, pleiotrophin, rantes (regulatory activation proteins expressed and secreted), stem cell factor, stromal cell derived factor 1B, thrombopoietin (thrombopoietin), transforming growth factors- (. alpha.,. beta.1, 2, 3,4, 5), tumor necrosis factor (A. and. beta.), vascular epithelial growth factor and bone morphogenetic protein, enzymes that alter the expression of hormones and hormone antagonists, such as 17B-estradiol, adrenocorticotropic hormone, adrenomedullin, alpha-melanocyte hormone, chorionic gonadotropin, corticosteroid binding globulin, corticosterone, dexamethasone, estriol, follicle stimulating hormone, gastrin 1, glucagon, gonadotropin, L-3, 3 ', 5' -triiodothyronine, luteinizing hormone (leutinizing hormone), L-thyroxine, melatonin, MZ-4, oxytocin, parathyroid hormone, PEC-60, pituitary growth hormone, progesterone, prolactin, secretin, sex hormone binding globulin, thyroid hormone, thyrotropin releasing factor, thyroxine (thyroxin) binding globulin and vasopressin, extracellular matrix components such as fibronectin, proteolytic fragments of fibronectin, laminin, tenascin, thrombospondin (thrombospondin), and proteoglycans such as aggrecan, heparan sulfate proteoglycans, chondroitin sulfate proteoglycans, and cohesin. Other inducers include cells from defined tissues or components derived from cells that are used to provide an induction signal to differentiated cells derived from reprogrammed cells of the invention. Such inducer cells may be derived from humans, non-human mammals or birds, such as Specific Pathogen Free (SPF) embryonic or adult cells.

In certain embodiments of the invention, clonal cells are introduced into tissues in which they normally reside to exhibit therapeutic utility. For example, a clonally generated population of cells derived by the methods of the invention can be introduced into a tissue. In certain other embodiments, the clonal cells are introduced systemically or at a distance from the site of desired therapeutic utility (cite). In such embodiments, the clonal cell can act at a distance from or at the desired site.

In certain embodiments of the invention, the clonal cells derived by the methods of the present invention are used to induce differentiation of other pluripotent stem cells. The generation of a single cell-derived cell population capable of proliferation in vitro while maintaining embryonic gene expression patterns can be used to induce differentiation of other pluripotent stem cells. Cell-cell induction is a common method of directing differentiation of early embryos. Many potentially medically useful cell types are affected by induction signals during normal embryonic development, including spinal cord neurons, heart cells, pancreatic beta cells, and terminal hematopoietic cells. A single cell-derived cell population capable of proliferation in vitro while maintaining embryonic gene expression patterns can be cultured under a variety of in vitro, in ovo (in ovo) or in vivo culture conditions that induce differentiation of other pluripotent stem cells into desired cell or tissue types.

The subject embryonic or stem-like cells can be used to obtain any desired differentiated cell type. The therapeutic use of such differentiated human cells is unparalleled. For example, human hematopoietic stem cells may be used in medical treatments requiring bone marrow transplantation. Such procedures are useful in the treatment of a number of diseases, e.g., advanced cancers, such as ovarian cancer and leukemia, and diseases that compromise the immune system, such as AIDS. Hematopoietic stem cells can be obtained, for example, by fusing adult somatic cells (e.g., epithelial cells or lymphocytes) of a cancer or AIDS patient with an enucleated oocyte (e.g., bovine oocyte), obtaining embryonic or stem cell-like cells as described above, and culturing such cells under conditions conducive to differentiation until hematopoietic stem cells are obtained. Such hematopoietic cells are useful in the treatment of diseases including cancer and AI DS.

Alternatively, adult somatic cells of a patient with a neurological disorder can be fused with an enucleated animal oocyte (e.g., a primate or bovine oocyte), thereby obtaining a human embryonic or stem cell-like cell, and culturing such cells under differentiation conditions to generate a neural cell line. Specific diseases that can be treated by transplantation of such human neural cells include, for example, parkinson's disease, alzheimer's disease, ALS, and cerebral palsy, among others. In the specific case of parkinson's disease, transplanted fetal brain nerve cells have been shown to connect correctly with peripheral cells and produce dopamine. This can cause long-term reversal of parkinson's disease symptoms.

To allow specific selection of differentiated cells, donor cells can be transfected with a selectable marker expressed by an inducible promoter, thereby allowing selection or enrichment of a particular cell lineage upon induction of differentiation. For example, CD34-neo can be used to select hematopoietic cells, Pw1-neo for muscle cells, Mash-1-neo for sympathetic neurons, Mal-neo for CNS neurons of gray matter of the human cerebral cortex, etc.

It is a great advantage of the present invention to provide an essentially unlimited supply of isogenic or isogenic (synegenic) human cells suitable for transplantation. It will therefore eliminate the significant problem associated with current transplantation procedures, namely rejection of the transplanted tissue, which may occur due to host-versus-graft or graft-versus-host rejection. Conventionally, rejection is prevented or reduced by administering an anti-rejection drug such as cyclosporine. However, such drugs have significant adverse side effects, such as immunosuppression, carcinogenic properties, and are very expensive. The present invention should eliminate or at least greatly reduce the need for antirejection drugs such as cyclosporine, azathioprine, FK-506, glucocorticoids, and rapamycin and its derivatives.

Other diseases and conditions treatable by isogene cell therapy include, for example, spinal cord injury, multiple sclerosis, muscle atrophy, diabetes, liver disease, i.e., hypercholesterolemia, heart disease, cartilage replacement, disability, foot ulcers, gastrointestinal disease, vascular disease, kidney disease, urinary tract disease, and aging-related diseases and conditions.

Methods for cloning mammals from cloned embryos are well known in the art. The two main procedures used to clone mammals are the Roslin method and the Honolulu method. These programs were named after the production of sheep Dolly (Campbell, K.H. et al (1996) Nature 380: 64-66) studied by Roslin, Scotland, 1996 and mouse Cumulina, Hawaii university, honolulu, 1998 (Wakayama, T. et al (1998) Nature 394: 369-.

In other embodiments, the methods of the invention can be used to produce cloned split-stage or morula-stage embryos that can be used as parent embryos. Such maternal embryos can be used to produce ES cells. For example, blastomeres (1, 2, 3,4 blastomeres) can be removed or dissected in vivo from such maternal embryos and such blastomeres can be used to derive ES cells.

Blastomere culture

Previous attempts to induce the development of isolated human blastomeres into pluripotent embryonic stem cells failed (Geber S. et al, hum. reprod.10: 1492-1496 (1995)). The present invention is based in part on the following findings: using the novel methods disclosed herein, stem cells can be produced from an embryo without affecting the viability of the embryo. In one embodiment, these methods utilize in vitro techniques related to those currently used in pre-implantation genetic diagnosis (PGD) to isolate individual blastomeres from embryos without destroying the embryos or otherwise significantly altering their viability. As demonstrated herein, pluripotent human embryonic stem (hES) cells and cell lines can be generated from a single blastomere taken from an embryo without interfering with the normal development of the embryo to birth.

The methods described herein have many important uses that will facilitate the fields of stem cell research and developmental biology. ES cells, ES cell lines, TS cells and cell lines and cells differentiated therefrom are useful for studying fundamental developmental biology and are therapeutically useful in the treatment of a number of diseases and disorders. In addition, these cells can be used in screening assays to identify factors and conditions that can be used to modulate the growth, differentiation, survival or migration of these cells. The identified agents can be used to modulate cellular behavior in vitro and in vivo, and can form the basis of cellular or cell-free therapies.

The following detailed description is presented to provide a thorough understanding of the invention described herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

All publications, patents, patent publications and applications, and other documents mentioned herein are incorporated by reference in their entirety.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

The term "blastomere" always refers to at least one blastomere (e.g., 1, 2, 3,4, etc.) obtained from an embryo. The term "cluster of two or more blastomeres" is used interchangeably with "blastomere-derived growth" and refers to cells produced during in vitro culture of blastomeres. For example, after a blastomere is obtained from an embryo and initially cultured, it typically splits at least once to produce clusters of two or more blastomeres (also known as blastomere-derived growths). The clusters may be further cultured with embryonic or fetal cells. Eventually, the blastomere-derived growth will continue to divide. From these structures, ES cells, TS cells and partially differentiated cell types will develop during the course of the culture process.

As summarized above, the present invention provides methods for deriving ES cells, ES cell lines and differentiated cell types from a single blastomere of an early embryo without destroying the embryo. Various features of the method are described in detail below. All combinations of the various aspects and embodiments of the invention described above and in detail below are contemplated.

Blastomere removal

Blastomeres may be removed from the embryo at various developmental stages prior to implantation, including but not limited to: before morula densification, during morula densification, after morula densification, before blastocyst cavity formation or during the blastocyst stage. In certain embodiments, blastomeres (one blastomere, two blastomeres, or more than two blastomeres) are removed from the embryo at the 4-16 cell stage, or the 4-10 cell stage, or the 4-8 cell stage.

In one embodiment, the invention provides a method of biopsiing blastocysts that will give rise to embryonic stem cells, while the remainder of the blastocyst is implanted and causes pregnancy and later live birth. In one example of such a situation: the zona pellucida is removed from the blastocyst by any method known to one of ordinary skill in the art, and the blastocyst is then biopsied.

In another embodiment, the contention related to derivation of human ES cells is addressed by using a technique similar to that used in pre-implantation genetic diagnosis (PGD), in which a single blastomere is removed from the embryo. In one embodiment, the single blastomere is removed prior to morula densification. The dissected blastomeres may be allowed to undergo cell division and one progeny cell used for genetic testing while the remaining cells are used to produce human stem cells. The biopsied embryos can also be implanted at the blastocyst stage, or frozen for implantation at a later time.

In certain embodiments, the biopsy (e.g., removal of a blastomere from an embryo) consists of two stages. The first stage is to punch a hole in the transparent tape surrounding the embryo or in some cases to remove it completely. After punching, the cells (preferably one or both) can then be removed from the human embryo. In certain preferred embodiments, the method comprises removing the zona pellucida or creating extraction holes therein, and the method may be performed by one or more techniques, such as physical manipulation, chemical treatment, and enzymatic digestion. Exemplary techniques that may be used include:

partial band cleavage (PZD): partially cutting the transparent band using a micropipette;

tape drilling (Zona drilling): chemical opening on the clear band by partial digestion with Tyrode acid;

a belt drilling method: enzymatic opening on the clearing zone by partial digestion with pronase or other protease;

thinning method of transparent belt: thinning the transparent belt by Tyrode acid or laser;

carrying out point-shaped opening on the transparent belt by using laser;

the transparent band was mechanically opened in a punctiform fashion using a Piezo micromanipulator.

To briefly illustrate one embodiment, the procedure is performed using 8-10 cell stage embryos. The embryos are placed in a drop of biopsy medium under mineral oil by holding them with a holding pipette. Zona pellucida was digested locally by releasing acidified Tyrode solution (Sigma, st. louis, mo.63178) via assisted incubation pipette (assisted incubation pipette). After punching, cells (blastomeres) can be aspirated through the hole.

To illustrate another embodiment, the zona pellucida of the blastocyst may be at least partially digested by treatment with one or more enzymes or a mixture of enzymes, such as pronase. Brief pronase (Sigma) treatment of blastocysts with intact zona pellucida resulted in the removal of zona pellucida. Other types of proteases having the same or similar protease activity as pronase can also be used.

Can also be prepared by adding Ca in the absence of Ca⁺⁺/Mg⁺⁺The zona pellucida-stripped embryos were disaggregated in PBS to obtain individual blastomeres.

The present invention also provides a novel and more efficient method of isolating individual blastomeres. The embryos are fixed and then tapped until the individual blastomeres are released from the blastocyst. The method is not limited to human embryos and can be performed on embryos of other species, including, without limitation, non-human embryos, such as non-human mammals, mice, rabbits, pigs, cows, sheep, dogs, and primates.

Embryos can be fixed by any method known to those skilled in the art. In one embodiment, the embryo is fixed using a micropipette and the micropipette handle is gently stroked to isolate the blastomere. In another embodiment, the embryos are cultured in a medium that is free of calcium and magnesium. The embryo may be in the 2-cell stage to the 16-cell stage. In one embodiment, the embryo is at the 4-10 cell stage. In another embodiment, the embryo is a 6-8 cell stage embryo. In yet another embodiment, the embryo is an 8-10 cell stage embryo. In certain embodiments, tapping comprises generating a force sufficient to remove at least one blastomere without substantially reducing the viability of the remainder of the embryo. The maintenance of viability may be demonstrated, for example, by culturing the remaining embryos for at least one day and confirming that the remaining embryos can continue to divide under culture.

Any of the foregoing methods may be used to obtain a blastomere (one blastomere or more than one blastomere) from an embryo. The specific method may be used alone or in combination with another method that facilitates blastomere removal.

In certain embodiments, the embryo is a mammalian embryo. In certain embodiments, the mammalian embryo is a human embryo. Exemplary mammals include, but are not limited to, mice, rats, rabbits, cows, dogs, cats, sheep, hamsters, pigs, non-human primates, and humans.

In some of any of the foregoing embodiments, the blastomere is removed from the embryo without damaging the remainder of the embryo. The remaining embryos (embryos minus the removed blastomeres) may be cultured and/or cryopreserved. In certain embodiments, the remaining embryos are cultured for a sufficient time to confirm that the remaining embryos can continue to divide (e.g., remain viable), and then when viability is confirmed, the remaining embryos are cryopreserved. In certain other embodiments, the remaining embryos are immediately cryopreserved.

In certain other embodiments, multiple blastomeres are removed from a single embryo, and the embryo is destroyed during or after the removal of the multiple blastomeres. Multiple blastomeres may be used together in an experiment, for example, by aggregating multiple blastomeres in an initial blastomere culture. Alternatively, multiple blastomeres may be used in separate experiments in an effort to maximize the number of lines or cell types that can be produced from a single embryo.

The embryo from which the blastomere is obtained may be produced by sexual or asexual methods. In certain embodiments, the embryo is produced by fertilizing an egg with a sperm. In certain other embodiments, the embryo is produced by somatic cell nuclear transfer, parthenogenesis, parthenogenetic development, or other asexual techniques. Note that embryos derived from asexual techniques may not look identical to embryos produced by fertilization. However, despite any differences in appearance, the term embryo is intended to include products of asexual reproduction and products of fertilization or other sexual reproduction methods.

Production of cultured blastomeres and ES cells

After removal from the embryo, the isolated blastomeres can be initially cultured on any type of medium, such as embryo culture medium, such as Quinn's division medium (Cooper surgical inc. cat # ART 1529). Any medium that supports embryo growth may be used, including without limitation any commercial formulation. As used herein, the term "embryo culture medium" is used to refer to a medium that promotes the survival of blastomeres (particularly human blastomeres) in culture. In certain embodiments, the embryo culture medium is a medium containing less than 5mM glucose. In certain embodiments, the embryo culture medium is a medium having an osmolality of less than 310 mosm. In certain other embodiments, the embryo culture medium is a medium containing less than 5mM glucose and having an osmolality of less than 310 mosm. In certain embodiments, the medium used to initially cultivate the blastomeres has an osmolality of less than 300mosm, less than 280mosm, or less than 260mosm, and optionally contains less than 5mM glucose. In certain embodiments, the medium used to initially culture the blastomeres has an osmolality of about 260-. Note that regardless of the osmolality and specific concentration of glucose in the medium used to initially culture the blastomeres, the medium may also be supplemented with antibiotics, minerals, amino acids and other factors commonly found in commercial medium formulations.

Blastomeres may not grow well initially in standard ES cell culture media. However, as described in detail herein, after the blastomeres are cultured and/or allowed to divide one or more times in the presence of certain embryonic or fetal cells, the blastomere clusters may optionally be cultured in the ES cell culture medium or may be slowly transferred from the embryo culture medium to the ES cell culture medium by gradually replacing the medium. As used herein, the term "ES cell culture medium" is used to refer to a medium that facilitates the maintenance of ES cells in culture, and that can be used to culture blastomere clusters as they continue to divide and produce ES cells, ED cells, and the like. Such media are optimized, at least to some extent, for ES cells. In certain embodiments, the ES cell culture medium contains at least 5mM glucose (relatively high glucose). In certain other embodiments, the ES cell culture medium has an osmolality of at least 310 mosm. In certain other embodiments, the medium contains at least 5mM glucose and has an osmolality of at least 310mo sm. In certain embodiments, the medium has an osmolality of at least 320mosm or at least 330mosm, and optionally contains at least 5mM glucose. In certain embodiments, the medium has an osmolality of about 310-340mosm, and optionally contains at least 5mM glucose. The ES cell culture medium may also be supplemented with factors known in the art to promote ES cell growth, and the medium may contain antibiotics, minerals, amino acids, and other factors commonly found in commercial medium preparations. In certain embodiments, the pre-nuclear stage human embryos are cultured in Quinn's division medium (Cooper Surgical).

In certain embodiments, the pronuclear stage human embryos are cultured up to the 8-cell stage. In certain embodiments, the pronuclear stage embryos can be cultured at up to about the 2-cell stage, 4-cell stage, or 16-cell stage. In certain embodiments, the pronuclear stage embryos can be cultured up to the 2-cell stage and 4-cell stage, the 2-cell stage and 8-cell stage, the 2-cell stage and 16-cell stageThe cellular phase, the 4-cell phase and the 8-cell phase, the 4-cell phase and the 16-cell phase or between the 8-cell phase and the 16-cell phase. In certain embodiments, Ca-free supplemented with 0.05% PVA⁺⁺And Mg⁺⁺Pre-incubating embryos in phosphate buffered saline. In certain embodiments, the embryo is preincubated at room temperature for about 5, 10, 15, 20, 25, 30, 5-10, 5-15, 5-30, 10-15, 10-30, or 15-30 minutes. In certain embodiments, embryos are transferred to Quinn's hepes medium for manipulation.

In certain embodiments, the PIEZO is used to isolate individual blastomeres from an embryo. In certain embodiments, a hole (500 μm in diameter) is punched in the tape prior to insertion of the biopsy pipette. In certain embodiments, the holes may be made by applying several PIEZO pulses using a small (20 μm) pipette. In certain embodiments, a biopsy pipette (500 μm) is inserted through the hole to grasp the blastomere using mild negative pressure. In certain embodiments, the blastomere (blastomeres) is pulled away when its 2/3 is inside the pipette. In certain embodiments, the blastomere 1/3, 1/2, or 3/4 is inside the pipette. In certain embodiments, the blastomere 1/3 to 1/2, 1/3 to 2/3, 1/3 to 3/4, 1/2 to 2/3, 1/2 to 3/4, or 2/3 to 3/4 is inside the pipette.

In certain embodiments, after biopsy, maternal embryos and blastomeres can be returned to the original culture drops (Quinn's split medium) and cultured together for 12 to 18 hours. In certain embodiments, after biopsy, the maternal embryos and blastomeres can be returned to the original culture drop (Quinn's split medium) and cultured together for about 6 to 12, 6 to 18, 6 to 24, 12 to 18, 12 to 24, or 18 to 24 hours. In certain embodiments, the maternal embryo is transferred to blastocyst medium (Quinn's blastocyst medium). In certain embodiments, blastomeres are transferred to a small culture drop (50 μ l) containing MEF. In certain embodiments, the blastomere medium may be supplemented with laminin, fibronectin, or Matrigel (Matrigel). In certain embodiments, the blastomeres are cultured for about 3,4, 5,6, 7, or 8 days. In certain embodiments, the blastomeres are cultured in the same medium until they form a cell mass comprising about 20 cells. In certain embodiments, GFP ES cell culture drops may be combined with blastomere culture drops to allow the two media to mix together. In certain embodiments, some or all of the blastomere masses may be removed and plated on the same culture drop after about 12, 18, 24, 36, or 48 hours.

In certain embodiments, blastomeres are obtained from human or other mammalian embryos and cultured in embryo culture media. Preferably, the blastomeres are cultured in embryo culture medium for at least one day or until the blastomeres divide at least once. However, the blastomeres may be cultured in embryo culture media for more than 1 day (at least 2, 3,4, etc.), and/or the blastomeres may be cultured in contact with embryonic or fetal cells prior to cleavage to produce blastomere (blastoemre) clusters. When cultured in embryo culture media, blastomeres may split one or more times, or a cluster of two or more blastomeres is produced. Further culturing the cluster of blastomeres comprises culturing the blastomeres with their progeny. In certain embodiments, the blastomeres divide and their progeny are cultured as aggregates.

In one embodiment, the blastomeres may be cultured in microdroplets. Each droplet may contain a single blastomere or multiple blastomeres. After about at least 1 day, at least 2-3 days, or at least 4 days, the cultured blastomeres may divide and form vesicles or aggregates. A benefit of culturing blastomeres prior to direct or indirect contact with embryonic cells is to prevent the embryonic cells from overgrowing the blastomeres.

After initial culture of the blastomeres to produce clusters of two or more blastomeres, the cultured clusters of two or more blastomeres are contacted with the embryonic or fetal cells, either directly or indirectly, or alternatively, with a medium that promotes further maturation of the blastomeres in the absence of the embryonic or fetal cells. Such media include media conditioned with embryonic or fetal cells (conditioned media) or media supplemented with growth factors or cytokines that promote blastomere maturation. In certain embodiments, the medium is supplemented with ACTH (adrenocorticotropic hormone).

For embodiments in which direct or indirect culture with embryonic or fetal cells is used, the embryonic or fetal cells may be derived from, for example, a mammal. In certain embodiments, the embryonic or fetal cell is a mouse or human cell. Exemplary embryonic or fetal cells include, but are not limited to, Embryonic Stem (ES) cells (whether derived from blastocysts, blastomeres, or by other means, and whether derived using somatic cell nuclear transfer or other asexual propagation), embryonic germ cells, embryonic carcinoma cells, placental cells, trophoblast/trophectoderm cells, trophoblast stem cells, primordial germ cells, amniotic fluid cells, amniotic membrane stem cells, placental stem cells, and umbilical cord cells. In certain embodiments in which the blastomeres are contacted directly or indirectly with embryonic or fetal cells, the medium in which the blastomeres are cultured is further supplemented with ACTH or other growth factors or cytokines that promote blastomere maturation.

When used, embryonic or fetal cells can be cultured in the presence or absence of a feeder layer of cells. Feeder cells can be used to help maintain embryonic or fetal cells and prevent them from differentiating. The particular feeder cells may be selected based on the particular embryonic or fetal cells used. Exemplary feeder cells include, but are not limited to, fibroblast feeder cells. Such fibroblast feeder cells may be derived from the same species as the embryonic or fetal cells, or they may be derived from a different species. Similarly, feeder cells and embryonic or fetal cells may be derived from the same species as blastomeres or from a different species. In certain embodiments, feeder cells are irradiated or otherwise treated to prevent overgrowth with respect to embryonic or fetal cells. Exemplary feeder cells include, but are not limited to, mouse embryonic fibroblasts (MEF cells), human embryonic fibroblasts, human foreskin fibroblasts, human skin fibroblasts, human endometrial fibroblasts, human fallopian tube fibroblasts, and placental cells. Similar cell types derived from other animals (mammals, chickens, etc.) are also contemplated.

In one embodiment, the feeder cells and/or embryonic cells are human cells that are autologous cells derived from the same embryo as the blastomere.

Embryonic or fetal cells are grown in ES cell culture medium or any medium that supports the growth of embryonic or fetal cells, such as Knockout DMEM (Invitrogen Cat # 10829-018). Exemplary embryonic or fetal cells include, but are not limited to, embryonic stem cells (such as from established lines), embryonic carcinoma cells, murine embryonic fibroblasts, other embryonic-like cells, cells of embryonic origin, or cells derived from embryos, many of which are known in the art and available from the american type culture collection (Manassas, VA 20110-.

The embryonic or fetal cells may be added directly to the cultured blastomeres or may be grown in close proximity to, but not in direct contact with, the cultured blastomeres. Various direct and indirect co-culture systems may be useful to provide factors or signals from embryonic or fetal cells to cultured blastomeres. As used herein, "contacting a cluster of two or more blastomeres in culture" refers to any method of direct or indirect contact or co-culture.

In certain embodiments, contacting clusters of two or more blastomeres comprises aggregating the clusters of blastomeres with embryonic or fetal cells. In certain other embodiments, contacting comprises co-culturing a cluster of two or more blastomeres such that the cells are in direct contact with but do not aggregate with embryonic or fetal cells. In other embodiments, contacting comprises co-culturing a cluster of two or more blastomeres with embryonic or fetal cells such that the cells are in indirect contact, e.g., held in the same culture vessel without direct contact of the cells, or as a continuous droplet.

In certain embodiments, the method comprises the step of contacting a cultured cluster of two or more blastomeres with an embryonic or fetal cell, either directly or indirectly, provided that the contacting is not performed by aggregating the cultured blastomeres with embryonic cells, such as Chung et al, Nature (2006) 439: 216-9. Alternatively, the culture of blastomeres and the culture of embryonic or fetal cells are indirectly linked or combined. This can be accomplished by any method known in the ART including, for example, drag manipulating the pipette between two drops under a light mineral oil such as Cooper Surgical ACT # ART4008, paraffin oil or Squibb's oil. The connection may be made by using a glass capillary or similar device. Such indirect linkage between the cultured blastomeres and the embryonic cells allows for gradual mixing of the embryo culture medium in which the blastomeres are cultured and the ES cell culture medium in which the human embryonic cells are cultured. In another embodiment, blastomeres may be co-cultured with the remaining embryos. For example, the blastomeres and remaining embryos are co-cultured in a microdroplet culture system or other culture system known in the art that does not allow cell-cell contact but allows cell-secreted factors and/or cell-matrix contact. The volume of the droplet may be reduced (e.g., from 50. mu.l to about 5. mu.l) to enhance the signal. In another embodiment, the embryonic cells can be from a species other than human, such as a non-human primate or mouse.

In certain embodiments, the particular medium formulation used to culture the blastomeres, clusters of two or more blastomeres, and embryonic or fetal cells may vary slightly depending on the species. In addition, whether the initial blastomere culture benefits from a different medium formulation than that used to culture the clusters of blastomeres or embryonic cells may also vary slightly from species to species.

In certain embodiments, the medium used to culture blastomeres alone and the medium used to culture embryonic or fetal cells need not be the same. In embodiments where the medium is different, it will take a period of time for the blastomere or cluster of blastomeres to be initially exposed to a medium different from the medium in which the blastomere was initially cultured (e.g., the cells will be slowly exposed to the medium in which the embryonic or fetal cells are cultured). In such embodiments, clusters of two or more blastomeres, which have now divided multiple times to produce a population of cells and a halo of cell growth, may be gradually transferred (e.g., by exchanging media) and cultured in media having the characteristics of ES cell media.

After about 3-4 days, blastomeres exhibited characteristics of ES cells. In particular, as cells continue to divide and blastomere progeny cluster, various cell types emerge and can be identified phenotypically. The cell types that occur include trophectoderm-like cells, ES cells, and partially or terminally differentiated ED cells. Likewise, these methods can be used to generate ES cells, TS or other trophectodermal cells, or ED cells. While not wishing to be bound by any particular theory, it is believed that after several days or weeks, cultured blastomeres exhibit ES cell growth, possibly as a result of factors secreted by embryonic or fetal cells or by the extracellular matrix. Further, the progeny of the disrupted blastomere clusters are similar in some respects to the changes observed during development of the blastocyst prior to implantation. Thus, the cell types that appear in these cultures recapitulate to some extent the cell types observed when plating intact blastocysts or ICMs.

In certain embodiments, blastomere culture conditions may include exposure of the cells to factors that inhibit or otherwise enhance cell differentiation, such as preventing differentiation of the cells into non-ES cells, trophectoderm, or other cell types. Such conditions may include contacting the cultured cells with heparin or introducing Oct-4 into the cells (such as by including Oct-4 in the culture medium) or activating endogenous Oct-4 in the cells. In yet another embodiment, expression of CDX-2 is prevented by any method known in the art, including, without limitation, introducing CDX-2RNAi into the blastomere, thereby inhibiting differentiation of the blastomere into a TS cell.

In certain embodiments, the blastomere medium is supplemented with factors that inhibit differentiation into non-ES cells. In certain embodiments, laminin is added to the culture medium to inhibit differentiation into non-ES cells. In certain embodiments, the medium is supplemented with about 2.5, 5, 7.5, 10, 15, or 20 μ g/ml laminin. In certain embodiments, the medium is supplemented with 1-5, 1-10, 5-10, 10-20, or 1-20 μ g/ml laminin.

In certain embodiments, the medium is supplemented with a factor that disrupts tight junctions. In certain embodiments, laminin is added to the culture medium to disrupt tight junctions.

In certain embodiments, the medium is supplemented with factors that inhibit the trophectoderm differentiation pathway. In certain embodiments, laminin is added to the culture medium to inhibit the trophectoderm differentiation pathway.

In certain embodiments, the medium is supplemented with a factor that depolarizes the cell. In certain embodiments, laminin is added to the culture medium to depolarize the cells. In certain embodiments, depolarization is determined by the absence of microvilli on the cell surface. In certain embodiments, depolarization is determined by stacking cells to form a multi-layered structure.

As detailed above, the invention provides methods for producing ES cells, ED cells and TS cells from blastomeres obtained from embryos. This method can be used to generate ES cells, ED cells and TS cells and cell lines without disrupting the embryo from which the blastomere is obtained.

Production of cultured blastomeres and ED cells

Long-term culture of inner cell mass cells has been used in the past to generate embryonic stem cell lines. Embryonic stem cells are then cultured, conditionally genetically modified, and induced to differentiate to produce cells for therapeutic use. U.S. patent application No. 11/025,893 (published as US 2005/0265976a1), incorporated herein in its entirety, describes methods of directly inducing differentiation of inner cell mass cells or morula-derived cells to generate differentiated progenitor cells from these cells without the need to generate embryonic stem cell lines and the use of the differentiated cells, tissues and organs in transplantation therapies. Since these cells are derived from cells of the embryo, rather than the ES cell line, we have named such cells as embryo-derived (ED) cells. Blastomere-derived ED cells have a wider differentiation potential than human ES cells produced using methods known in the art, since ED cells can be readily differentiated into germline cells using techniques known in the art, such as using methods for differentiating murine ES cell lines into germline cells. In contrast, human ES cell lines derived from inner cell masses (inner mass cells) are not expected to be capable of differentiating into germline cells. This phenomenon has been observed in ES cells derived from inner cell masses (inner mass cells) in animals such as pigs, cattle, chickens and rats, and it is likely that the germline is one of the first cell lineages that branch in differentiation.

In some methods of the invention, blastomeres from an embryo having at least two cells are induced to differentiate directly into differentiated progenitor cells prior to the embryo entering the stage of densified morula development, which are then used for cell therapy and to generate cells, tissues and organs for transplantation. If desired, the genetic modification may be introduced, for example, into somatic cells prior to nuclear transfer to produce morulae or blastocysts, or into somatic cells prior to reprogramming said somatic cells into undifferentiated cells by juxtaposing (juxtaposition) their DNA with a factor capable of reprogramming said somatic cells, or into ES cell lines prepared using these methods. See U.S. patent application No. 10/831,599 (published as US 2004199935), PCT/US06/30632 filed on 8/3 2006, and U.S. provisional patent application nos. 60/705,625, 60/729,173 and 60/818,813, the disclosures of which are incorporated herein by reference in their entirety. Thus, the differentiated progenitor cells of the invention do not possess the pluripotency of embryonic stem cells or embryonic germ cells and are tissue-specific, partially or fully differentiated cells in nature. These differentiated progenitors can give rise to cells from any of the three embryonic germ layers (i.e., endoderm, mesoderm, and ectoderm). For example, differentiated progenitors can differentiate into bone, cartilage, smooth muscle, skin with a prenatal gene expression pattern and capable of promoting scar-free wound repair, and hematopoietic or angioblasts (mesoderm), terminal endoderm, liver, primitive digestive tract, pancreatic beta cells, and respiratory epithelium (endoderm); or neurons, glial cells, hair follicles, or eye cells (including retinal neurons and retinal pigment epithelium).

Furthermore, the differentiated progenitor cells of the invention need not express the catalytic component of telomerase (TERT) and need not be immortalized, or the progenitor cells need not express cell surface markers found in embryonic stem cells, such as the cell surface markers characteristic of primate embryonic stem cells: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, alkaline phosphatase activity is positive, while SSEA-1 is negative. Furthermore, the differentiated progenitor cells of the invention are distinct from embryoid bodies, i.e., embryoid bodies are derived from embryonic stem cells, whereas the differentiated stem cells of the invention are derived from blastomeres.

Preferably, the differentiated cells of the invention are produced by culturing blastomere-derived cells in the absence of embryonic stem cells. For example, the growth of undifferentiated embryonic stem cells may be prevented by culturing blastomeres in the presence of a differentiation-inducing agent or by introducing a genetic modification to the cells such that the growth of the embryonic stem cells is prevented.

Any vertebrate embryo can be used as a source of blastomeres or cells that are developmentally equivalent to mammalian blastomeres. In particular, human blastomeres have important utility in generating human cell-based therapies. The primary embryo can be produced by in vitro fertilization, derived by fertilization in the reproductive tract by normal sexual reproduction, artificial insemination or gamete intrafallopian transfer (GIFT) and subsequent restoration, and derived by somatic cell nuclear transfer.

Differentiation

Methods of isolating blastomeres have been described herein. Isolated blastomeres can be induced, directly or via cells or cell lines, to differentiate in the presence of differentiation-inducing conditions including various combinations of growth factors, serum, hormones, extracellular matrix useful in producing a particular desired differentiated cell type, as known in the art (for a list of exemplary molecules, see table 1), or as disclosed in the following pending applications: PCT/US2006/013573, filed on day 11, 2006, US 60/835,779, filed on day 3, 8, 2006, 60/792,224, filed on day 14, 4, 2006, 60/801,993, filed on day 19, 5, 2006, PCT/US 2006/013519, filed on day 11, 4, 2006, US 11/025,893 (published as US 20050265976), WO2005/070011, published on day 4, 2005, and WO 2006/080952, published on day 3, 8, 2006, the disclosures of which are incorporated herein by reference. For example, blastomere or ES cells can be cultured on various inducer cell types, such as those isolated as single cell-derived cell populations, or on specific extracellular matrix components and other differentiation inducing factors, such as the factors or combinations of factors shown in Table 1 below.

TABLE 1

Culture variables

EGF ligands

1) Amphiregulin

2) Beta animal cellulose

3)EGF

4) Watch source (Epigen)

5) Epidermal regulin (Epireglu in)

6)HB-EGF

7) Neuregulin-3

8) NRG1 isoform GGF2

9) NRG1 isoform SMDF

10)NRG1-α/HRG1-α

11)TGF-α

12)TMEFF1/Tomoregul in-1

13)TMEFF2

14) EGF ligand set (1-13 above)

EGF R/ErbB receptor family

15) EGF receptor

16)ErbB2

17)ErbB3

18)ErbB4

19) EGF/ErbB receptor sets (15-18 above)

FGF ligands

20) FGF acidity

21) Basic of FGF

22)FGF-3

23)FGF-4

24)FGF-5

25)FGF-6

26)KGF/FGF-7

27)FGF-8

28)FGF-9

29)FGF-10

30)FGF-11

31)FGF-12

32)FGF-13

33)FGF-14

34)FGF-15

35)FGF-16

36)FGF-17

37)FGF-18

38)FGF-19

39)FGF-20

40)FGF-21

41)FGF-22

42)FGF-23

43) FGF ligand set (20-38 above)

FGF receptors

40)FGF R1

41)FGF R2

42)FGF R3

43)FGF R4

44)FGF R5

45) FGF receptor set (40-44 above)

FGF modulators

46)FGF-BP

Hedgehog factor (Hedgehogs)

47) Desert hedgehog factor

48) Sonic hedgehog factor

49) Indian hedgehog factor

50) Hedgehog factor set (above 47-49)

Hedgehog modulators

51)Gas1

52)Hip

53) Hedgehog regulator set (above 51-52)

IGF ligands

54)IGF-I

55)IGF-II

56) IGF ligand set (54-55 above)

IGF-I receptor (CD221)

57)IGF-IR

GF binding protein (IGFBP) family

58)ALS

59IGFBP-4

60)CTGF/CCN2

61)IGFBP-5

62) Endocan (endothelial cell specific molecule)

63)IGFBP-6

64)IGFBP-1

65)IGFBP-rp1/IGFBP-7

66)IGFBP-2

67)NOV/CCN3

68)IGFBP-3

69) GF binding protein family Collection (58-68 above)

Receptor tyrosine kinases

70)Ax1

71)C1q R1/CD93

72)DDR1

73)Flt-3

74)DDR2

75)HGF R

76)Dtk

77)IGF-II R

78)Eph

79) Insulin R/CD220

80)EphA1

81)M-CSF R

82)EphA2

83)Mer

84)EphA3

85)MSP R/Ron

86)EphA4

87)MuSK

88)EphA5

89)PDGF R α

90)EphA6

91)PDGF R β

92)EphA7

93)Ret

94)EphA8

95)ROR1

96)EphB1

97)ROR2

98)EphB2

99)SCF R/c-kit

100)EphB 3

101)Tie-1

102)EphB4

103)Tie-2

104)EphB6

105)TrkA

106)TrkB

107)TrkC

108)VEGF R1/Flt-1

109)VEGF R2/Flk-1

110)VEGF R3/Flt-4

111) Receptor tyrosine kinase pool (70-110 above)

Proteoglycans

112) Aggrecan

113) Luminal proteoglycans

114) Biglycan proteoglycan

115)Mimecan

116) Decorin proteoglycans

117)NG2/MCSP

118)Endocan

119)Osteoadherin

120)Endorepellin

121) syndecan-1/CDl 38

122) Glypican 2

123) Syndecan-3

124) Glypican 3

125) Testosterone proteoglycan 1/SPOCK1

126) Glypican 5

127) Testosterone proteoglycan 2/SPOCK2

128) Glypican 6

129) Testosterone proteoglycan 3/SPOCK3

130) Heparan sulfate proteoglycans

131) Heparin

132) Chondroitin sulfate proteoglycan

133) Hyaluronic acid

134) Dermatan sulfate proteoglycan

Proteoglycan modulators

135) Arylsulfatase A/ARSA

136)HAPLN1

137) Exostosin-like 2

138)HS6ST2

139) Exostosin-like 3

140)IDS

141) Proteoglycan modulator set (135-140 above)

SCF, F1t-3 ligand & M-CSF

142)Flt-3

143)M-CSF R

144) Flt-3 ligands

145)SCF

146)M-CSF

147)SCF R/c-kit

148) Aggregate factor (142-

Activating element

149) Activin A

150) Activin B

151) Activin AB

152) Activin C

153) Collection activin (149-

BMP (bone morphogenetic protein)

154)BMP-2

155)BMP-3

156)BMP-3b/GDF-10

157)BMP-4

158)BMP-5

159)BMP-6

160)BMP-7

161)BMP-8

162)Decapentaplegic

163) Aggregated BMP (154-162 above)

GDF (growth differentiation factor)

164)GDF-1

165)GDF-2

166)GDF-3

167)GDF-4

168)GDF-5

169)GDF-6

170)GDF-7

171)GDF-8

172)GDF-9

173)GDF-10

174)GDF-11

175)GDF-12

176)GDF-13

177)GDF-14

178)GDF-15

179) GDF Assembly (164 + 178 above)

GDNF family ligands

180)Artemin

181)Neurturin

182)GDNF

183)Persephin

184) GDNF ligand set (180 + 183 above)

TGF-β

185)TGF-β

186)TGF-β1

187)TGF-β1.2

188)TGF-β2

189)TGF-β3

190)TGF-β4

191)TGF-β5

192)LAP(TGF-β1)

193) Latent TGF-. beta.1

194) TGF-. beta.Collection (185 + 193 above)

Other TGF-beta superfamily ligands

195)Lefty

196)Nodal

197)MIS/AMH

198) Other TGF-. beta.ligand sets (195-197 above)

TGF-beta superfamily receptors

199) Activin RIA/ALK-2

200)GFR α-1

201) Activin RIB/ALK-4

202)GFR α-2

203) Activin RIIA

204)GFR α-3

205) Activin RIIB

206)GFR α-4

207)ALK-I

208)MIS RII

209)ALK-7

210)Ret

211)BMPR-IA/ALK-3

212)TGF-βR1/ALK-5

213)BMPR-1B/ALK-6

214)TGF-βRII

215)BMPR-II

216)TGF-βRIIb

217) Endoglin/CD 105

218)TGF-βRIII

219) TGF-. beta.family receptor Collection (199- & 218, above)

TGF-beta superfamily modulators

220)Amnionless

221)GASP-2/WFIKKN

222)BAMBI/NMA

223)Gremlin

224)Caronte

225)NCAM-1/CD56

226)Cerberus 1

227) Noggin

228) Notochord genesis element

229)PRDC

230) Chordin-like 1

231) Chordin-like 2

232)Smad1

233)Smad4

234)Smad5

235)Smad7

236)Smad8

237)CRIM1

238)Cripto

239)Crossveinless-2

240)Cryptic

241)SOST

242)DAN

243) Latent TGF-beta bp1

244)TMEFF1/Tomoregulin-1

245)FLRG

246)TMEFF2

247) Follistatin

248)TSG

249) Follistatin-like 1

250)Vasorin

251)GASP-1/WFIKKNRP

252) Set of TGF modulators (220 + 251 above)

VEGF/PDGF family

253) Neuropilin-1

254)P1GF

255)P1GF-2

256) Neuropilin-2

257)PDGF

258)VEGF R1/Flt-1

259)PDGF R

260)VEGF R2/Flk-1

261)PDGF R β

262)VEGF R3/Flt-4

263)PDGF-A

264)VEGF

265)PDGF-B

266)VEGF-B

267)PDGF-C

268)VEGF-C

269)PDGF-D

270)VEGF-D

27I)PDGF-AB

272) VEGF/PDGF family Collection (253-271, supra)

Dickkopf protein & Wnt inhibitors

273)Dkk-1

274)Dkk-2

275)Dkk-3

276)Dkk-4

277)Soggy-1

278)WIF-1

279) Aggregate factor (273 one above 278)

Frizzled & related proteins

280)Frizzled-1

281)Frizzled-2

282)Frizzled-3

283)Frizzled-4

284)Frizzled-5

285)Frizzled-6

286)Frizzled-7

287)Frizzled-8

288)Frizzled-9

289)sFRP-1

290)sFRP-2

291)sFRP-3

292)sFRP-4

293)MFRP

294) Factor set (280-293 above)

Wnt ligands

295)WnM

296)Wnt-2

297)Wnt-3

298)Wnt-3a

299)Wnt-4

300)Wnt-5

301)Wnt-5a

302)Wnt-6

303)Wnt-7

304)Wnt-8

305)Wnt-8a

306)Wnt-9

307)Wnt-10a

308)Wnt-10b

309)Wnt-11

310Wnt ligand set (295-

Other Wnt-related molecules

311) Beta-catenin

312)LRP-6

313)GSK-3

314)ROR1

315)Kremen-1

316)ROR2

317)Kremen-2

318)WISP-1/CCN4

319)LRP-1

320) Aggregate factor (above 311-319)

Other growth factors

321)CTGF/CCN2

322)NOV/CCN3

323)EG-VEGF/PK1

324) Ossein (Osteocrin)

325)Hepassocin

326)PD-ECGF

327)HGF

328) Granule protein precursor (Progranulin)

329)β-NGF

330) Thrombopoietin

331) Aggregate factor (321 above and 330)

Steroid hormones

332)17 beta-estradiol

333) Testosterone

334) Cortisone (Cortisone)

335) Dexamethasone

Extracellular/membrane proteins

336) Plasma fibronectin

337) Tissue fibronectin

338) Fibronectin fragments

339) Type I collagen (gelatin)

340) Type II collagen

341) Type III collagen

342) Tenascin

343) Matrix Metalloproteinase 1

344) Matrix Metalloproteinase 2

345) Matrix Metalloproteinase 3

346) Matrix Metalloproteinase 4

347) Matrix Metalloproteinase 5

348) Matrix Metalloproteinase 6

349) Matrix Metalloproteinase 7

350) Matrix Metalloproteinase 8

351) Matrix Metalloproteinase 9

352) Matrix Metalloproteinase 10

353) Matrix Metalloproteinase 11

354) Matrix Metalloproteinase 12

355) Matrix Metalloproteinase 13

356)ADAM-1

357)ADAM-2

358)ADAM-3

359)ADAM-4

360)ADAM-5

361)ADAM-6

362)ADAM-7

363)ADAM-8

364)ADAM-9

365)ADAM-10

366)ADAM-11

367)ADAM-12

368)ADAM-13

369)ADAM-14

370)ADAM-15

371)ADAM-16

372)ADAM-17

373)ADAM-18

374)ADAM-19

375)ADAM-20

376)ADAM-21

377)ADAM-22

378)ADAM-23

379)ADAM-24

380)ADAM-25

381)ADAM-26

382)ADAM-27

383)ADAM-28

384)ADAM-29

385)ADAM-30

386)ADAM-31

387)ADAM-32

388)ADAM-33

389)ADAMTS-1

390)ADAMTS-2

391)ADAMTS-3

392)ADAMTS-4

393)ADAMTS-5

394)ADAMTS-6

395)ADAMTS-7

396)ADAMTS-8

397)ADAMTS-9

398)ADAMTS-10

399)ADAMTS-11

400)ADAMTS-12

401)ADAMTS-13

402)ADAMTS-14

403)ADAMTS-15

404)ADAMTS-16

405)ADAMTS-17

406)ADAMTS-18

407)ADAMTS-19

408)ADAMTS-20

409)Arg-Gly-Asp

410)Arg-Gly-Asp-Ser

411)Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro

412)Arg-Gly-Glu-Ser

413)Arg-Phe-Asp-Ser

414)SPARC

415)Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg

416)

Cys-Ser-Arg-Ala-Arg-Lys-Gln-Ala-Ala-Ser-Ile-Lys-Val-Ser-Ala-ASp-Arg

417) Elastin

418) Tropoelastin (tropielastin)

419)Gly-Arg-Gly-Asp-Ser-Pro-Lys

420)Gly-Arg-Gly-Asp-Thr-Pro

421) Laminin

422)Leu-Gly-Thr-Ile-Pro-Gly

423)Ser-Asp-Gly-Arg-Gly

424) Vitronectin

425) Super fibronectin (Superfibronectin)

426) Thrombospondin

427)TIMP-1

428)TIMP-2

429)TIMP-3

430)TIMP-4

431) Fibriodulin

432)Flavoridin

433) Collagen IV

434) Collagen V

435) Collagen VI

436) Collagen VII

437) Collagen VIII

438) Collagen IX

439) Collagen X

440) Collagen XI

441) Collagen XII

442) Nestin

443) Myofibrillar proteins

444) Syndecan-1

445) Keratan sulfate proteoglycan

Ambient oxygen

446) 0.1-0.5% oxygen

447) 0.5-1% oxygen

448) 1-2% oxygen

449) 2-5% oxygen

450) 5-10% oxygen

451) 10-20% oxygen

Animal serum

452) 0.1% bovine serum

453) 0.5% bovine serum

454) 1.0% bovine serum

455) 5.0% bovine serum

456) 10% bovine serum

457) 20% bovine serum

458) 10% horse serum

Interleukins

459)IL-1

460)IL-2

461)IL-3

462)IL-4

463)IL-5

464)IL-6

465)IL-7

466)IL-8

467)IL-9

468)IL-10

469)IL-11

470)IL-12

471)IL-13

472)IL-14

473)IL-15

474)IL-16

475)IL-17

476)IL-18

Protease enzyme

477)MMP-1

478)MMP-2

479)MMP-3

480)MMP-4

481)MMP-5

482)MMP-6

483)MMP-7

484)MMP-8

485)MMP-9

486)MMP-10

487)MMP-11

488)MMP-12

489)MMP-13

490)MMP-14

491)MMP-15

492)MMP-16

493)MMP-17

494)MMP-18

495)MMP-19

496)MMP-20

497)MMP-21

498)MMP-22

499)MMP-23

500)MMP-24

501) Cathepsin B

501) Cathepsin C

503) Cathepsin D

504) Cathepsin G

505) Cathepsin H

506) Cathepsin L

507) Trypsin

508) Pepsin

509) Elastase

510) Carboxypeptidase A

511) Carboxypeptidase B

512) Carboxypeptidase G

513) Carboxypeptidase P

514) Carboxypeptidase W

515) Carboxypeptidase Y

516) Chymotrypsin

517) Plasminogen

518) Fibrinolysin

519) u-type plasminogen activators

520) t-type plasminogen activators

521) Plasminogen activator inhibitor-1

522) Carboxypeptidase Z

Amino acids

522) Alanine

523) Arginine

524) Asparagine

525) Aspartic acid

526) Cysteine

527) Glutamine

528) Glutamic acid

529) Glycine

530) Histidine

531) Isoleucine

532) Leucine

533) Lysine

534) Methionine

535) Phenylalanine

536) Proline

537) Serine

538) Threonine

539) Tryptophan

540) Tyrosine

541) Valine

Prostaglandin

542) Prostaglandin A1

543) Prostaglandin A2

544) Prostaglandin B1

545) Prostaglandin B2

546) Prostaglandin D2

547) Prostaglandin E1

548) Prostaglandin E2

549) Prostaglandin F1 alpha

550) Prostaglandin F2 alpha

551) Prostaglandin H

552) Prostaglandin I2

553) Prostaglandin J2

554) 6-keto-prostaglandin F1a

555)16, 16-dimethyl-prostaglandin E2

556)15 d-prostaglandin J2

557) Prostaglandin collection (542 and 556 above)

Retinoid receptor agonists/antagonists

558) Methoprene Acid (Methoprene Acid)

559) Total Retinoic acid

560) 9-cis retinoic acid

561) 13-cis retinoic acid

562) Retinoid agonist set (558. 561, supra)

563) Retinoid antagonists

564) Retinoic acid receptor isoform RAR alpha

565) Retinoic acid receptor isoform RAR beta

566) Retinoic acid receptor isoform RAR gamma

567) Retinoid (Retinoic) X receptor isoform RXR alpha

568) Retinoid X receptor isoform RXR beta

569) Retinoid X receptor isoform RAR gamma

Other inducers

570) Plant agglutinin

571) Bacterial lectins

572) Maohuosu for treating trichomadesis

573) Phorbol myristate acetate

574) poly-D-lysine

575)1, 25-dihydroxy vitamin D

576) Inhibin

577) Opsonin

578) Glycogen

579) Progesterone

580)IL-1

581) Serotonin

582) Fibronectin-45 kDa fragment

583) Fibronectin-70 kDa fragment

584) Glucose

585) Beta mercaptoethanol

586) Heparanase

587) Pituitary extract

588) Chorionic gonadotropin

589) Corticotropin

590) Thyroxine preparation

591) Bombesin peptides

592) Neuregulin B

593) Gastrin releasing peptides

594) Adrenalin

595) Isoproterenol

596) Ethanol

597)DHEA

598) Nicotinic acid

599)NADH

600) Oxytocin

601) Vasopressin

602) Pressurized oxytocin

603) Angiotensin I

604) Angiotensin II

605) Angiotensin I converting enzyme

606) Angiotensin I converting enzyme inhibitor

607) Chondroitinase AB

608) Chondroitinase C

609) Brain natriuretic peptides

610) Calcitonin

611) Calcium ionophore I

612) Calcium ionophore II

613) Calcium ionophore III

614) Calcium ionophore IV

615) Bradykinin

616) Albumin

617)Plasmonate

618)LIF

619) PARP inhibitors

620) Lysophosphatidic acid

621) (R) -METH arachidonic acid ethanolamide

622)1, 25-dihydroxy vitamin D3

623)1, 2-didecanoyl-glycerol (10: 0)

624)1, 2-dicaprylyl-SN-glycerol

625)1, 2-dioleoyl-glycerol (18: 1)

626) 10-hydroxycamptothecin

627)11, 12-epoxy eicosatrienoic acid

628)12(R)-HETE

629)12(S)-HETE

630)12(S)-HPETE

631) 12-Methoxydodecanoic acid

632)13(S)-HODE

633)13(S)-HPODE

634)13, 14-dihydro-PGE 1

635) 13-ketooctadecadienoic acid

636)14, 15-epoxy eicosatrienoic acid

637)1400W

638)15(S)-HETE

639)15(S)-HPETE

640) 15-keto eicosatetraenoic acid

641) 17-allylamino-geldanamycin

642) 17-octadecynoic acid

643) 17-phenyl-TRINOR-PGE 2

644) 1-acyl-PAF

645) 1-hexadecyl-2-arachidonoyl-522) 646) glycerol

647) 1-hexadecyl-2-methyl glyceryl-3 PC

648) 1-hexadecyl-2-O-acetyl-glycerol

649) 1-hexadecyl-2-O-methyl-glycerol

650) 1-octadecyl-2-methylglyceryl-3 PC

651) 1-oleoyl-2-acetyl-glycerol

652) 1-stearoyl-2-linoleoyl-glycerol

653) 1-stearoyl-2-arachidonoyl-glycerol

654)2, 5-di-tert-butylhydroquinone

655)24(S) -hydroxycholesterol

656)24, 25-dihydroxy vitamin D3

657) 25-hydroxy vitamin D3

658) 2-arachidonoyl glycerol

659) 2-Fluoropalmitic acid

660) 2-Hydroxymyristic acid

661) 2-methoxy antimycin A3

662)3, 4-dichloro isocoumarin

663) Granzyme B inhibitors

664) 4-aminopyridines

665) 4-hydroxyphenyl retinoamides

666) 4-oxa-tetralauric acid

667)5(S)-HETE

668)5(S)-HPETE

669)5, 6-epoxy eicosatrienoic acid

670)5, 8, 11, 14-eicosatetraynoic acid

671)5, 8, 11-eicosatriynoic acid

672) 5-hydroxydecanoic acid

673) 5-iodotubercidin

674) 5-keto eicosatetraenoic acid

675) 5' -N-Ethylamide adenosine (NECA)

676)6，7-ADTN HBr

677) 6-formylindolo [3, 2-B ] carbazole

678)7, 7-dimethyleicosadienoic acid

679)8, 9-epoxy eicosatrienoic acid

680) 8-methoxymethyl-IBMX

681)9(S)-HODE

682)9(S)-HPODE

683)9, 10-Octadecenamide

684)A-3

685)AA-861

686) Acetyl (N) -s-farnesyl-1-cysteine

687) Acetyl-farnesyl-cysteine

688)Ac-Leu-Leu-Nle-CHO

689) Aconitine

690) Actinomycin D

691) Adrenaline (22: 4, n-6)

692)1mM

693)AG-1296

694)AG1478

695) AG213 (tyrosine phosphorylation inhibitor 47)

696)AG-370

697)AG-490

698)AG-879

699)AGC

700)AGGC

701)Ala-Ala-Phe-CMK

702) Procalotide

703) Abastatin

704)AM 92016

704)AM-251

706)AM-580

707) Amantadine (AMANTIDINE)

708) Aminopyrazinamidines

709) Amino-1, 8-naphthalimide [ 4-amino-1, 8-522) naphthalimide ]

710) Aminobenzamide (3-ABA) [3-522) aminobenzamide (3-ABA) ]

711) Amiodarone

712) Arachidonic acid ethanolamide (18: 2, n-6)

713) Arachidonic acid ethanolamide (20: 3, n-6)

714) Arachidonic acid ethanolamide (20: 4, n-6)

715) Arachidonic acid ethanolamide (22: 4, n-6)

716) Anisomycin

717) Aphidicolin

718) Arachidonic acid amides

719) Arachidonic acid (20: 4, n-6)

720) Peanut tetraalkenoyl-PAF

721) Aristolochic acid

722)Arvanil

723) Ascomycin (FK-520)

724)B581

725)BADGE

726) Barfosfomycin (bafilomycin) A1

727)BAPTA-AM

728)BAY 11-7082

729)BAY K-8644

730)BENZAMIL

731)BEPRIDIL

732) Phenobenzenestatin

733) Beta-lapachone (lapachone)

734) Betulinic acid

735) Bezafibrate (bezafibrate)

736)Blebbistatin

737)BML-190

738)Boc-GVV-CHO

739) Rice ferment acid

740) Brefeldin A

741) Bromo-7-nitroindazole [ 3-bromo-7-nitroindazole ]

742) bromo-cAMP [ 8-bromo-cAMP ]

743) bromo-cGMP [ 8-bromo-cGMP ]

744) Bumetanide (bumetanide)

745)BW-B 70C

746) C16 ceramide

747) C2 ceramide

748) C2 dihydroceramide

749) C8 ceramide

750)C8CERAMINE

750) C8 dihydroceramide

751)CA-074-Me

753)calpeptin

754) Inhibin C

755) Calyculin A

756) Camptothecin

757) Cantharidin

758)CAPE

759) Capsaicin (capsacin (E))

760) Anti-capsaicin

761) CARBACYCLIN (CARBACYCLIN)

762) Castanea mollissima alkali

763)CDC

764) Cerulenin

765)CGP-37157

766) Sanguinarine

767) CIGLITAZONE (CIGLITAZONE)

768) CIMATEROL (CIMATEROL)

769)CinnGEL 2Me

770) Salad oxazoline (CIRAZOLINE)

77I)CITCO

772) Chlorobenzene butyl ester

773) Chlorine press

774) Cloprepoprostenol sodium (CLOPROSTENOL Na)

775) Clozapine

776)C-PAF

777) Curcumin (curcumin)

778) Cyclo [ Arg-Gly-Asp-D-Phe-Val ]

779) Actinomycenone

780) Protein synthesis inhibitors

781) Actinolinone-N-acetic acid ethyl ester

782) Cyclopamine (cyclopamine)

783) Cyclopiazonic ACID (CYCLOPIAZONIC ACID)

784) Cyclosporin A

785) Cypermethrin (cypermethrin)

786) Cytochalasin B

787) Cytochalasin D

788) D12-prostaglandin J2

789)D609

790) Damnacanthal

791) Nitrobenzofuran hydantoin

792) Dequamycin

793) Decyl ubiquinone

794) Deoxymannose nojirimycin (deoxymanojirimycin) (1)

795) Deoxynojirimycin (deoxynojirimycin) (1)

796) Propynyl amphetamine (Deprenyl)

797) DIAZOXIDE (DIAZOXIDE)

798) Dibutyryl cyclic AMP

799) Dibutyryl cyclic GMP

800) Dichloro benzamine (DICHLOROBENZAMIL)

801) Dihomo-gamma-linolenic acid

802) Sphinganine

803) Diindolylmethane

804) Thiazone

805) Diphenylene iodine chloride

806) Dipyridamole (dipyridamole)

807) DL-dihydrosphingosine

808)DL-PDMP

809)DL-PPMP

810) Docosahexaenoic acid (22: 6n-3)

811) Docosapentaenoic acid

812) Docosatrienoic acid (22: 3n-3)

813) Adriamycin

814)DRB

815)E-4031

816) E6 Berberine

817)E-64-d

818) Benzoselenazolone

819)EHNA HCl

820) Eicosa-5, 8-dienoic acid (20: 2n-12)

821) Eicosadienoic acid (20: 2n-6)

822) Eicosapentaenoic acid (20: 5n-3)

823) Eicosatrienoic acid (20: 3n-3)

824) enantiomer-PAF C16

825) Epibatidine (epibatidine) (+/-)

826) Epipodophyllotoxin glucopyranoside

827) Farnesylthioacetic acid

828)FCCP

829) FIPRONIL (FIPRONIL)

830)FK-506

831) Flucanini (FLECAINIDE)

832) Flufenamic ACID (FLUFENAMIC ACID)

833) Flunarizine (FLUNARIZINE)

834) Fluprostenol (FLUPROSTENOL)

835) Flusterine (FLUSPIRININE)

836)FPL-64176

837) Fusarium moniliforme B1

838) Oxofurazan (Furoxan)

839) Gamma-linolenic acid (18: 3n-6)

840) Geldanamycin

841) Genistein

842)GF-109203X

843) Gingerol

844) Gliotoxin

845) GLIPIZIDE (GLIPIZIDE)

846) Glibenclamide (GLYBURIDE)

847)GM6001

848)Go6976

849) Chenopodium quinotoxin III

850)GW-5074

851)GW-9662

852)H7]

853)H-89

854)H9

855)HA-1004

856)HA1077

857)HA14-1

858)HBDDE

859) Medicine 'Tuxiangling' for curing elecampane

860) 4-isopropyl cycloheptadiene phenol ketone

861) Histamine

862)HNMPA-(AM)3

863) Hoechst 33342 (cell permeable) (diphenylimide)

864) Huperzine A [ (-) -huperzine A ]

865)IAA-94

866)IB-MECA

867)IBMX

868)ICRF-193

869) Vernodiamycin

870) Indirubin

871) Indirubin-3' -monoxime

872) Indometacin

873) Juglone

874)K252A

875) Kavain (+/-)

876)KN-62

877)KT-5720

878)L-744,832

879) Latrunculin B

880) Fumigant A

881) L-cis-azothiones

882) Leukotoxin A (9, 10-EODE)

883) Leukotoxin B (12, 13-EODE)

884) Leukotriene B4

885) Leukotriene C4

886) Leukotriene D4

887) Leukotriene E4

888) Enzyme inhibiting aldehyde peptides

889)LFM-A 13

890) Lidocaine

891) Linoleamides

892) Linoleic acid

893) Linolenic acid (18: 3n-3)

894) Lipoxin A4

895)L-NAME

896)L-NASPA

897) Chlorophthalamide (LOPERAMIDE)

898)LY-171883

899)LY-294002

900)LY-83583

901) Lycorine

902)LYSO-PAF C1 6

903)Manoalide

904) Manumycin A

905) The concentration of D-red-MAPP,

906) the concentration of L-red-MAPP,

907) mast degranulation peptide

908)MBCQ

909)MCI-186

910)MDL-28170

911) Honey acid (20: 3n-9)

912) Honey ETHANOLAMIDE (MEAD ETHANOLAMIDE)

913) Methotrexate (MTX)

914) Methoxy verapamil

915) Compactin (lovastatin)

916)MG-132

917) Milrinone (R)

918) MINOXIDIL (MINOXIDIL)

919) Minoxidil sulfate

920) MISOPROSTOL (MISOPROSTOL), free acid

921) Mitomycin C

922)ML7

923)ML9

924)MnTBAP

925) Single star essence

926) Monensin (Moonex)

927)MY-5445

928) Mycophenolic acid

929) N, N-dimethyl sphingosine

930) N9-isopropyl kinase inhibitor

931) N-acetyl-leukotriene E4

932)NapSul-Ile-Trp-CHO

933) N-arachidonoylglycine

934) Nitropyramine methyl ester

935) Nifedipine pyridine

936) Fluorometyr

937) Nigericin

938) NIGULDIPINE (NIGULDIPINE)

939) Nimesulide (Nimesulide)

940) NIMODIPINE (NIMODIPINE)

941) Nitrendipine (NITRENDIPINE)

942) N-linoleoyl glycine

943) Noconazol

944) N-Phenylanthranilic acid (CL)

945)NPPB

946)NS-1619

947)NS-398

948)NSC-95397

949)OBAA

950) Okadaic acid

951) Oligomycin A

952) Kinase inhibin

953) Ouabain

954)PAF C16

955)PAF C18

956)PAF C18∶1

957) Palmitoyl ethanolamide

958) Parthenolide (Parthenolide)

959) Mushroom penicillin

960)PCA 4248

961)PCO-400

962)PD 98059

963) Penicillium tremorine (PENITEM) A

964) Pepsin inhibitors

965)PHENAMIL

966) Phenanthrene diketone [6(5H) -phenanthrene diketone ]

967) Phenoxybenzylamine

968) Phentolamine

969) Phenytoin

970) Dipalmitoyl phosphatidic acid

971) Piceatannol (Piceatannol)

972) P53 inhibin (pifithrin)

973) Perishanqing (PIMOZIDE)

974) Pinacidil (PINACIDIL)

975) Piroxicam (piroxicam)

976)PP1

977)PP2

978)prazoc in

979) Pregnenolone 16 alpha nitrile

980)PRIMA-1

81) Procainamide

982) PROPAFENONE (PROPAFENONE)

983) Propidium iodide

984) Heart-derived tranquilization (S-)

985) Puromycin

986) Quercetin

987) Quinidine (I)

988) Quinine (quinine)

989)QX-314

990) Rapamycin

991) Resveratrol

992) Retinoic acid, all trans

993)REV-5901

994)RG-14620

995)RHC-80267

996)RK-682

997)Ro 20-1724

998)Ro 31-8220

999) Rolipram (Rolipram)

1000)roscovitine

1001) Tung mycin (Rottlerin)

1002)RWJ-60475-(AM)3

1003) Lannuodine

1004)SB 202190

1005)SB 203580

1006)SB-415286

1007)SB-431542

1008)SDZ-201106

1009) S-farnesyl-L-cysteine ME

1010) Shikonin medicine

1011) Cyaniguanidine zolan (siguazodan)

1012)SKF-96365

1013)SP-600125

1014) Sphingosine

1015)Splitomycin

1016)SQ22536

1017)SQ-29548

1018) Staurosporine

1019)SU-4312

1020) Suramin

1021) Swainsonine

1022) Triphenoxy amine

1023) Salvianoline IIA

1024) Taxol (taxol) ═ paclitaxel (paclitaxel)

1025) Tetrahydrocannabinol-7-oic acid

1026) Tetrandrine

1027) Thalidomide (thalidomide)

1028) Thapsigargin

1029) Thioocitrulline [ L-Thioocitrulline HCl ]

1030)Thiorphan

1031)TMB-8

1032) TOLAZAMIDE (TOLAZAMIDE)

1033) Tolbutamide

1034) tosyl-Phe-CMK (TPCK)

1035)TPEN

1036) Quinaxine (Trequinsin)

1037) nystatin-A

1038) Trifluoperazine

1039)TRIM

1040) Triptolide

1041)TTNPB

1042) Tunicamycin

1043) Tyrphostin 1

1044) Tyrphostin 9

1045) Tyrosine phosphorylation inhibitor AG-126

1046) Tyrosine phosphorylation inhibitor AG-370

1047) Tyrosine phosphorylation inhibitor AG-825

1048) Tyrosine phosphorylation inhibitor-8

1049)U-0126

1050)U-37883A

1051)U-46619

1052)U-50488

1053)U73122

1054)U-74389G

1055)U-75302

1056) Valinomycin

1057) Valproic acid

1058) Counterpulsation

1059) Veratridine

1060) Catharanthine

1061) Vinpocetine (vinpocetin)

1062)W7

1063)WIN 55，212-2

1064)Wiskostatin

1065) Wortmannin

1066)WY-14643

1067)Xestospongin C

1068)Y-27632

1069)YC-I

1070) Yohimbine

1071) Zaprast (zaprinast)

1072) Zadavirin (Zardaverine)

1073)ZL3VS

1074)ZM226600

1075)ZM336372

1076) Z-prolyl-prolinaldehyde

1077)zVAD-FMK

1078) Ascorbic acid

1079) 5-azacytidine

1080) 5-azadeoxycytidine

1081) Hexamethylene bisamide (HMBA)

1082) Sodium butyrate

1083) Dimethyl sulfoxide

1084)Goosecoid

1085) Glycogen synthase kinase-3

1086) Galectin-1

1087) Galectin-3

Cell adhesion molecules

1086) Cadherin 1 (E-cadherin)

1087) Cadherin 2 (N-cadherin)

1088) Cadherin 3 (P-cadherin)

1089) Cadherin 4 (R-cadherin)

1090) Cadherin 5 (VE-cadherin)

1091) Cadherin 6 (K-cadherin)

1092) Cadherin 7

1093) Cadherin 8

1094) Cadherin 9

1095) Cadherin 10

1096) Cadherin 11 (OB-cadherin)

1097) Cadherin 12 (BR-cadherin)

1098) Cadherin 13 (H-cadherin)

1099) Cadherin 14 (identical to cadherin 18)

1100) Cadherin 15 (M-cadherin)

1101) Cadherin 16 (KSP-cadherin)

1102) LI cadherins

The foregoing are exemplary factors and conditions that may be used to promote differentiation of ES cells or ED cells along a particular developmental lineage. Partially or terminally differentiated endodermal, mesodermal or ectodermal cell types can be used in screening assays to study development and stem cell biology or to produce therapeutic agents. The partially or terminally differentiated cell type may optionally be substantially purified, formulated into a pharmaceutical product, and/or cryopreserved.

Pluripotency of ES cells

The pluripotency of a human ES cell or cell line produced by any of the methods of the invention can be determined by detecting the expression of a human ES cell marker protein. Examples of such proteins include, but are not limited to, octamer binding protein 4(Oct-4), Stage Specific Embryonic Antigen (SSEA) -3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase. In some embodiments, the putative ES cell line retains pluripotency after more than 13, 20, 30, 40, 50, 60, 70, 80, 90, or 100 generations. Maintenance of normal karyotype of ES cells can also be determined. Pluripotency can also be confirmed using methods known in the art to differentiate ES cells produced by the methods of the invention into cells of the ectodermal, endodermal and mesodermal lineages. Pluripotency can also be tested by transplanting ES cells in vivo, for example, to immunodeficient mice (such as SCID mice) and assessing teratoma formation.

In certain embodiments, the ES cells or cell lines that are produced from blastomeres express one or more ES cell marker proteins. Additionally or alternatively, in certain embodiments, the cell maintains a normal karyotype. Additionally or alternatively, in certain embodiments, the cell maintains pluripotency after more than 13, 20, 30, 40, 50, 60, 70, 80, 90, or 100 generations.

For any of the foregoing, ES cells or cell lines produced from blastomeres can be produced without destroying the embryo from which the blastomere used to produce the cell or line was obtained. This property of the cells distinguishes these cells from currently available ES cells and lines, which are produced using methods that must disrupt the underlying embryo.

Production of TS cells

The invention also provides methods of directly differentiating cell types from isolated blastomeres prior to and without the generation of an ES cell line. In one example, human trophoblast stem ("TS") cells are produced by contacting a blastomere growth halo, which is morphologically similar to trophoblasts and/or extraembryonic endoderm, but not to ES cells, with FGF-4. For example, FGF-4 is added to the halo-growing medium. TS cells can be detected by measuring the expression of proteins such as cdx-2, fgfr 2, PL-1, and human chorionic gonadotropin (hCG) using procedures standard in the art. TS cell identification can also be demonstrated by the absence of expression of proteins such as, but not limited to, Oct-4 and alpha-fetoprotein.

Production of purified preparations and cell lines

In certain embodiments, a cell line can be generated. For example, after a particular cell type comprising a cluster of two or more blastomeres (blastomere-derived outgrowth) is identified in a culture, the cell can be separated from the remainder of the culture for further use. After separation, the desired cell can be propagated as a purified or substantially purified population, or it can be maintained as a cell line.

In certain embodiments, the ES cells produced from culturing blastomeres obtained from the embryo are separated from the blastomere-derived culture of the growth halo, and the ES cell line is established using standard techniques developed in establishing ES cell lines from blastocyst-stage embryos. In other embodiments, partially differentiated ED cells of interest may be selected based on, for example, morphology, and the cells may be separated from the culture and purified or impure for further analysis.

Exemplary cell lines include stable cell lines. ES cell lines established in this way may have characteristics of existing ES cell lines, such as differentiation potential, protein expression, karyotype, and the like. Alternatively, an ES cell line established in this way may differ from existing ES cell lines in one or more ways.

Therapeutic uses of ES and ED cells

The ES or ED cells of the invention are suitable for any use for which ES cells are useful. The invention provides methods of treating a condition using cell therapy comprising administering to an affected subject a therapeutically effective amount of ES cells.

In one embodiment, the method of the invention is used to remove blastomeres prior to implantation into a human embryo, after which the blastomeres will be cultured as described above to derive and store human ES cells for therapeutic use using cell therapy if progeny derived from a human embryo are needed in the future, e.g., disease treatment, tissue repair, transplantation, treatment of cell viability or treatment of cellular dysfunction.

In another embodiment of the invention, cells derived from blastomeres, pre-densified morulae, densified morulae or sectioned blastocysts are differentiated directly in vitro or in vivo to produce differentiated (differentiating) or differentiated cells, without producing embryonic stem cell lines. See U.S. patent publication No. 20050265976, published on 12/1/2005, and international patent publication No. WO0129206, published on 4/26/2001, the disclosures of which are incorporated herein by reference, for direct differentiation methods. The cells of the invention are useful in medical, veterinary and biological research as well as in the treatment of diseases by providing cells for use in cell therapy, such as allogeneic cell therapy.

In another embodiment, the ES cell or cell line is derived from a blastomere, and said ES cell or cell line is induced to differentiate to produce one or more mesodermal, endodermal or ectodermal cell types. Exemplary cell types include, but are not limited to, RPE cells, hematopoietic stem cells, hematopoietic cell types (e.g., RBCs, platelets, etc.), pancreatic beta cells, skin cells, cardiac muscle cells, smooth muscle cells, epithelial cells, liver cells, neurons, glia, skeletal muscle cells, vascular cells, and the like. Although ES cells themselves are useful in the treatment of diseases or disorders, the present invention also contemplates the generation of differentiated cell types useful in treatment.

The methods of the invention can be used to generate stem cells from blastomeres, wherein the stem cells are either hemizygous or homozygous for MHC antigens. These cells can be used to reduce immunogenicity during transplantation and cell therapy. The stem cells so produced can be assembled into a library with reduced MHC gene complexity. Blastomeres of the invention may be derived from embryos that are hemizygous or homozygous for MHC antigens. These embryos can be selected to be either hemizygous or homozygous for MHC antigens, or processed to be either hemizygous or homozygous for MHC antigens by any method known in the art. Alternatively, stem cells derived from blastomeres can be processed to be either hemizygous or homozygous for MHC antigens, e.g., by gene targeting. See, e.g., WO 03/018760 and U.S. provisional patent application No. 60/729,173, published 3/6/2003, the disclosures of which are incorporated herein in their entirety.

ES cells and human embryo-derived cells produced by the above-mentioned new technologies are used in cell biology, drug discovery related research and cell therapy, including, but not limited to, the production of hematopoietic and angioblasts for the treatment of blood disorders, vascular disorders, heart disease, cancer and wound healing, the production of pancreatic beta cells for the treatment of diabetes, the production of retinal cells such as nerve cells and retinal pigment epithelial cells for the treatment of retinal diseases such as retinitis pigmentosa and macular degeneration, the production of neurons for the treatment of Parkinson's disease, Alzheimer's disease, chronic pain, stroke, psychiatric disorders and spinal cord injury, the production of cardiomyocytes for the treatment of heart diseases such as heart failure, the production of skin cells for the treatment of wounds (scarless wound repair), burns, promotion of wound repair and the treatment of skin aging, the production of hepatocytes to treat liver diseases such as cirrhosis diseases, kidney cells to treat kidney diseases such as kidney failure, cartilage to treat arthritis, lung cells to treat lung diseases and bone cells for treating bone disorders such as osteoporosis.

Such cell therapy methods may include the combined use of the ES cells of the invention with a proliferation factor, lineage-directing factor, or gene or protein of interest. Methods of treatment may include providing stem cells or appropriate progenitor cells for direct use in transplantation, where the tissue is regenerated in vivo, or remodeled in vitro into a desired tissue, and then providing the tissue to the affected subject.

Pharmaceutical product

The present invention provides methods for producing ES cells, ES cell lines, TS cells, and various partially and terminally differentiated cells and cell lines. The cells and cell lines thus generated can be studied in vitro and in vivo. In certain embodiments, the study of these cells provides information about the biology of the underlying development and the biology of the stem cells. In certain other embodiments, research into these cells and/or factors that can be used to manipulate proliferation, differentiation, and survival of these cells can be used to develop stem cell-based therapies to treat or ameliorate any of a variety of diseases or conditions. In other embodiments, the cells and cell lines produced by these methods can be used in screening assays to identify agents and conditions useful for therapy. The identified therapeutic agents may be used to develop cell therapies, or may themselves be useful when delivered to a patient.

In certain embodiments, the ES cells, ES cell lines, TS cells, TS cell lines, or partially or terminally differentiated cells may be formulated into a pharmaceutical preparation by combining said cells with a pharmaceutically acceptable carrier or excipient. In certain embodiments, the pharmaceutical product contains a number of cells per unit volume of the carrier such that cell therapy can be administered to deliver a particular dose of cells. For example, the pharmaceutical product may be formulated to allow delivery within a volume of carrier appropriate to the condition being treated and the route of administration, e.g. 1x10⁵、1x10⁶、2x10⁶、3x10⁶、4x10⁶、5x10⁶、1x10⁷Or greater than 1x10⁷And (4) cells.

Methods of conducting the study

As detailed above, embryonic stem cell research has been partially hampered by political and ethical objections to the destruction of embryos. The present invention not only provides an alternative method for efficiently producing cells and cell lines, including ES cells and cell lines, but the invention also provides a method that does not require the destruction of new embryos as part of the ES cell-derived process. The remaining embryos may be cryopreserved and permanently stored or stored for other, future research uses.

For some, the ability to derive ES cells and cell lines (either differentiated from ES cells, or partially or terminally differentiated cell types differentiated directly from embryos) without destroying new embryos would provide important benefits beyond the significant technical advantages reflected in these methods. Thus, the present invention provides a novel method for conducting embryonic stem cell studies without destroying the human embryo. The method can obtain human ES cells or ES cell lines derived from human embryos, but without destroying the human embryos. The ES cells or cell lines can be produced from blastomeres obtained from human embryos using any of the methods disclosed herein. After derivation of the ES cells or cell lines, the method may further be used for embryonic stem cell studies using human ES cells or ES cell lines. The method provides a way to conduct ES cell studies without destroying new embryos.

In certain embodiments, embryonic stem cell studies include studies examining the differentiation potential of ES cells or cell lines. For example, the study may comprise contacting human ES cells or ES cell lines with one or more factors and identifying factors that promote differentiation of said ES cells or ES cell lines into one or more mesodermal, endodermal or ectodermal cell types. In other embodiments, embryonic stem cell studies include studies of possible therapeutic uses of ES cells or cells differentiated therefrom.

Regardless of the specific research use, this approach may extend the opportunity to collaborate with researchers around the world, particularly those working in countries where there is legal regulation of embryo destruction.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present specification, including definitions, will control.

Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

In general, the nomenclature used and the techniques described herein relating to cell and tissue culture, molecular biology, immunology, microbiology, genetics, developmental biology, cell biology are those well known and commonly employed in the art.

Exemplary methods and materials are described below, although those similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

All publications, patents, patent publications, and other references mentioned herein are incorporated by reference in their entirety.

Throughout the specification and claims, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

The following examples are intended to be illustrative, and not limiting in any way.

Description of the examples

Example 1 delaying the effect of time between core injection and enucleation on the Pronuclear (PN) stage zygotes.

Nuclear injection was performed on 64 PN stage embryos. GFP positive mouse ES nuclei were transplanted to PN stage zygotes. The embryos are then cultured for 3, 6, 9, or 12 hours before enucleating the original pronuclei. The cloned embryos are then cultured and their development observed. A high percentage of embryos at all time points reached the 2-cell stage, but only embryos enucleated 3 hours after nuclear transfer reached the 4-cell stage (see table 2).

TABLE 2 Effect of delaying the time between nuclear injection and enucleation on the zygotes of mice at the PN stage.

Example 2 sequential cloning of PN stage zygotes and 2-cell stage embryos was used.

To achieve further development of the cloned embryos, serial cloning was performed. The nuclear injection was performed as described in example 1. The embryos were then cultured for 3 hours before enucleating the original pronuclei. The cloned embryos are then cultured to the 2-cell stage.

18 hours after the first cloning, the dissociated individual cloned embryo cells were transplanted into normal fertilized 2-cell stage mouse embryos. Recipient embryos are enucleated prior to nuclear transfer. Each cloned blastomere cell was transplanted into the perivitellin space (perivitellin space) of an enucleated 2-cell stage embryo. Electrofusion of the transplanted blastomeres and the enucleated embryos was performed by a single pulse of 150V DC given for 15 microseconds.

Serial cloned embryos were cultured in KSOM medium and further development was monitored. Two of the six embryos developed into blastocysts (FIG. 1-B). As a control, PN-stage zygotes were injected with mouse ES nuclei (GFP positive), and then their nuclei were enucleated after 3 hours and cultured under 5% CO 2. None of these embryos continued to develop into blastocysts.

TABLE 3 Effect of serial cloning on cloned embryo development.

Example 3 somatic cloning of mouse embryos using 2-cell stage.

It was hypothesized that cloned blastomeres in mosaic embryos could be stimulated by non-cloned cells for further development. One of the two blastomeres of a 2-cell stage mouse embryo is enucleated, and the enucleated blastomere is injected with an ES nucleus immediately after enucleation. Embryos are cultured in KSOM without any further manipulation.

The cloned blastomeres divide the next day and promote the formation of mosaic embryos by GFP positive cells (FIGS. 2A-C). When these embryos developed to the 8-cell stage, at least 3 blastomeres were obtained from the cloned blastomeres (FIG. 2B). Four of these cloned embryos developed into blastocysts (see table 4). GFP positive blastomeres were incorporated into a portion of the blastocyst.

Table 4.2-Effect of cloned embryos at cell stage helper cells located inside the same zona pellucida as the cloned blastomeres.

Example 4. materials and methods of examples 1-3.

All experiments were performed using mouse strain CD-1. The operating medium used was CZB. The medium used was KSOM. All nuclear donor cells were GFP positive mouse ES cells (CD-1XSv 129F 1). The nuclear injection was performed using a PIEZO drill. The blastomeres were dissociated using a glass pipette. Enucleation was performed using micropipette microsurgery to remove the polar body in the PN stage embryo and the visible nucleus of the adjacent cytoplasmic or 2-cell stage embryo.

Example 5 development of clonal blastocysts.

The developmental rate is significantly affected by the cloning method. Tables 5 and 6 record the pre-implantation development of F2GFP NT embryos derived from single clones and serial nuclear transfer when using 2-cell stage embryos in vivo. The most significant developmental differences were found at the 2-cell to 4-cell transition: single and sequential clones developed 59% and 97%, respectively. Abrasion (abrasion) was also observed in inbred lines. In addition, almost all F2 GFP-split serial cloned embryos developed to the expanded or hatched blastocyst stage within 4 days after the initial cloning. This developmental rate was the same as in vivo fertilized B6D2F1 embryos (95%) cultured in KSOM. Clones derived from inbred lines DBA2 and C57BL/6 showed poor efficient development compared to F2 GFP; but blastocyst rate was significantly increased compared to the single NT group (P < 0.001).

Table 5 development of F2GFP serial cloned embryos.

Different superscripts in the same column represent significant differences (P < 0.01).

B6D2F1 embryos fertilized in vivo.

TABLE 6 development of inbred mouse cloned embryos after serial cloning.

Different superscripts in the same column represent significant differences (P < 0.01).

B6D2F1 embryos fertilized in vivo.

DBA2 inbred strain. C57BL/7 inbred strain.

Example 6. development of live pups.

To assess the capacity for terminal development, a total of 35-cell stage embryos constructed by serial nuclear transfer using F2GFP cumulus nuclei were transferred to 4 pseudopregnant females (0.5 d.p.c.). A total of 6 surviving pups were recovered by caesarean section on day 19.5 of gestation. All 6 pups were successfully housed in replacement mothers and grew and matured normally (fig. 3). In contrast, the clonal generation from a single transplantation technique using the same F2GFP cumulus nuclei yielded only 1 pup in 98 transplanted embryos (table 7).

To investigate whether the same technique can be used to generate inbred strains of mice, we used DBA2 and C57BL/6 inbred mice. 2 pups (1.6%) were recovered by continuous cloning from DBA2 by caesarean section on day 19.5 of gestation, but no viable pups were found in C57BL/6 clone regardless of the cloning method used. Of the 2 DBA2 pups, 1 pup died from respiratory defects within a few minutes after recovery. The remaining pups did not show any sign of dyspnea, but were discarded and partially eaten several hours after being directed to foster mothers (foster mothers) (table 7).

To confirm that cloned mice were derived from cumulus cells of F1GFP and DBA2 mice, we confirmed the presence of the two mouse microsatellite markers D1MIT46 previously described and analyzed Nd 3C9461T polymorphism by Restriction Fragment Length Polymorphism (RFLP) of mitochondrial DNA (19) (fig. 4& 5). These studies confirmed that the cloned mice are genetically identical to donor mice that made cumulus cells. The mitochondrial RFLP of cloned mice was identical to that of the cytoplasmic donor B6D2F1 strain (fig. 4&5), providing direct evidence for the source (recipient) of the cytoplasm. The F2GFP clone emits green fluorescence under UV light, providing further evidence of the genetic origin of the clone (FIG. 3).

TABLE 7 results of cloned embryo transfer.

No gross abnormalities in postnatal growth and behavioral development of the cloned animals were observed. Interestingly, animals using nuclear reimplantation clones did not express the obesity phenotype recorded in adult cloned mice. Obesity in adult cloned mice is due to abnormal gene expression and epigenetic modification during NT and embryo culture and reflects an increase in adipose tissue in addition to the larger body types starting at 8-10 weeks of age. The mean weight (+ -SD) of mice using nuclear reimplantation clones was 34.9 + -0.8 g at 6 months, which was not different from that of normal control animals (33.6 + -1.9) (P > 0.1). In contrast, animals using traditional SCNT clones weighed 54.8 ± 2.6 grams at three to six months of age (table 8).

Table 8 body weights of mice cloned using conventional SCNT and nuclear reimplantation.

Differences (P < 0.01) exist in the mean values with different superscripts.

One clone was 3 months old, another 6 months old. The mice of the individual clones and normal mice are at least 6 months old.

Example 7 Gene expression profiles of cloned blastocyst stage embryos.

Many studies strongly suggest imperfect epigenetic reprogramming in reconstructed embryos, which may account for their poor performance in vitro culture and in vivo development after transplantation. Since our serial cloning results in an abnormal increase in the development of cloned embryos to the blastocyst stage of hatching, we hypothesized that the gene expression pattern of several genes could be identical to that of normal in vivo fertilized embryos. To examine our hypothesis, we analyzed the expression of two imprinted genes H19 and I GF2 and one pluripotency-associated gene OCT-4 at the 4-cell, 8-cell and blastocyst stages. As presented in fig. 6-8, expression of all three genes in the serial cloned embryos was more similar to the B6D2F1 control embryos in vivo, compared to single cloned embryos. In particular, H19 gene expression in serial cloned blastocysts was significantly different from the single clone's equivalent and closer to the B6D2F1 control (fig. 6). Serial cloned 8-cell stage embryos significantly upregulated IGF2 expression compared to single cloned embryos, more closely to levels in B6D2F1 controls (fig. 7). A similar trend in OCT-4 expression was also found in all developmental stages of the study (FIG. 8).

Example 8 cell number count of blastocyst stage embryos.

Abnormal gene expression patterns in blastocyst stage clones were consistent with less than half the normal number of cells, and higher cell numbers were associated with increased cloning efficiency and correct expression of OCT-4. Nuclear replanting significantly increased both the total cell number of the cloned blastocysts as well as the Inner Cell Mass (ICM) cell number (table 9). Conventional SCNT (n ═ 14 embryos) yielded 32.3 ± 4.6 cells (8.3 ± 5.9 ICM cells)/blastocyst, compared to 67.4 ± 6.5 cells (28.7 ± 4.8 ICM cells) of normal in vivo fertilized embryos (n ═ 15, P < 0.001). Blastocysts generated by nuclear replant (n ═ 15) contained a total of 49.8 ± 6.9 cells and 16.2 ± 7.1 ICM cells, representing approximately 1.5-fold and 2-fold increases, respectively. The average ICM/Trophectoderm (TE) cell ratio also increased from 0.33 to 0.48 (45%) (P < 0.01) (Table 9). Higher ICM/TE ratios and cell numbers may at least partially account for significant improvements in post-implantation development and survival birth following transplantation into surrogate mothers.

TABLE 9 blastocyst quality analysis by differential staining of Inner Cell Mass (ICM) and Trophectoderm (TE) cells of blastocysts following single or serial nuclear transfer.

Example 9 embryonic stem cells derived from cloned embryos and their characterization.

A total of 35 single cloned F2GFP blastocysts and 30 consecutive cloned blastocysts were subjected to ES cell isolation. Of these, 1ES cell line from a single cloned embryo (3%) and 5 ES cell lines from consecutive cloned embryos (17%) were established (Table 10). All established cell lines were positive for alkaline phosphatase, OCT-4 and SSEA1 (FIG. 9A). In addition, all of these nuclear transfer ES cell lines formed embryoid bodies in vitro and teratomas when injected intramuscularly to the hind limbs (fig. 9B). Two samples showed differentiated tissues originating from all three germ layers. Furthermore, when these ntES cells were injected into 8-cell stage CD-1 embryos and transplanted into replacement females (2.5d.p.c), several chimeric mice were generated with defined punctate gray coat and emitting green fluorescence (indicative of F2GFP genotype) when irradiated with ultraviolet light (FIG. 9C).

TABLE 10 efficiency of derivation of embryonic stem cells from cloned embryos.

Example 10. materials and methods of examples 5-9.

Animal(s) production

Female DBA2, C57BL/6, and F2GFP hybrid mice were used as nuclear donors. To generate F2GFP mice, female 129/SV mice were crossed with 129/CD F1 males (129/Sv x CD-1) carrying and expressing the Green Fluorescent Protein (GFP) gene. For initial and sequential cloning, enucleated BDF1(C57BL/6 x DBA/2) oocytes and enucleated 2-cell stage BDF1 embryos were used as recipients, respectively. CD-1 females were used as surrogate mothers to inoculate cloned embryos.

Culture medium

Oocytes and embryos were incubated in KSOM (Specialty Media, USA) containing amino acids, glucose and 1mg/ml Bovine Serum Albumin (BSA) at 37 ℃ in a high humidity incubator (incubator) containing 5% CO₂The culture was carried out in the air. Enucleation of oocytes was performed in M2 medium (Specialty Media, USA), and for primary cloning, cell injection was performed in Hepes buffered CZB medium at room temperature. Activation of the reconstructed oocytes in the presence of 10mM SrCl₂And 5ug/ml cytochalasin B in Ca-free²⁺CZB to prevent the discharge of the electrode bodies. Nuclear reimplantation was performed at 37 ℃ in M2 medium (Specialty Media, USA) supplemented with 7.5ug/ml cytochalasin B and 0.4ug/ml noconazole (a microtubule polymerase inhibitor) to facilitate micromanipulation. The reconstituted embryos were subjected to pulsing in a mannitol-based fusion medium comprising 0.27mM mannitol, 100 μm MgSO₄And 50 μm CaCl₂And supplemented with 0.3% bovine serum albumin.

Isolation of cumulus cells, oocytes, and 2-cell stage embryos.

Superovulation was performed using 8-10 week old mature BDF1, DBA2, C57BL/6 and F2GFP female mice. Mice were injected with equine chorionic gonadotropin (eCG) (5IU) and human chorionic gonadotropin (hCG) (5IU) at 48 hour intervals. Cumulus-oocyte complexes were collected from the oviduct 13 hours after hCG injection and cumulus cells were dispersed by treatment with 0.1% (w/v) bovine testicular hyaluronidase (150USP units/ml) in M2 for 5 minutes at 37 ℃. The dispersed cumulus cells were washed in fresh M2 and resuspended in 3% polyvinylpyrrolidone (PVP; Mr 360,000, ICN Biochemicals, USA) and stored in the refrigerator (4 ℃) until use. 36 hours after hCG injection, 2-cell stage embryos were collected from the plugged females (in M2 medium) by washing the oviducts with a 1ml syringe connected to a 27G blunt needle.

Cloning and sequential cloning

Nuclear transfer was performed according to the method reported by Chung et al (29). Enucleation was performed using a Nikon inverted microscope (TE300, japan) equipped with a Narshige syringe (Narshige, japan). Metaphase II spindles of B2DF1 oocytes were removed in M2 medium drops containing 5ug/ml cytochalasin B by aspiration using a Piezo micromanipulation controller PMM 150(PrimeTech, Japan) using a 10-12um pipette. The enucleated oocytes were washed well in CZB medium and stored in an incubator for use. After removal of cytoplasm in 3% PVP using a small bore syringe pipette (internal diameter 7um), cumulus cell nuclei were injected separately. Cytoplasmic removal is performed so that only a small amount of cytoplasm remains around the denuded nucleus. Activation of the reconstructed oocytes in the presence of 10mM SrCl₂And 5. mu.g/ml cytochalasin B in Ca-free form²⁺CZB 5.5% CO at high humidity₂The incubation was performed in an incubator for 6 hours. Following activation, the reconstructed oocytes are cultured in KSOM medium.

The procedure for serial cloning is depicted in FIG. 4. For the second clone, one nucleus (with a trace amount of cytoplasm) of the 2-cell stage cloned embryo was removed and transferred to an enucleated 2-cell stage in vivo fertilized B6D2F1 embryo (in M2 medium supplemented with 7.5ug/ml cytochalasin B and 0.4 μ g/ml noconazole). The nucleus was placed between two 2-cell stage cytoplasts. Fusion of 2-cell stage cytoplasts and clonal nuclei was performed in the above mannitol-based fusion medium using a BTX 2001 electrofusion apparatus with 2 DC pulses of 2.4kV/cm for 15 usec. The 1st pulse was given after aligning the reconstructed eggs so that the implanted nuclei were directed to the negative lead and the two blastomeres were directed to the positive lead. After the first pulse, the reconstructed egg was rotated 90 degrees by a 5 volt AC alignment pulse to orient the two blastomeres to opposite poles and a secondary pulse was applied. The pulsed reconstructed embryos were cultured in KSOM after extensive washing and their fusion was confirmed 30 minutes after the second pulse. We typically observed more than 98% fusion after the first pulse. Only the fused embryos were cultured for an additional three days.

Generation of cloned offspring.

When some cloned embryos developed to the 2-cell stage, they were transplanted into the oviduct of a pseudopregnant CD-1 foster mother (0.5 day post-mating) that had been mated one day ago with vasectomized CD-1 males. Recipient females were euthanized 19.5 days post-coital (d.p.c) and examined for the presence of fetuses in their uterus and implantation sites. The surviving pups were bred from a foster mother (CD-1) delivered on the same day.

Cell count in blastocysts

Total cell number of blastocysts and TE and ICM cell number were counted after differential staining with nucleotide specific fluorescent dyes as previously described. Briefly, embryos that had developed to enlarged blastocysts 4.5 days after initial cloning were exposed to acidic Tyrode's solution (pH 2.1) to remove the zona pellucida. Naked blastocysts were washed in M2 medium and then labeled for 10 minutes at 4 ℃ for trinitrobenzene sulfonic acid (TNBS; Sigma P-2297) in M2. After removal of excess TNBS, the blastocysts were exposed to anti-dinitrophenol antibody dissolved in M2 for 10 minutes at 37 ℃. Excess antibody was then removed by extensive washing before exposure to guinea pig complement diluted 1: 4 with 2ug/ml Propidium Iodide (PI) in M2 for 10 minutes at 37 ℃. The blastocysts were then washed rapidly in protein-free Hepes CZB medium supplemented with 5ug/ml propidium iodide and then fixed in ice-cold absolute ethanol for 5 min. The blastocysts were then transferred to 10ug/ml Hoechst 33258 in ethanol for at least 10 minutes at 4 ℃. The stained blastocysts were fixed in 100% glycerol and evaluated by fluorescence microscopy (Nikon TE200, japan). The blue nuclei were counted as Inner Cell Mass (ICM) and the red nuclei were regarded as Trophoblast (TE) cells.

Establishment of ntES cell line

When the F2GFP cloned embryos had developed to the blastocyst stage, they were used to establish the ntES cell line, as described previously with minor modifications. Briefly, the zona pellucida was removed by brief exposure to acidic Tyrode's solution and vigorous washing prior to plating. A set of 3 or 4 naked blastocysts was placed in a mitomycin-C treated Mouse Embryonic Fibroblast (MEF) monolayer grown in one well of a 4-well dish (Nunclon, USA) with 500ul mouse ES Cell culture medium supplemented with 2000 units/ml leukemia inhibitory factor (chemicon. USA) and 50 μ M MEK-1 inhibitor (Cell Signaling Tech, USA). When the inner cell mass formed an initial outgrowth (typically within 3 days), the cell mass was divided into small pieces by treating it with 0.05% trypsin/EDTA and pipetting them with a small bore pipette. The segmented cell mass and dispersed cells originating from the same embryo were transferred to fresh MEFs grown in 50ul drops of mouse ES cell culture medium overlaid with tissue culture mineral oil. The culture medium drops were observed daily for the presence of halo of ES cell growth. The growth halo was then transferred to the wells of a 4-well plate containing fresh MEF.

DNA isolation

DNA was isolated from the tail tips of the following animals using DNeasy tissue kit (QIAgen, USA) according to the manufacturer's recommendations: the nurseries CD-1, the oocyte donor lines B6D2F1, the nuclear donor lines (F2GFP and DBA2) and the somatic cell nuclear transfer derived animals (clone 1-6 for F2GFP and clone 1 for DBA 2). DNA was quantified using a Nanodrop spectrophotometer (Nanodrop, USA).

Mitochondrial DNA RFLP analysis

The Nd 3C9461T polymorphism was confirmed by Restriction Fragment Length Polymorphism (RFLP) analysis (19) as previously described. Briefly, a 204bp fragment containing 9461 sites was amplified by PCR. The mutation made by the primer together with the C9461 wild type form creates a recognition site for BclI. As a result, the recognition site was disrupted by the presence of the T9461 polymorphism. Fragments were analyzed by electrophoresis in a 4% agarose gel.

Nuclear GFP DNA PCR analysis

Genomic DNA of the F2GFP clone was isolated from the tail tip as described above and 100ng of each reaction was used to amplify the GFP gene by PCR. We used the forward (5'-ttgaattcgccaccatggtgagc-3') and reverse (5'-ttgaattcttacttgtacagctcgtcc-3') primers for the GFP gene with reaction parameters of 95 ℃ for 9 minutes (1 cycle), and 94 ℃ for 45 seconds, 59 ℃ for 1 minute, 72 ℃ for 1.5 minutes, 37 cycles. The PCR products were separated on a 1.5% agarose gel and visualized by ethidium bromide staining.

DNA typing

Nuclear DNA from tail tip DNA was genotyped using the D1Mit46 Simple Sequence Repeat (SSR) polymorphism by using MapPairs to determine B219 forward and reverse unlabeled primers (Invitrogen, USA). The reaction parameters included 20ng template, 1 XFailailSafe Premix (Premix) K (EpiCentre, USA), 0.2. mu.M of each primer, 0.5 units of FailSafe PCR enzyme mix (EpiCentre, USA), and cycling conditions of 96 ℃ for 2 minutes (1 cycle), 94 ℃ for 45 seconds, 55 ℃ for 45 seconds, 72 ℃ for 1 minute (30 cycles), and 72 ℃ for 7 minutes. The PCR products were separated on a 4% agarose gel and visualized by ethidium bromide staining. PCR was performed for 30 cycles and the products were separated by 3% agarose gel and visualized by ethidium bromide staining.

Blot gene expression analysis

Total RNA was isolated from 20 to 30 embryos at 4-cell, 8-cell and hatching blastocyst stage using TRIzol extraction column and PureLink purification column (Invitrogen, USA). Total RNA was reverse transcribed using a supplier recommended cDNA library kit (applied biosystems.usa) and used as a template for quantitative real-time PCR using TaqMan chemistry, gene expression assays for predetermined H19, IGF2, OCT-4 and endogenous housekeeping gene GAPDH, and a supplier recommended ABI SDS 7900HT instrument (applied biosystems.usa). Relative quantification of gene expression was performed using the comparative threshold cycle method (comparative threshold cycle method) described previously and using RQ Manager and Excel as recommended by the manufacturer (Applied biosystems.

Statistical analysis

Results were evaluated using the chi-square test with correction according to persistence.

Example 11. human embryonic stem cell lines produced under embryos are not disrupted.

A series of nine experiments were performed using residual embryos generated by IVF for clinical purposes. Embryos were obtained with complete informed consent using an advanced cell technology ethical council (EAB) and an Institutional Review Board (IRB). Pronuclear stage embryos were thawed and cultured to 8-cell stage in Quinn's division medium under 6% CO 2. Embryos were scored using a standard system and a total of 41 grade I or II embryos were used in both sets of experiments (table 11). Just one (or in a few [7/41] cases, two) blastomeres were removed from each embryo using the previously described (Klimanskaya et al Nature 2006; 444 (7118): 481-. In the first set of experiments, both maternal embryos and blastomeres were cultured together in the original microdroplets for 12 hours, then transferred to Quinn's blastocyst medium for another 48 hours. 22 of the 26 biopsied embryos (85%) continue to develop to the blastocyst stage and most (21/31) single blastomeres divide, forming cell masses or "ovules" containing 4-8 cells. They were transferred to blastocyst medium droplets supplemented with laminin and fibronectin and inoculated with mitotically inactivated Mouse Embryonic Fibroblasts (MEFs). The next day, the droplets were combined with droplets containing hES cells expressing Green Fluorescent Protein (GFP) as described previously. Most of the single blastomere-derived cell aggregates formed cavitated oocysts, which were forced to join by teasing them with a 26G needle if they did not spontaneously join within 28 hours of plating.

In a second set of experiments, maternal embryos and blastomeres were co-cultured together less than 12 hours after the biopsy procedure. Maternal embryos are transferred to Quinn's blastocyst medium where they are allowed to continue to develop to the blastocyst stage. Biopsied blastomeres (whether cell dividing or not) were transferred to blastomere microdroplets as described above and cultured for approximately 5 days without pooling with droplets containing other GFPES cells. Importantly, no "ovules" were formed under these conditions, but nearly all (9/11) of the blastomere-derived cell aggregates produced outgrowth (table 11).

In both sets of experiments, maternal embryos were allowed to develop to the blastocyst stage and frozen. 80-85% of the biopsied embryos formed healthy blastocysts (Table 11 and FIG. 11C), which ratio was consistent with or higher than the ratio previously reported for both biopsied and non-biopsied embryos (Geber and Sampaio. Hum Reprod 1999; 14 (3): 782-.

29 out of 33 (88%) blastomere-derived aggregates produced cellular outgrowths that were 4/20 (20%) and 4/9 (44%) from the first and second groups were morphologically similar to hES cells (table 11 and fig. 11 a). In the first set of experiments, only 1 out of 26 embryos (3.8%) produced stable hES cell lines, consistent with the low efficiencies previously reported (Klimanskaya et al Nature 2006; 444 (7118): 481-485). However, in the second set of experiments, 3 out of 15 embryos (20%) produced stable hES cell lines when the dissected blastomeres were placed under conditions favorable for hES cell growth, with a derivation rate comparable to that obtained using blastocysts.

When blastomere-derived (hES cell-like) colonies reached about 50 cells or more, they were mechanically dispersed and the dispersed cells were plated beside the initial growth halo. Secondary colonies were also allowed to grow to similar sizes and were mechanically passaged to fresh MEFs every 3-5 days until they were suitable for routine passage with trypsin and could be frozen (usually after 7-10 passages) as previously described (Klimanskaya and McMahon. handbook of StemCells. San Diego: Elsevier Academic Press; 2004p. 437-449). At each passage, colonies were screened under a fluorescent microscope for the absence of GFP-positive cells. All 4 hES cell lines were Oct-4, nanog, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and alkaline phosphatase positive (FIG. 11 b). In vitro differentiation confirmed the presence of derivatives from all three germ layers, hematopoietic and epithelial cells, neurons, Retinal Pigment Epithelium (RPE), beating cardiomyocytes, and other cell types of therapeutic importance (fig. 12). To assess differentiation potential in vivo, cells were injected under the renal capsule of NOD-SCID mice, where they would form teratomas, differentiating into structures of all three germ layers, after approximately 6 to 8 weeks.

All 4 newly established hES cell lines possess normal karyotype (no embryo disruption [ NED ]1, 46 XY; NED2, 46 XY; NED3, 46 XX; and NED 446 XY (FIG. 14). PCR analysis confirmed the absence of GFP DNA, this eliminates the possibility of cross contamination or fusion with GFP + hES cells used in co-culture (fig. 13a) further genotyping analysis showed the unique identity of the new hES cell line, excluding any possibility of cross contamination with other hES cell lines currently maintained in our laboratory (fig. 13b) in the first series of experiments, 1 blastomere was dissected from 21 live embryos, while 5 (from experiment numbers 1 & 3) had two single blastomeres removed, in the second series of experiments, of the embryos 13 biopsies had 1 blastomere, while 2 embryos (from experiments 8&9) had two single blastomeres removed.

TABLE 11 hES cell derivation from single blastomeres without destroying the embryo

hES cell line derived from in vivo dissected single blastomere embryos

hES cell line derived from one of two in vivo dissected blastomeres

Example 12 blastomere biopsy and culture.

In another set of experiments, double doses of laminin were used and blastomeres were grown in blastocyst medium with MEF cells for extended periods of time (5 days) to prevent blastomere formation.

Pre-nuclear stage human embryos in Quinn's division Medium (Cooper Surgical) at 5% CO₂Incubate to 8-cell stage. Individual blastomeres were isolated from embryos as previously described using pinezo. Briefly, 8-cell embryos were treated in Ca-free supplemented with 0.05% PVA⁺⁺And Mg⁺⁺Pre-incubated in phosphate buffered saline at room temperature for 15 minutes to facilitate separation of individual blastomeres. Embryos were then transferred to Quinn's hepes medium for manipulation. Before inserting the biopsy pipette, a small (20 μm) pipette was used to punch a hole (500 μm diameter) in the tape by applying several PIEZO pulses. To separate individual blastomeres, a pipette (50O μm) was inserted through the hole and a gentle negative pressure was applied to grasp the blastomeres. When 2/3 of the blastomere is inside the pipette, the blastomere is pulled out. After biopsy, the maternal embryos and blastomeres were returned to the original culture drop (Quinn's division medium) and cultured together for 12 to 18 hours. The blastomeres and maternal embryos were then separated: maternal embryos were transferred to blastocyst medium (Quinn's blastocyst medium) to allow them to develop into blastocysts, while blastomeres were transferred to small culture drops (50 μ l) containing MEF. The blastomere medium was supplemented with laminin (10. mu.l/ml), fibronectin (10. mu.l/ml) or matrigel (10. mu.l/ml). They were cultured in the same medium for 5 days, or until they formed a cell pellet comprising about 20 cells. The adjacent GFP ES cell culture drops were then merged with the blastomere culture drops to allow the two media to mix together. After approximately 24 hours, 1/2 blastomeres were removed and plated on the same culture drop. ES colony formation was checked daily and 1/2 medium was replaced daily with fresh medium. The ES colonies were then separated and mechanically transferred to fresh ES cell culture dishes up to passage 4, and then the ES cells were gradually trypsinized for large-scale culture.

After the blastomeres develop into blastomeres, almost all of the blastomeres become trophoblast-like cells, which have no potential to become ES cells. Most 8-cell blastomeres were directed to become ES cells by preventing the formation of oocysts and adding higher laminin, which interferes with cell polarity (table 12).

TABLE 12 ES cell lines derived from individual blastomeres

Example 13 hESC co-culture is not necessary for hESC line derivation.

In another set of experiments, GFP-hESC co-cultures were examined to determine whether co-culture was necessary for successful derivation. The experiment was performed using 2 frozen split-stage embryos that were thawed and cultured in blastocyst medium for 2 hours prior to dissection of the blastomeres in vivo. A single blastomere was removed from one embryo and two blastomeres were removed from a second embryo. The remaining biopsy embryos were allowed to continue to develop and were frozen at the blastocyst stage. The blastomeres removed were cultured under the same conditions as described in the second set of experiments of example 11, but in the absence of GFP-hESC. Both blastomere-derived aggregates produced halos of cell growth, while one of the two embryos (50%) produced stable hESC lines. Immunostaining of stable hESC lines established from this colony (NED5) confirmed the expression of pluripotency markers including Oct-4, Nanog, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and alkaline phosphatase (fig. 11C). The newly established hESC lines possessed a normal male (46XY) karyotype (fig. 14), and derivatives were differentiated from all three germ layers, including immunostaining with antibodies directed against tubulin β III (ectoderm), smooth muscle actin (mesoderm), alpha-fetoprotein (primitive endoderm).

Example 14 laminin directs the differentiation of blastomeres into ICM.

Separate studies were performed to investigate the mechanism of enhanced blastomere differentiation into ICM. In the absence of laminin and fibronectin, the dissociated individual blastomeres uniformly differentiated into trophectoderm (16/16[ 100% ] the blastomere growth of blastomeres contained trophectoderm cells, whereas 0/16[ 0% ] contained ICM-like cells). In contrast, when laminin was added to the medium, only 1/14 (7%) of blastomere-derived aggregates produced trophectoderm-like vesicles, while 13/14 (93%) produced ICM-like cells. Addition of fibronectin alone had little or no effect on lineage specification (5/6[ 83% ] outgrowth contains trophectoderm cells, whereas 1/6[ 17% ] contains ICM-like cells). This suggests that laminin may play a key role in directing the differentiation of blastomeres into ICMs. To test this hypothesis, we performed immunostaining of trophectoderm markers and ICM/ESC markers, respectively, on blastomere-derived vesicles formed in the absence of laminin (FIG. 15A), and ICM-like cells derived in the presence of laminin (FIG. 15D). As expected, blastocyst-derived vesicles formed in the absence of laminin expressed key trophectoderm markers, including cytokeratin 8 and cdx2 (fig. 15B, C), while ICM-like outgrowth formed in the presence of laminin expressed Oct-4 (fig. 15E). Interestingly, immunostaining of the tight junction marker ZO-1 and ultrastructural analysis by transmission electron microscopy and half-thin sections (FIG. 15G-I) revealed that addition of laminin to the medium of established hESC lines disrupted the tight junctions and depolarized the ESCs, inducing them to assume an ICM-like phenotype. In addition, ZO-1 staining confirmed that addition of laminin to the culture medium of blastomeres disrupted tight junctions and inhibited the trophectoderm differentiation pathway.

EXAMPLE 15 materials and methods of examples 11-14

Single blastomere biopsy

Residual embryos produced by IVF for clinical purposes were obtained with complete informed consent using the advanced cell technology ethics council board (EAB) and the Institutional Review Board (IRB). Donated pre-nuclear stage embryos were thawed using the embryo thawing kit (Cooper Surgical, CAT # ART-8016) according to the manufacturer's instructions. All procedures were performed at room temperature. Briefly, embryos are unloaded into 0.5M sucrose melt medium and held there for 10 minutes before being transferred to the mediumIt was thawed in air for 2 minutes followed by 3 minutes at 37 ℃. Embryos were then transferred to 0.3M sucrose melt medium and incubated for 10 minutes followed by several washes in embryo melt diluent before being re-transferred to pre-equilibrated embryo medium. The thawed embryos were cultured in 20ul drops of Quinn's split medium in a high humidity incubator containing 6% CO2 in air at 37 ℃. Only embryos that developed to the 8-cell stage within 48 hours post-thaw underwent single blastomere biopsy. Before biopsy, a small hole (50 uM in diameter) was made in the clear zone of all embryos using a PIEZO drill, followed by Ca-free supplemented with 0.05% PVA (polyvinyl alcohol)⁺⁺And Mg⁺⁺Incubated in PBS for 15 minutes. Blastomere biopsies were performed at 37 ℃ in Quinn's Hepes medium supplemented with 5% SPS (serum protein substitute) using a PIEZO drill as described previously. Only a single blastomere (in a few cases, two blastomeres) was removed from each embryo. In experimental group 1, maternal embryos and biopsied blastomeres were co-cultured for 12-24 hours, then transferred to Quinn's blastocyst medium and co-cultured for another 48 hours. After co-culture, the maternal embryos were frozen and the blastomere masses were transferred to MEF drops for further growth as described below. In experimental group 2, maternal embryos and blastomeres were co-cultured for less than 12 hours. They were then separated and embryos were cultured in Quinn's blastocyst medium for an additional 48 hours before freezing. Blastomeres in experiment 2 were cultured on MEF cells for 5 days in Quinn's blastocyst medium supplemented with laminin (Sigma) and fibronectin (from human plasma, Sigma) from human placenta.

Blastocyst freezing

After confirmation of blastocoel formation, the maternal embryos were frozen using a blastocyst freezing kit (CooperSurgical, Cat # ART-8015) according to the manufacturer's instructions. All procedures were performed at room temperature. Briefly, embryos were rinsed and incubated in dilute medium for 5 minutes before being transferred to the final 9% glycerol plus 0.2M sucrose freezing solution, and then transferred to 5% glycerol freezing medium for 10 minutes. Each embryo was then loaded into a 0.25ml embryo freezing pipette (IMV-ZA475, France) prior to freezing. Embryo freezing was performed using an embryo freezer (FreezeControl CL-869, Australia). Embryos were brought from an initial temperature of 25 ℃ to-6.5 ℃ at 2 ℃/min. They were then inoculated manually and held at-6.5 ℃ for 10 minutes before being cooled to-45 ℃ at 0.3 ℃/minute and transferred to liquid nitrogen storage tanks.

ESC derivatization

hESC culture was performed as previously described (Klimanskaya et al, 2006; Klimanskaya et al, 2007; Klimanskaya and McMahon, 2004). In experimental group 1, two days before egg plating, MEF cells were plated in 50ul drops lined up in gelatin-treated 60mm cell culture dishes. MEF cell drops are arranged as 2 or 3 drops ("auxiliary drops") around one "blastomere" (the drop designated for the blastomere outgrowth). The following day, small clumps of GFP + hES cells were transferred to "helper drops" and cultured overnight, and MEF plating medium in these drops was replaced with hESC medium. On the third day, the medium in the "blastocyst drops" was replaced with freshly prepared Quinn's blastocyst medium supplemented with 5ug/ml fibronectin and 2.5ug/ml laminin. The dishes were preincubated in a 6% CO2 incubator for at least 3 hours prior to transfer of the ovules. The day after transfer of the ovules, each ovule culture drop was connected to 2 or 3 surrounding GFP-hESC drops by dragging the medium between the two drops with a small glass pipette. On the other day, the connecting channel was widened with a pipette and the blastocyst medium was replaced with the derivative medium as described above two days later. After the initial growth halo forms a colony large enough to disperse (typically 3-5 days after plating), it is cut into two pieces and replated to the same drop.

In Experimental group 2, after the first 12 hours of co-culture with the maternal embryo, blastomeres were cultured in 50ul drops of Quinn's blastocyst medium supplemented with 5ug/ml fibronectin and 5ug/ml laminin and containing MEF cells (as prepared in Experimental group 1). Blastomere culture drops and surrounding GFP ESC culture drops were not connected for 5 days. During this time, most blastomeres form a cell mass containing 20-30 cells similar to the inner cell mass. On day 6 after plating, blastomere culture drops and surrounding GFP ESC culture drops were connected as in experimental group 1 and blastocyst medium was replaced with the derivative medium on day 7. The initial growth halo was examined daily and proliferation of the growth halo was performed as in experimental group 1. Half of the original volume of medium was replaced every other day. When stable growth of hescs was observed, serum was removed from the medium. Detailed procedures are described in Klimanskaya et al, 2007(Klimanskaya et al, 2007).

Experimental group 3 followed the same procedure as group 2, but without hESC-GFP co-culture.

Immunostaining

Immunostaining was performed using standard staining protocols. Briefly, samples were fixed with 2% paraformaldehyde for 10 minutes (cells) or 40 minutes (vesicles), permeabilized with 0.1% NP-40, blocked with PBS containing 10% each of goat and donkey serum for 1 hour, and incubated with primary antibody overnight at 4 ℃. After each antibody incubation, 3 washes were performed, each for 10 minutes. Fluorescently labeled or biotinylated secondary antibody (Jackson Immunoresearch or Molecular Probes) was added for 1 hour, followed by fluorescently labeled streptavidin (Molecular Probes) for 15 minutes to visualize the biotinylated secondary antibody. Samples were fixed in DAPI to Vectashield (Vector Laboratories, Burlingame, Calif.) and photographed using an inverted fluorescence microscope (Nikon). Peroxidase staining of teratoma sections was performed using standard protocols. Briefly, slides were dewaxed three times in xylene. Xylene was removed with 100% ethanol, endogenous peroxidase activity was blocked with 3% H2O2, and slides were incubated for one hour in blocking agent containing 0.1% Triton X-100 as described above, followed by overnight incubation at 4 ℃ in primary antibody diluted with the same blocking agent. Primary antibodies against the following antigens were used: oct-4(Santa Cruz Biotechnology, Santa Cruz, Calif.), SSEA-3, SSEA-4, TRA-1-60, TRA-1-81(Chemicon, Temecula, Calif.), Nanog (Kamiya), beta III tubulin (Covance), alpha fetoprotein, smooth muscle actin (Dako), cdx2(Abcam), ZO-1(Zymed), cytokeratin 8(development Studies Hybridoma Bank)

Teratoma formation

6-8 week old NOD-SCID male mice (Jackson Laboratories, BarHarbor, ME) were used. Small pellets of 50-100 cells were injected under the kidney capsule and mice were sacrificed 7-12 weeks post-transplantation, kidneys were fixed overnight in 4% paraformaldehyde, embedded in paraffin, sectioned, immunostained or hematoxylin-eosin stained, and analyzed for the presence of derivatives of all three germ layers.

PCR analysis of GFP sequences

Genomic DNA of WA01-GFP (H1-GFP), NED1, NED2, NED3 and NED4 cells WAs isolated using the MicroDNA kit (Qiagen) and the GFP-specific PCR reaction WAs performed as described previously (Klimanskaya et al, 2006). As an internal control for the PCR reactions, myogenic primers were included in all PCR reactions, which produced the (Klimanskaya et al, 2006)245bp fragment. The PCR products were separated on a 3% agarose gel and visualized by ethidium bromide staining.

Generating blast cells from NED hES cells

The performance of generating blasts from NED hescs with both hematopoietic and epithelial potential was previously reported (Lu et al, 2007). Briefly, 3.5 day old Embryoid Bodies (EBs) were generated from hescs cultured in StemLine II serum-free medium supplemented with a combination of morphogens and early hematopoietic cytokines, early stage EBs were dissociated, and individual cells were plated on serum-free semi-solid maternal cell colony (blast-colony) growth medium for 7 days. Grape-like blast colonies (grape-like blast colonies) were picked and plated for differentiation of both hematopoietic and epithelial cells.

Epithelial progenitor assay

For epithelial progenitor assays, the blast cells were plated on fibronectin coated plates (BDbioscience) in EGM-2 complete medium (Lonza) for 3-5 days. For Ac-LDL uptake, hES-BC cells were cultured in fibronectin-coated wells for 3-5 days and incubated with 10. mu.g/ml Alexa Fluor 594-labeled Ac-LDL (Invitrogen) for 6-8 hours. Cells were then washed 3 times with 1X PBS and fixed with 4% paraformaldehyde for 30 minutes. The uptake of Ac-LDL was visualized under a fluorescence microscope. For vwf (dako), PECAM-1(CD31) (cell signaling Technologies), VE-cadherin (R & D Systems) and kdr (cell signaling Systems) expression, cells were permeabilized, then incubated with primary antibodies overnight at 4 ℃ and then with FI TC-labeled corresponding secondary antibodies (jackson laboratory) for 30-60 minutes. After the final wash, the cells were examined under a fluorescent microscope.

Karyotyping analysis

Cells were plated on gelatin at a 1: 6 ratio. When the cells were approximately 50% confluent, 0.12ug/ml colchicamide (Invitrogen) was added to the medium for 40 minutes. Cells were harvested by trypsin, incubated in 0.075M KCl at 37 ℃ for 12 minutes, and fixed with 3: 1 methanol and acetic acid. Diffusion analysis was performed by Cell Line Genetics, inc. (NED 1-NED4 for the hESC Line) and the cytogenetic laboratory at the auckland children hospital using the G-banding technique.

Genotyping

Identification of newly derived hESC lines was performed by Seq Wright, inc using the AmpF1STRIdentifiler kit (Applied Biosystems).

RNA isolation and Gene expression analysis by PCR

Total RNA was isolated from approximately 100 hESCs and eluted in 80ul DEPC-H2O using the RNAeasy Micro Kit (Qiagen, Valencia, Calif.) following the supplier's recommended procedure. First strand cDNA synthesis of RNA was performed using Superscript II reverse transcriptase (Clontech) with SMART IIA and SMARTCDS primer IIA (Clontech), and cDNA pools were constructed using the SuperSMART cDNA Synthesis kit (Clontech) according to the supplier's recommendations. The 5ul cDNA library was used to analyze OCT-4 and Nanog expression with hypoxanthine phosphoribosyl transferase (HPRT) gene as a positive control. Total RNA isolated from H1ES cells was used as a positive control, and H2O was used as a negative control. 10 μ l of PCR product was separated on a 1.5% agarose gel and visualized by ethidium bromide staining.

Reference to the literature

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Claims (84)

1. A method for reprogramming the nucleus of a differentiated cell comprising the steps of:

(a) providing a differentiated cell, an enucleated MII stage egg of an animal, and an enucleated 2-cell stage embryo of an animal, wherein said MII stage egg and said embryo are synchronized;

(b) injecting nuclei of said differentiated cells into said enucleated egg;

(c) activating the egg comprising the nucleus;

(d) allowing the activated egg comprising the nucleus to develop to a 2-cell stage;

(e) removing at least one nucleus and at least a portion of the surrounding cytoplasm of the activated 2-cell stage egg of step (d); and

(f) fusing said at least one nucleus removed in step (e) into said enucleated 2-cell stage embryo by placing said nucleus between 2 cells of said 2-cell stage embryo to produce a single cell containing a reprogrammed nucleus of said differentiated cell.

2. The method of claim 1, wherein in step (c) the egg is actinone, CsCl₂Calcium ionophore, ionomycin, sperm factor, 6-DMAP, SrCl₂Cytochalasin B, or any combination thereof.

3. The method of claim 1, wherein in step (f), the fusing step is performed electrically.

4. The method of claim 3, wherein the electrofusion is performed in two steps: a first step in which the nucleus is aligned with the anode and electrically shocked, and a second step in which the embryo and the nucleus are rotated by about 90 degrees and electrically shocked.

5. The method of claim 1, further comprising the step of (g) culturing the single cell obtained from step (f) to the blastomere, morula, or blastocyst stage.

6. The method of claim 1, wherein the MII stage egg is a human egg.

7. The method of claim 1, wherein the enucleated 2-cell stage embryo is a human embryo.

8. The method of claim 1, wherein the differentiated cell is a human cell.

9. The method of claim 1, wherein the MII stage egg and the enucleated 2-cell stage embryo are from the same species.

10. The method of claim 1, wherein the differentiated cells and the MII stage eggs are from the same species.

11. The method of claim 1, wherein said differentiated cell and said enucleated 2-cell stage embryo are from the same species.

12. The method of claim 1, wherein the differentiated cell, the MII stage egg, and the enucleated 2-cell stage embryo are from the same species.

13. The method of claim 10, wherein the same species is human.

14. A method of producing an animal comprising:

(a) providing a differentiated cell, an enucleated MII stage egg of an animal, and an enucleated 2-cell stage embryo of an animal, wherein said MII stage egg and said embryo are synchronized;

(b) injecting nuclei of said differentiated cells into said enucleated egg;

(c) activating the egg comprising the nucleus;

(d) allowing the activated egg comprising the nucleus to develop to a 2-cell stage;

(e) removing at least one nucleus and at least a portion of the surrounding cytoplasm of the 2-cell stage egg of step (d);

(f) fusing said at least one nucleus removed in step (e) into said enucleated 2-cell stage embryo to produce a single cell; and

(g) culturing said individual cells from step (f) to allow development into an animal.

15. The method of claim 14, wherein in step (c) the egg is actinone, CsCl₂Calcium ionophore, ionomycin, sperm factor, 6-DMAP, SrCl₂Cytochalasin B, or any combination thereof.

16. The method of claim 14, wherein in step (f), the fusing step is performed electrically.

17. The method of claim 16, wherein the electrofusion is performed in two steps: a first step in which the nucleus is aligned with the anode and electrically shocked, and a second step in which the embryo and the nucleus are rotated by about 90 degrees and electrically shocked.

18. The method of claim 14, wherein the MII stage egg is a human egg.

19. The method of claim 14, wherein the enucleated 2-cell stage embryo is a human embryo.

20. The method of claim 14, wherein the differentiated cell is a human cell.

21. The method of claim 14, wherein the MII stage egg and the enucleated 2-cell stage embryo are from the same species.

22. The method of claim 14, wherein the differentiated cell and the MII stage egg are from the same species.

23. The method of claim 14, wherein the differentiated cell and the enucleated 2-cell stage embryo are from the same species.

24. The method of claim 14, wherein the differentiated cell, the MII stage egg, and the enucleated 2-cell stage embryo are from the same species.

25. The method of claim 22, wherein the same species is human.

26. A method of producing embryonic stem cells comprising the steps of:

(a) providing a differentiated cell, an enucleated MII stage egg of an animal, and an enucleated 2-cell stage embryo of an animal, wherein said MII stage egg and said embryo are synchronized;

(b) injecting nuclei of said differentiated cells into said enucleated egg;

(c) activating the egg comprising the nucleus;

(d) allowing the activated egg comprising the nucleus to develop to a 2-cell stage;

(e) removing the nucleus and surrounding cytoplasm of the 2-cell stage egg of step (d);

(f) fusing the nucleus removed in step (e) into the enucleated 2-cell stage embryo by placing the nucleus between 2 cells of the 2-cell stage embryo to produce a single cell; and

(g) culturing said individual cells from step (f) to a stage of development wherein embryonic stem cells can be derived.

27. The method of claim 26, wherein in step (c) the egg is actinone, CsCl₂Calcium ionophore, ionomycin, sperm factor, 6-DMAP, SrCl₂Cytochalasin B, or any combination thereof.

28. The method of claim 26, wherein in step (f), the fusing step is performed electrically.

29. The method of claim 28, wherein the electrofusion is performed in two steps: a first step in which the nucleus is aligned with the anode and electrically shocked, and a second step in which the embryo and the nucleus are rotated by about 90 degrees and electrically shocked.

30. The method of claim 26, wherein the MII stage egg is a human egg.

31. The method of claim 26, wherein the enucleated 2-cell stage embryo is a human embryo.

32. The method of claim 26, wherein the differentiated cell is a human cell.

33. The method of claim 26, wherein the MII stage egg and the enucleated 2-cell stage embryo are from the same species.

34. The method of claim 26, wherein the differentiated cell and the MII stage egg are from the same species.

35. The method of claim 26, wherein the differentiated cell and the enucleated 2-cell stage embryo are from the same species.

36. The method of claim 26, wherein the differentiated cell, the MII stage egg, and the enucleated 2-cell stage embryo are from the same species.

37. The method of claim 34, wherein the same species is human.

38. The method of claim 26, wherein the embryonic stem cells are hemizygous or homozygous for an MHC allele, wherein the differentiated cells are hemizygous or homozygous for an MHC allele, or the embryonic stem cells are engineered to be hemizygous or homozygous for an MHC allele by homologous recombination or by loss of heterozygosity, or both, and wherein the same species is human.

39. The method of claim 38, wherein the method is repeated a plurality of times to produce a pool of embryonic stem cells, each embryonic stem cell being hemizygous or homozygous for a different MHC allele from the other embryonic stem cells of the pool.

40. The method of claim 14, wherein step (g) comprises implanting the cultured cells into the uterus of an animal.

41. The method of claim 40, wherein the implanted cells and the animal into which they are implanted are of the same species.

42. The method of claim 26, wherein step (g) comprises:

(i) culturing the single cells from step (f) to the morula stage;

(ii) isolating blastomeres from the morula;

(iii) culturing the blastomeres to produce clusters of two or more blastomeres;

(iv) contacting the cultured cluster of two or more blastomeres with an embryonic or fetal cell, directly or indirectly; and

(v) (iv) culturing the cluster of two or more blastomeres of (iv) until ES cells are produced.

43. A method of producing Embryonic Stem (ES) cells, comprising:

(a) culturing blastomeres dissected from a mammalian maternal embryo in vivo with the maternal embryo for 12 to 18 hours;

(b) transferring the blastomeres to blastocyst medium further comprising laminin and inoculated with Mouse Embryonic Fibroblasts (MEFs), and

(c) culturing the blastomeres of (b) until ES cells are produced.

44. The method of claim 43, wherein MEF is meiotically inactivated.

45. The method of claim 43, wherein step (c) comprises culturing under conditions that reduce egg ball formation.

46. The method of claim 45, wherein the blastocyst medium comprises 2.5 μ g/ml laminin.

47. The method of claim 45, wherein step (c) comprises culturing for 5 days in blastocyst medium seeded with MEF cells.

48. The method of claim 43, wherein step (c) further comprises: culturing to form a cell pellet of about 20 cells in the shape of the blastomere, and transferring the cell pellet to a medium seeded with ES cells.

49. The method of claim 48, wherein the ES cells express a marker or are labeled.

50. The method of claim 43, wherein the maternal embryo is transferred to blastocyst medium and allowed to develop into a blastocyst.

51. The method of claim 43, wherein the blastomere is isolated from an embryo, comprising:

(a) fixing the embryo; and

(b) the fixed embryos were flicked until blastomere separation.

52. A method of producing embryonic stem cells comprising the steps of:

(a) providing a differentiated cell, an enucleated MII stage egg of an animal, and an enucleated 2-cell stage embryo of an animal, wherein said MII stage egg and said embryo are synchronized;

(b) injecting nuclei of said differentiated cells into said enucleated egg;

(c) activating the egg comprising the nucleus;

(d) allowing the activated egg comprising the nucleus to develop to a 2-cell stage;

(e) removing the nucleus and surrounding cytoplasm of the 2-cell stage egg of step (d);

(f) fusing the nucleus removed in step (e) into the enucleated 2-cell stage embryo by placing the nucleus between 2 cells of the 2-cell stage embryo to produce a single cell;

(g) culturing the single cell from step (f) to produce morula, which can be used as a maternal embryo;

(h) isolating blastomeres from the morula;

(i) culturing the blastomeres and the maternal embryos together for 12 to 18 hours;

(j) transferring the blastomeres to blastocyst medium further comprising laminin and inoculated with Mouse Embryonic Fibroblasts (MEFs), and

(k) culturing the blastomeres of (j) until ES cells are produced.

53. The method of claim 52, wherein MEF is meiosis inactivated.

54. The method of claim 52, wherein step (k) comprises culturing under conditions that reduce egg ball formation.

55. The method of claim 54, wherein the blastocyst medium comprises 2.5 μ g/ml laminin.

56. The method of claim 54, wherein step (k) comprises culturing in blastocyst medium seeded with MEF cells for 5 days.

57. The method of claim 52, wherein step (k) further comprises: culturing to form a cell pellet of about 20 cells in the shape of the blastomere, and transferring the cell pellet to a medium seeded with ES cells.

58. The method of claim 57, wherein the ES cells express a marker or are labeled.

59. The method of claim 52, wherein the maternal embryo is transferred to a blastocyst medium and allowed to develop into a blastocyst.

60. The method of claim 52, wherein the blastomeres are isolated from morulae, comprising:

(a) fixing the morula; and

(b) gently beat the fixed morula until the blastomeres separate.

61. The method of claim 52, wherein in step (c) the egg is actinone, CsCl₂Calcium ionophore, ionomycin, sperm factor, 6-DMAP, SrCl₂Cytochalasin B, or any combination thereof.

62. The method of claim 52, wherein in step (f), the fusing step is performed electrically.

63. The method of claim 62, wherein the electrofusion is performed in two steps: a first step in which the nucleus is aligned with the anode and electrically shocked, and a second step in which the embryo and the nucleus are rotated by about 90 degrees and electrically shocked.

64. The method of claim 52, wherein the MII stage egg is a human egg.

65. The method of claim 52, wherein the enucleated 2-cell stage embryo is a human embryo.

66. The method of claim 52, wherein the differentiated cell is a human cell.

67. The method of claim 52, wherein the MII stage egg and the enucleated 2-cell stage embryo are from the same species.

68. The method of claim 52, wherein the differentiated cells and the MII stage eggs are from the same species.

69. The method of claim 52, wherein said differentiated cell and said enucleated 2-cell stage embryo are from the same species.

70. The method of claim 52, wherein said differentiated cell, said MII stage egg, and said enucleated 2-cell stage embryo are from the same species.

71. The method of claim 70, wherein the same species is human.

72. The method of claim 52, wherein the embryonic stem cells are hemizygous or homozygous for an MHC allele, wherein the differentiated cells are hemizygous or homozygous for an MHC allele, or the embryonic stem cells are engineered to be hemizygous or homozygous for an MHC allele by homologous recombination or by loss of heterozygosity, or both, and wherein the same species is human.

73. The method of claim 72, wherein the method is repeated a plurality of times to produce a pool of embryonic stem cells, each embryonic stem cell being hemizygous or homozygous for a different MHC allele than the other embryonic stem cells of the pool.

74. The method of claim 52, wherein said blastocyst medium comprises a factor capable of inhibiting differentiation into a non-ES cell.

75. The method of claim 52, wherein said blastocyst medium comprises a factor capable of disrupting tight junctions.

76. The method of claim 52, wherein the blastocyst medium comprises a factor capable of inhibiting the trophectoderm differentiation pathway.

77. The method of claim 52, wherein said blastocyst medium comprises a factor capable of depolarizing a cell.

78. A method of producing Embryonic Stem (ES) cells, comprising:

(a) culturing blastomeres dissected from mammalian maternal embryos in vivo;

(b) transferring the blastomeres to blastocyst media further comprising laminin; and

(c) culturing the blastomeres of (b) until ES cells are produced.

79. The method of claim 78, wherein step (c) comprises culturing under conditions that reduce egg ball formation.

80. The method of claim 78, wherein the blastocyst medium comprises 2.5 μ g/ml laminin.

81. The method of claim 78, wherein the maternal embryo is transferred to a blastocyst medium and allowed to develop into a blastocyst.

82. The method of claim 78, wherein the blastomere is isolated from an embryo, comprising:

(a) fixing the embryo; and

(b) the fixed embryos are flicked until the blastomeres separate.

83. The method of claim 78, wherein the blastomeres of (a) are cultured with the mammalian maternal embryo.

84. The method of claim 78, wherein the blastomeres are not cultured with previously obtained human stem cells.