HK1222890B - Conditionally immortalized long-term stem cells and methods of making and using such cells - Google Patents

Description

Conditionally immortalized long-term stem cells and methods of making and using the same

The present application is a divisional application of an invention patent application filed on October 18, 2006, with application number 200680045545.8 and the invention name being “Conditionally Immortalized Long-term Stem Cells and Methods for Preparing and Using the Same”.

Field of the Invention

The present invention generally relates to conditionally immortalized long-term stem cells, methods of making the cells, and methods of using the cells, including therapeutic methods and drug discovery methods.

Background of the Invention

The ability to control the bone marrow output of different blood cells has become an important tool in the treatment of a variety of diseases. Some of the best new therapies for hematological malignancies are based on the development of compounds that encourage leukemic cells to differentiate into a desired lineage before the transformation event. One such example is acute promyelocytic leukemia. Treatment of patients with arsenic trioxide has been shown to drive malignant cells down the myelomonocytic pathway, leading to remission of these tumors. Another example is the successful engraftment of transplanted bone marrow stem cells (long-term reconstituting hematopoietic stem cells, or lt-HSCs) in irradiated individuals. Systemic administration of cytokines known to specifically induce red blood cell development (erythropoietin, or Epo) or myeloid cell development (granulocyte-macrophage colony-stimulating factor, or GM-CSF) can promote the emergence of differentiated blood cells. Finally, the harvesting of lt-HSCs from donors has been greatly simplified using a "mobilization" approach, in which these cells are induced to move from their normal location in the bone marrow into the peripheral blood through systemic administration of a cytokine called G-CSF. The stem cells can then be harvested from the peripheral blood, thus avoiding the painful and complex collection of bone marrow biopsy tissue.All of these approaches rely on the ability to program and control the biological behavior of lt-HSCs.

Accordingly, bone marrow (stem cell) transplantation is an invaluable therapeutic tool for the blood and immune reconstitution of individuals who have undergone radiation and/or chemotherapy (e.g., cancer patients, or those who have been exposed to high levels of radiation), and is also an important method for treating immunodeficiency and hematological malignancies. In addition, bone marrow transplantation will be a very useful therapy to combat the adverse effects of aging on the immune system and other cells and tissues. It is estimated that stem cell transplantation will benefit more than 35,000 children and adults each year.

Bone marrow transplants work by replacing radiosensitive hematopoietic stem cells (HSCs) that produce all blood cell types. Recent studies suggest that bone marrow transplants may be valuable for treating heart disease. Although the basis for this effect is unknown, this and other findings raise the possibility that hematopoietic stem cells (HSCs) can be reprogrammed to produce other tissues. If this is true, HSCs could have broader applications and offer an alternative to controversial embryonic stem cell therapies.

The major obstacle faced by the clinical application of bone marrow transplantation is first the identification of appropriate histocompatibility bone marrow donors. This is usually completed by an institution with more than 6 million potential donors registered. The donor of choice must undergo a rigorous treatment of inducing stem cell mobilization into the blood and subsequent 4 to 5 days of leukocyte clearance to separate rare lt-HSCs. After these cells are transplanted, the recipient must be carefully monitored and treated to minimize the graft-versus-host reaction caused by passing lymphocytes.

The related cell colony of low frequency makes it difficult to illustrate the molecular basis of the impaired development of hematopoietic lineage in history, and this has stopped the biochemical analysis to signal transduction and downstream reaction.In fact, this is a main limiting factor in all hematopoietic research.In addition, the limited availability of long-term hematopoietic stem cell (LT-HSC) is also a major obstacle in many types of cancers and several immunodeficiencies for the treatment of the mankind. As far as the inventors know, there is no available similar lt-HSC spontaneous generation and can be differentiated into the cell line of normal lineage or the human reconstruction of lethally irradiated mice or sublethally irradiated, nor is there any method of recording that can be expected to produce the cell line. Moreover, there is no feasible technology of continuous amplification lt-HSC at present, so that these cells need to be obtained from the donor at each time when needed.

There is also an urgent need for the additive method for treating hematological malignancies and immunodeficiency, and the new cytokine that improves transplanting lt-HSC output. In addition, there is no suitable platform for target identification and drug discovery at present. The factor of disappearance is the cell line representing the different development stages of hematopoietic pedigree. Ideally, the cell should remain on the ability of further differentiation in the specific pedigree. For the evaluation of gene product, therefore for the new pharmaceutically useful target relevant with the regulation of cell development, proliferation and survival, these cell lines are important. In addition, in new drug development, the cell line is important for the screening of the cDNA library for the small molecule and shRNA library that are used for loss of function research and for the function increase research.

Current drug discovery obstacles in this area include: (a) isolating sufficient numbers of cells from a specific stage of development; (b) propagating cells in vitro for a sufficient period of time; and (c) the ability to use conditional oncogenes to screen for drugs that affect leukemia cells without affecting normal HSCs or progenitor cells.

Therefore, there is a great need in the art for a method to generate lt-HSC lines that can be extensively expanded, frozen, and readily reused when needed without the need for subsequent harvesting from a donor.

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to a method for producing conditionally immortalized adult stem cells. The method comprises the following steps: (a) obtaining a population of expanded adult stem cells; (b) transfecting the stem cells with a nucleic acid molecule comprising a proto-oncogene or a biologically active fragment or homolog thereof that promotes cell survival and proliferation, wherein the proto-oncogene is inducible; (c) transfecting the stem cells with a nucleic acid molecule encoding a protein that inhibits apoptosis in the cells; and (d) expanding the transfected cells in the presence of a combination of stem cell growth factors and under conditions that maintain the activity of the proto-oncogene to produce conditionally immortalized adult stem cells. In one aspect of this embodiment, the nucleic acid molecule of (b) and/or (c) is contained in an integrating vector. In one aspect, the nucleic acid molecule of (b) and/or (c) is transfected into the cells using a virus or viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, a parvovirus, a vaccinia virus, a coronavirus, a calicivirus, a papillomavirus, a flavivirus, an orthomyxovirus, a togavirus, a picornavirus, an adenoviral vector, and a modified and attenuated herpesvirus. In one aspect, the nucleic acid molecules of (b) and/or (c) are transfected into cells using direct electroporation. In one aspect, the nucleic acid molecules of (b) and/or (c) are contained in a vector comprising a nucleic acid sequence encoding a drug-sensitive protein. In one aspect, the nucleic acid molecules of (b) and/or (c) are contained in a vector that flanks the nucleic acid molecules of (b) or (c) and comprises a nucleic acid sequence encoding a recognition substrate sequence for a recombinase.

In one aspect, this embodiment includes the following additional steps: (e) removing the conditions in (d) that maintain the proto-oncogene activity; and (f) culturing the cells in (e) in a medium containing a growth factor that induces cell differentiation. This method may further include: (g) subjecting the cells in (f) to the conditions in (d) that maintain the proto-oncogene activity to produce conditionally immortalized cells at an intermediate stage of cell differentiation.

Another embodiment of the present invention relates to a method for producing conditionally immortalized adult stem cells, comprising: (a) obtaining an expanded population of adult stem cells; (b) culturing the stem cells in the presence of: (1) a combination of stem cell growth factors; (2) a first Tat-fusion protein, wherein Tat is fused to a protein encoded by a proto-oncogene, or a biologically active fragment or homolog thereof, that promotes cell survival and proliferation; and (3) a second Tat-fusion protein, wherein Tat is fused to a protein that inhibits apoptosis of the stem cells.

Another embodiment of the present invention relates to a method for producing conditionally immortalized embryonic stem cells, comprising: (a) obtaining an expanded population of embryonic stem cells; (b) transfecting the stem cells with a nucleic acid molecule comprising a proto-oncogene, or a biologically active fragment or homolog thereof, that promotes cell survival and proliferation, wherein the proto-oncogene is inducible; (c) transfecting the stem cells with a nucleic acid molecule encoding a protein that inhibits apoptosis of the cells; and (d) expanding the transfected cells in the presence of a combination of stem cell growth factors and under conditions that maintain the activity of the proto-oncogene to produce conditionally immortalized embryonic stem cells.

Another embodiment of the present invention relates to a method for producing conditionally immortalized stem cells, comprising: (a) obtaining an expanded stem cell population; (b) culturing the stem cells in the presence of: (1) a combination of stem cell growth factors; (2) a protein encoded by a proto-oncogene or a biologically active fragment or homolog thereof that promotes cell survival and proliferation; and (3) a protein that inhibits apoptosis in the stem cells. The proteins of (2) and (3) are delivered to the stem cells using any suitable delivery system, including but not limited to Tat fusion, aptamer technology, or CHARIOT ^™ technology.

Another embodiment of the present invention relates to a method for producing conditionally immortalized stem cells, comprising: (a) obtaining an expanded stem cell population; (b) delivering a protein encoded by a proto-oncogene or a biologically active fragment or homolog thereof that promotes cell survival and proliferation, or a nucleic acid molecule encoding the protein, into the cells, wherein the proto-oncogene is inducible; (c) inhibiting apoptosis of the stem cells by delivering into the cells a protein that inhibits apoptosis of the cells, a nucleic acid molecule encoding a protein that inhibits apoptosis of the cells, or a nucleic acid molecule or protein that inhibits a pro-apoptotic protein in the cells; and (d) expanding the cells in the presence of a combination of stem cell growth factors and under conditions that keep the proto-oncogene active to produce conditionally immortalized adult stem cells.

In any of the above embodiments, the proto-oncogene may be selected from, but not limited to, MYC-ER and ICN-I-ER. In any of the above embodiments, the protein that inhibits apoptosis may be selected from, but not limited to, members of the Bcl-2 family that inhibit apoptosis, such as Bcl-2, Bcl-X, Bcl-w, BclXL, Mcl-1, Dad-1, or hTERT. When the proto-oncogene is MYC-ER or ICN-I-ER, the conditions that maintain the activity of the proto-oncogene may include the presence of tamoxifen or an agonist thereof. In one aspect, the cells are transfected or delivered (as proteins) with the following substances: MYC-ER and Bcl-2; MYC-ER and hTERT; ICN-1-ER and Bcl-2; ICN-1-ER and hTERT; or MYC-ER and ICN-1-ER.

In any of the above embodiments, the expansion step can be performed in a medium including, but not limited to: (1) interleukin-6 (IL-6), IL-3 and stem cell factor (SCF); (2) a serum-free medium comprising stem cell factor (SCF), thrombopoietin (TPO), insulin-like growth factor 2 (IGF-2) and fibroblast growth factor 1 (FGF-1).

In any of the above embodiments, adult stem cells include but are not limited to: hematopoietic stem cells, intestinal stem cells, osteoblast stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, cardiomyocyte progenitor stem cells, skin stem cells, skeletal muscle stem cells, and liver stem cells. In one aspect, mesenchymal stem cells can be selected from lung mesenchymal stem cells and bone marrow stromal cells. In one aspect, epithelial stem cells are selected from: lung epithelial stem cells, breast epithelial stem cells, vascular epithelial stem cells and intestinal epithelial stem cells. In one aspect, skin stem cells are selected from: epidermal stem cells and vesicle stem cells (hair follicle stem cells). In one aspect, neural cells are selected from neuronal dopaminergic stem cells and motor neuron stem cells. In one aspect, stem cells are from fresh or refrigerated umbilical cord blood. In one aspect, stem cells are hematopoietic progenitor cells obtained from the peripheral blood of normal people or patients treated with granulocyte colony stimulating factor (G-CSF).

In any of the above embodiments, the method can further comprise genetically modifying the stem cells to correct a genetic defect in the cells, genetically modifying the stem cells to silence gene expression, and/or genetically modifying the stem cells to overexpress a gene.

In any of the above embodiments, the method may further comprise preserving the cells. In one aspect, the method further comprises recovering the cells from the preservation and culturing the cells.

Another embodiment of the invention relates to cells produced by any of the methods described above or elsewhere herein.

Another embodiment of the present invention relates to a method for providing adult stem cells or differentiated cells thereof to an individual, comprising: (a) providing a source of conditionally immortalized adult stem cells produced by any of the methods described above or elsewhere herein; (b) removing the conditions that conditionally immortalized the stem cells of (a), and (c) administering the stem cells or differentiated cells thereof to the individual. In one embodiment, the cells are previously obtained from the individual of (c). In one embodiment, the cells are obtained from a previously frozen stock of the cells. In one aspect, the cells are freshly obtained from the individual and conditionally immortalized by any of the methods described above or elsewhere herein. In one aspect, the individual has cancer. In another aspect, the individual has leukemia. In another aspect, the individual has an immunodeficiency disorder. In another aspect, the individual has an anemic disorder. In another aspect, the individual is undergoing plastic surgery. In another aspect, the individual is undergoing elective cosmetic surgery. In another aspect, the individual is undergoing transplant surgery. In one aspect, the individual is in need of stem cells or differentiated cells thereof selected from the group consisting of hematopoietic stem cells, intestinal stem cells, osteoblast stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, cardiomyocyte progenitor stem cells, skin stem cells, skeletal muscle stem cells, and liver stem cells. In another aspect, the individual is in need of improved immune cell function. In another aspect, the individual has a genetic defect that can be corrected by stem cells.

Another embodiment of the invention is directed to a method of identifying a compound that modulates lineage commitment and/or cell differentiation and development, comprising: (a) contacting adult stem cells produced by any of the methods described above or elsewhere herein; and (b) detecting at least one genotypic or phenotypic characteristic in the stem cells of (a), compared to in the absence of the compound, wherein a difference in the characteristic detected in the presence of the compound indicates that the compound affects a property of the stem cells.

Another embodiment of the present invention relates to a method for studying lineage commitment and/or cell differentiation and development, comprising evaluating adult stem cells or differentiated cells thereof produced by any of the methods described above or elsewhere herein to detect at least one genotypic or phenotypic characteristic of the cells.

Another embodiment of the invention relates to the use of cells produced by any of the methods described above or elsewhere herein in a medicament for the treatment of a disease or condition in which stem cell transplantation is beneficial.

Another embodiment of the present invention relates to a mouse model of acute myeloid leukemia (AML), comprising a mouse produced by a method comprising: (a) lethally irradiating the mouse; (b) transferring into the mouse conditionally immortalized long-term stem cells produced by any of the methods described above or elsewhere herein and whole bone marrow cells from Rag ^−/− mice; and (c) regularly injecting the mouse with tamoxifen or an agonist thereof until the mouse develops clinical signs of AML. In one embodiment, the cells are transfected with MYC-ER and Bcl-2 or delivered with these substances (as proteins).

Another embodiment of the present invention relates to tumor cells obtained from the above-mentioned AML mouse model.

Another embodiment of the present invention relates to the use of AML mouse models in preclinical testing of human protein-specific drug candidates, in identifying, developing and/or testing compounds for diagnosing, studying or treating AML, or in identifying, developing and/or testing targets for diagnosing, studying or treating AML.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the death curve after bone marrow transplantation of transduced cells and activation of MYC function with 4OHT in vivo.

Figure 2 is a scatter plot showing the scatter characteristics and GFP expression levels of HSCs derived from young and old mice after in vitro transduction. The dot plots represent flow cytometric analysis data of the forward scatter (FSC) and side scatter (SSC) characteristics of HSCs after 3 days of culture with IL-3, IL-6, and SCF. These two criteria are correlated with cell size (FSC) and granularity (SSC).

Figure 3 is a scatter plot showing a comparison of the phenotypes of cell lines derived from irradiated recipients reconstituted with BCL-2, MYC-ER, and EGFP-transduced hematopoietic stem cells from old (>60% ID ^- repertoire) and young 3-83μδ transgenic mice. The phenotypes of representative clones are shown 3 (young) and 3 (aged) months after the start of culture.

Figure 4 is a scatter plot showing spontaneous differentiation of a senescent LT-HSC line (ABM46) in vitro following withdrawal of tamoxifen (stem cell and B-lineage marker expression analyzed by flow cytometry).

Figure 5 is a scatter plot showing analysis of the hematopoietic cell compartment derived from the Lt-HSC line 6 weeks after adoptive transfer into young irradiated recipients. Data from three mice are shown, one mouse receiving the aged HSC line ABM42 and two mice receiving the aged HSC line ABM46.

Figure 6 is a scatter plot showing that the development of the B-cell compartment is affected in mice reconstituted with the ABM42 and ABM46 cell lines. Data from three mice are shown in this figure, one mouse receiving the aged HSC line ABM42 and two mice receiving the aged HSC line ABM46.

Figure 7 is a scatter plot showing T-cell development in mice reconstituted with the ABM42 and ABM46 cell lines. Data from three mice are shown, one mouse receiving the aged HSC line ABM42 and two mice receiving the aged HSC line ABM46.

Figure 8 is a scatter plot and a graph showing a phenotypic comparison of cell lines derived from HSCs obtained from young C57/BL6 mice that were retrovirally transduced with BCL-2 and MYC-ER and maintained in vitro for more than 90 days in continuous culture. Each figure shows flow cytometric analysis of the expression of virally expressed markers (GFP and Thy1.1) and four markers required for determining long-term HSCs in mice: Sca-1, c-kit, CD34, and Flk-2. The four cell lines contain subpopulations that maintain a lt-HSC phenotype (Sca-1+, c-kit+, CD34-, flk-2-).

Figure 9 is a scatter plot and graph showing a phenotypic comparison of cell lines derived from HSCs obtained from young C57/BL6 mice that were retrovirally transduced with different oncogene combinations and maintained in continuous culture in vitro for more than 90 days (pMIG-MYC and pMIT-Bcl-2 (top panel), pMIG-MYC.ER and pMIG-hTERT (middle panel), and pMIG-ICN.1.ER and pMIT-Bcl-2 (bottom panel)).

Figure 10 is a scatter plot and graph showing a phenotypic comparison of cell lines derived from HSCs obtained from young C57/BL6 mice that were retrovirally transduced with different oncogene combinations and maintained in continuous culture in vitro for more than 90 days (pMIG-ICN.1.ER and pMIT-Bcl-2 (top panel), pMIG-ICN.1 and pMIT-Bcl-2 (second row of panels), or pMIG-ICN.1 and pMIG-Bcl-2 (third row of panels), or pMIG-hTERT and pMIT-Bcl-2 (bottom panel)).

Figure 11 is a scatter plot and graph showing a phenotypic comparison of cell lines derived from HSCs obtained from young C57/BL6 mice that were retrovirally transduced with different oncogene combinations and maintained in continuous culture in vitro for more than 90 days (pMIG-MYC and pMIG-ICN.1 (top panel), pMIG-MYC.ER and pMIG-ICN.1 (middle panel), or pMIG-ICN.1.ER and pMIG-MYC (bottom panel)).

Figure 12 is a scatter plot showing in vivo reconstitution of the T and B cell compartments of cell lines derived from HSCs obtained from young C57/BL6 mice that were retrovirally transduced with different oncogene combinations and maintained in serial culture in vitro for over 90 days.

FIG13 is a schematic diagram showing the use of a recognition substrate sequence (RSS) for a recombinase to ensure excision of recombinant DNA from conditionally immortalized long-term stem cells of the present invention prior to transplantation.

FIG. 14 is a graph showing detection of NK and erythroid lineage cells differentiated from conditionally immortalized long-term stem cells of the present invention.

Detailed Description of the Invention

The present invention provides a solution to the problem of being able to generate, maintain, and manipulate stable cell lines derived from long-term stem cells, particularly long-term hematopoietic stem cells (Lt-HSCs), which, when placed under appropriate conditions, can give rise to all cell lineages normally generated by these cells. The present invention generally relates to methods for generating conditionally immortalized long-term stem cells, stem cells generated using such methods, and methods of utilizing such stem cells. More particularly, using long-term hematopoietic stem cells as an exemplary stem cell population, the inventors have established an efficient method for generating conditionally immortalized (e.g., reversibly immortalized or immortalized under specific conditions that are reversible upon removal of such conditions) stem cells that are capable of differentiating into normal cell lineages in vitro and in vivo and regenerating patients in need thereof. In fact, the present invention can eliminate the need for bone marrow donors because it provides the ability to harvest stem cells from a patient prior to a procedure (e.g., chemotherapy, radiation therapy, etc.), expand the cells, and return them to the patient. Furthermore, the stem cells can be expanded extensively, stored (e.g., frozen), and then recovered and re-expanded, manipulated, and/or reused as needed or desired. For example, the stem cells can be manipulated to correct a genetic defect or provide a benefit (therapeutic or preventative benefit) to a patient, or differentiate into a desired cell type. Finally, the cells can be used in a variety of experiments to identify new targets relevant to the regulation of cell development, proliferation, and survival, and to identify and develop drugs for improving or treating diseases and conditions that would benefit from regulation of cell development, proliferation, and/or survival.

The inventors have developed a new technology that allows the immortalization of long-term stem cell conditions (such as long-term hematopoietic stem cells (lt-HSC) in this case). The resulting cell lines can be exponentially expanded (propagated) and/or cryopreserved (stored) indefinitely in vitro and have the ability to rescue mice subjected to lethal irradiation and reconstruct all blood cell lineages in the animals. Moreover, the inventors have been able to produce differentiated blood cells in vitro by inhibiting the function of transforming oncogenes. The cells described herein and the methods for producing them will allow the production of transplantable human stem cells that do not carry recombinant DNA and therefore do not pose long-term risks to the recipient. These conditionally immortalized lt-HSCs of the present invention can be stabilized in their mature phenotype, and cell lines that maintain their mature phenotype after oncogene reactivation have been established. For example, the inventors have been able to develop CD4 ⁺ αβ ⁺ T cells, as well as dendritic cell lines.

This technology has the following advantages over bone marrow transplantation when applied in clinical settings:

1. Very few lt-HSCs are needed to establish clones;

2. Clones represent a renewable resource that can be stored indefinitely and accessed quickly;

3. The cost of this treatment should be lower than traditional bone marrow transplantation;

4. The use of lt-HSC clones can alleviate the threat of graft-versus-host disease and the associated costs;

5. In at least some cases, this technology may alleviate the need for bone marrow donors.

In addition, the invention provides the application of long-term stem cells such as lt-HSC cells in cells that produce the pedigree (such as, the hematopoietic pedigree of differentiation, including the intermediate stage of hematopoietic pedigree development) that represents differentiation. For example, except countless treatment and prevention applications, these cell lines will allow identification of new compounds that can induce malignant cell differentiation, stop its growth or induce apoptosis. These cells will also allow screening of new cytokines and growth factors that guide stem cells to differentiate along specific pathways. Described cell line does not exist and is important for drug discovery.

More specifically, to overcome limitations in the art regarding the availability and utilization of long-term populations of adult-derived stem cells (although, as described below, the present invention is not limited to adult-derived stem cells), the present invention has developed novel methods for generating conditionally transformed cell lines representing early hematopoietic stem cell progenitors. In a specific, non-limiting example of the present technology described and exemplified herein, the strategy involves transfection (e.g., by retroviral transduction) of bone marrow stem cells from 5-fluorouracil (5-FU)-treated 3-83μδ mice. The inventors used a pMSCV bicistronic retroviral vector with inserts encoding Bcl-2 and green fluorescent protein (GFP) (as reporter genes) and MYC-ER and GFP (also as reporter genes). MYC was selected because it has the ability to replace cytokine-generated survival and proliferation signals in lymphocytes. By restricting the target cells, the inventors hypothesized that stem cell tumors would be formed. Importantly, in this case, MYC-ER function is dependent on tamoxifen, and removal of tamoxifen from the animal or culture allows MYC function to terminate and transform. In cells transduced with MYC-ER, the fusion protein is produced, but the fusion protein remains in the cytoplasm until exposed to tamoxifen. Bcl-2 was chosen because it has the ability to inhibit apoptosis, which normally occurs due to exposure to MYC signaling, and more specifically, occurs when MYC is "inactivated" or removed by removing tamoxifen from the cells. This novel combination of gene types (as discussed in more detail below, the present invention is not limited to these specific genes) is partly responsible for the successful generation of the conditionally immortalized stem cells of the present invention and, as described below, can be easily expanded to other similar gene combinations.

Recipients of the above-mentioned transduced stem cells develop tumors (in the presence of 4OHT), and tumor cells from the bone marrow, spleen, and lymph nodes are harvested and placed in culture with tamoxifen and a mixture of stem cell growth factors. The inventors have found that in the absence of an appropriate combination of stem cell growth factors, the stem cells produced by this method will stop growing and die within a very short period of time. Therefore, the use of a "mixture" of stem cell growth factors (i.e., a combination of appropriate or suitable stem cell growth factors) after transfecting the cells with the above-mentioned gene combination is a second important aspect of the method of the present invention. This mixture has the general property of promoting and maintaining stem cell growth, is not limited to a specific combination of growth factors, and the parameters for selecting the factors are discussed in more detail below.

The stem cells generated by the methods of the present invention can be expanded in culture and are homogenously positive for, for example, scal, endoglin, and ckit, and negative for CD34, Flt3, B220, CD19, and mIgM, indicating a phenotype of lt-HSC, as is well known in the art. These cells can be frozen (cold-stored or stored) and then readily revived and cultured after freezing. Importantly, these revived cells are phenotypically homogenous and display the phenotype of lt-HSC (e.g., homogeneous GFP-bright cells are positive for scal, endoglin, and ckit, and negative for CD34, Flt3, B220, CD19, and mIgM). This phenotype corresponds well to the published characteristics of long-term regenerative pluripotent stem cells that provide all long-term reconstitution in mice (Reya et al., 2003, Nature 423:409-14).

The inventor has further developed this method so that it can complete in vitro (as mentioned above, the initial step is partly carried out in vivo). The inventor has also confirmed that other gene combinations with similar characteristics as mentioned above also cause the conditional immortalization of lt-HSC. Moreover, cell line can be differentiated into hematopoietic lineages in vitro by removing tamoxifen and providing appropriate growth factors, and is differentiated into all hematopoietic lineages in vivo in the recipient animal that tamoxifen is blocked. In addition, cell can be differentiated into the intermediate level of growth, and this intermediate level has stable phenotype and remains on the ability that further differentiates along their directional pathway after applying or removing appropriate signal (described herein). Described cell is invaluable for multiple therapeutic applications. All these experiments are described in detail below and in the examples.

The methods and cell lines of the present invention not only provide an opportunity to study in detail the molecular, biochemical and cellular events associated with the commitment of adult stem cells to different cell lineages, as well as to study the differentiation and development of stem cells into different cell lineages, but also provide unique therapeutic and drug discovery tools.

For example, the stem cell lines of the present invention provide a unique source of expandable stem cells that can be used in a variety of transplantation, therapeutic, and preventive strategies, including the treatment of cancer, particularly cancers treated with radiation. For example, in current treatments for leukemia, limited access to bone marrow donors and the limited supply of stem cells from these donors severely limit options for reconstructing patients after radiation therapy. The present invention addresses this problem by providing a method for generating a sustainably expandable and renewable supply of autologous stem cells or histocompatible stem cells that can be stored or restored as needed. This technology could ultimately eliminate the need for bone marrow donors. In addition, various immunodeficiency disorders and anemias (e.g., aplastic anemia or hemolytic anemia) would also greatly benefit from this technology because it provides the ability to regenerate individual hematopoietic cells based on individual needs. Moreover, the aging process is associated with several important changes in the hematopoietic compartment, particularly the increasing inability to achieve a productive immune response. Hematopoietic stem cells derived from aged mice have been shown to have higher levels of mRNAs that address DNA repair issues. This may ultimately affect their ability to self-renew, differentiate, proliferate, and survive in response to myeloid cytokines. Thus, aging individuals may also benefit from the present invention, as a continued supply of healthy hematopoietic cells may be provided to correct or alleviate the defect.

The technology of the present invention is not limited to bone marrow stem cells, but is applicable to virtually any type of stem cell and can be extended beyond cells derived from adults to embryonic stem cells.

In one embodiment, another application of the present invention involves the production of continuously expandable and renewable hair follicle stem cells. The development of conditionally immortalized stem cells derived from this lineage can be used in plastic surgery for burn victims, for any patient who has undergone chemotherapy and/or radiation therapy resulting in irreversible loss of hair growth, and for patients following any surgical procedure affecting the skull. Furthermore, the cells can be used in selective procedures involving inducing hair growth in individuals affected by hereditary alopecia. Similarly, the application of the stem cells of the present invention to the skin is invaluable for wound healing and treatment in burn victims, as well as for plastic surgery for trauma and other patients, and for selective surgical procedures (including but not limited to cosmetic surgery). Furthermore, the cells can be genetically manipulated to correct congenital or acquired genetic defects in young and aging individuals. Those skilled in the art will appreciate, based on this disclosure, that benefits can be derived from the application of the present invention to a variety of other stem cell populations, including but not limited to stem cells derived from the lung, breast, and intestinal epithelium, and stem cells derived from neural and cardiac tissue, to name a few. Other stem cell types are mentioned elsewhere herein.

In addition, the present invention provides the unique opportunity that obtains the expandable supply of autologous stem cells and its differentiated cells as needed in the individual's lifetime.For example, it is known that when the body is aging, immune function and immune memory deteriorate.But, utilizing technology provided by the present invention, it is possible to utilize new autologous stem cells that can be differentiated into all cells of the hematopoietic lineage to regenerate the individual, thereby providing a "young" immune system to the aging individual.In addition, if necessary (for example, in the event that the individual develops cancer or immunodeficiency disease or for the newborn autologous cells of any type in fact there are other needs), the stem cells produced by the inventive method can be stored and utilized as a part of a treatment regimen in the individual's lifetime.

The present invention also offers unique opportunities for gene therapy. Specifically, genetic defects can now be corrected or beneficial genetic modifications can be introduced into somatic cells by manipulating autologous stem cells obtained from an individual that have been conditionally immortalized and expanded using the methods of the present invention. These stem cells can then be reintroduced into the individual from whom they originated.

The stem cell produced by the inventive method can also be used in multiple drug discovery experiments. Because the homogeneous stem cell that is easy to store, recover, amplify and operate of actually unlimited supply can be produced now, described stem cell can be used as stem cell in test or be differentiated into multiple cell lineage and use, for testing the influence of multiple compounds on cell differentiation, gene expression and cellular process.Described cell can be operated before contacting with compound, for example, by genetic manipulation.The stem cell from the individuality with genetic defect can be evaluated with described experiment, to identify therapeutic compound (for example, cancer therapeutic agent) and evaluate gene replacement therapy.In fact, the technology of the present invention provides a kind of cell for specific individual to identify drug candidate and treatment candidate and the opportunity of the strategy of " being suitable for " individual cell.An example of described experiment has more detailed description below.

About research and discovery before the present invention for pedigree orientation and cell differentiation and development, the cell colony that cannot obtain and can not produce enough expectation carries out the experiment of expectation and seriously hinders described research.For example, in order to identify or screen the intermediate product in specific progenitor cell line differentiation, it is necessary to obtain enough cells to provide significant and repeatable result.Progenitor cell line also should remain on the ability of further differentiation in directed pedigree, therefore produced these new tools that do not exist at present, also do not have about other descriptions of the required technology of producing these cells.Utilize obtainable technology before the present invention, this is impossible.The present invention solves this problem by providing the homogeneous stem cell that can be used for multiple experiment that can be expanded and is basically unlimited in supply.This technology will greatly improve the research capability of cell differentiation and discovery field.

As described above, the method of conditionally immortalizing lt-HSCs of the present invention can be applied to other stem cells derived from other tissues. For example, by varying gene delivery and growth factors, if desired, the present invention can be applied to a variety of different stem cells as described below. The cells can also be expanded in vitro and continue to differentiate after oncogene inactivation, as described herein for hematopoietic stem cells. These cells can then be used for therapeutic purposes, including tissue repair and tissue regeneration/engineering. Accordingly, the combination of MYC-ER and Bcl-2 genes or any other combination described herein can be transfected into cells by any method described herein or deemed suitable by a person skilled in the art in light of the present disclosure (including a variety of viral-mediated methods), including but not limited to: mesenchymal stem cells (including but not limited to lung mesenchymal cells, bone marrow stromal cells), neural stem cells (including but not limited to neuronal dopaminergic stem cells and motor neuron stem cells), epithelial stem cells (including but not limited to lung epithelial stem cells, breast epithelial stem cells, and intestinal epithelial stem cells), cardiomyocyte progenitor stem cells, skin stem cells (including but not limited to epidermal stem cells and cystic stem cells), skeletal muscle stem cells, endothelial stem cells (e.g., lung endothelial stem cells), and liver stem cells to generate conditionally immortalized cell lines that can be expanded in vitro and continue to differentiate after oncogene inactivation. In addition to the therapeutic potential of the cell lines, these cell lines can be further modified in vitro (or ex vivo) to correct congenital genetic defects and to study the molecular basis of early lineage commitment and differentiation. These cells may be a new source of potentially relevant therapeutic targets, and these cell lines can also be used to screen small molecules that prevent or induce differentiation, and to identify new compounds and molecular targets for a variety of therapies, including but not limited to cancer therapy and immunodeficiency therapy.

General Definition

According to the present invention, the nucleic acid molecules mentioned herein refer to the nucleic acid molecules isolated from its natural environment (that is, having carried out artificial manipulation), and its natural environment is the genome or chromosome of the natural discovery of this nucleic acid molecule therein.Therefore, "isolated" does not necessarily reflect the degree to which nucleic acid molecules are purified, but represents that this molecule does not include the full genome or full chromosome of the natural discovery of this nucleic acid molecule therein. The nucleic acid molecules of isolation can include a gene. The nucleic acid molecules of isolation comprising a gene are not the chromosomal fragment comprising the gene, but include the coding region and regulatory region relevant to the gene, but do not have other genes naturally found on the same chromosome. The nucleic acid molecules of isolation can also include a specific nucleic acid molecule sequence, and the side of the sequence (that is, 5 ' and/or 3 ' end of the sequence) is other nucleic acids (that is, heterologous sequences) that are not usually on the side of this specific nucleic acid sequence under natural conditions. The nucleic acid molecules of isolation can include DNA, RNA (for example mRNA) or a derivative (for example, cDNA, siRNA, shRNA) of DNA or RNA. Although "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and "nucleic acid sequence" primarily refers to the nucleotide sequence on a nucleic acid molecule, the two terms can be used interchangeably, especially for nucleic acid molecules or nucleic acid sequences that can encode proteins or protein domains.

Preferably, the isolated nucleic acid molecules of the present invention are produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Isolated nucleic acid molecules include natural nucleic acid molecules and homologs thereof, including but not limited to natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted and/or inverted so that the modification provides a desired effect (e.g., providing an inducible proto-oncogene, as described herein).

Nucleic acid molecule homologs can be produced using a variety of methods known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press (1989)). For example, nucleic acid molecules can be modified using a variety of techniques, including but not limited to classical mutagenesis techniques and recombinant DNA techniques, such as site-directed mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, restriction enzyme digestion of nucleic acid fragments, ligation of nucleic acid fragments, PCR amplification and/or mutagenesis of selected regions of nucleic acid sequences, synthesis of oligonucleotide mixtures and ligation of mixed groups to "build" mixtures of nucleic acid molecules and combinations thereof. Nucleic acid molecule homologs can be selected from the modified nucleic acid mixture by screening for the function of the protein encoded by the nucleic acid and/or by hybridization with the wild-type gene.

The minimum size of a nucleic acid molecule or polynucleotide of the present invention is a size sufficient to encode a protein for use in the present invention, such as a protein encoded by a proto-oncogene or a functional fragment thereof (i.e., a portion having the biological activity of the full-length protein and sufficient for use in the methods of the present invention), or an anti-apoptotic protein or a functional fragment thereof (i.e., a portion having the biological activity of the full-length protein and sufficient for use in the methods of the present invention). Other nucleic acid molecules useful in the present invention may include nucleic acid molecules having a minimum size sufficient to form a probe or oligonucleotide primer that can form a stable hybrid with the complementary sequence of a nucleic acid molecule encoding a native protein (i.e., under moderate, high, or very high stringency conditions), generally at least 5 nucleotides in length, preferably ranging from 5 to about 50 or about 500 nucleotides or longer, including any length in between all numerical increments (e.g., 5, 6, 7, 8, 9, 10, ... 33, 34, ... 256, 257, ... 500). There is no limit to the maximum size of a nucleic acid molecule of the present invention, other than limitations of practicality, and a nucleic acid molecule may include a sequence sufficient for use in any embodiment of the present invention described herein.

As used herein, stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al. (supra) is incorporated herein by reference in its entirety (see, in particular, pages 9.31-9.62). In addition, for example, Meinkoth et al., 1984, Anal. Biochem. 138, 267-284 discloses formulas for calculating appropriate hybridization and wash conditions for achieving hybridizations that tolerate varying degrees of nucleotide mismatches; Meinkoth et al. (supra) is incorporated herein by reference in its entirety.

More specifically, moderately stringent hybridization and wash conditions, as referred to herein, refer to conditions that allow the isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity with the nucleic acid molecule used in the hybridization probe (i.e., conditions that tolerate about 30% or fewer nucleotide mismatches). Highly stringent hybridization and wash conditions, as referred to herein, refer to conditions that allow the isolation of nucleic acid molecules having at least about 80% nucleic acid sequence identity with the nucleic acid molecule used in the hybridization probe (i.e., conditions that tolerate about 20% or fewer nucleotide mismatches). Very highly stringent hybridization and wash conditions, as referred to herein, refer to conditions that allow the isolation of nucleic acid molecules having at least about 90% nucleic acid sequence identity with the nucleic acid molecule used in the hybridization probe (i.e., conditions that tolerate about 10% or fewer nucleotide mismatches). As described above, one skilled in the art can calculate the appropriate hybridization and wash conditions required to achieve a specific level of nucleotide mismatches using the formula in Meinkoth et al. (supra). The conditions will vary depending on whether a DNA:RNA hybrid is formed or a DNA:DNA hybrid is formed. The calculated melting temperature for a DNA:DNA hybrid is 10°C lower than that for a DNA:RNA hybrid. In certain embodiments, stringent hybridization conditions for DNA:DNA hybrids comprise hybridization at an ionic strength of 6X SSC (0.9 M Na ⁺ ) at a temperature between about 20°C and about 35°C (lower stringency), more preferably between about 28°C and about 40°C (more stringent), and even more preferably between about 35°C and about 45°C (even more stringent), with appropriate wash conditions. In particular embodiments, stringent hybridization conditions for DNA:RNA hybrids comprise hybridization at an ionic strength of 6X SSC (0.9 M Na ⁺ ) at a temperature between about 30°C and 45°C (lower stringency), more preferably between about 38°C and about 50°C (more stringent), and even more preferably between about 45°C and about 55°C (even more stringent), with similar stringent wash conditions. These values are based on calculations of melting temperatures for molecules having greater than about 100 nucleotides, 0% formamide, and a G+C content of about 40%. Alternatively, the _Tm can be calculated empirically as described in Sambrook et al. (supra), pages 9.31 to 9.62. Wash conditions should generally be as stringent as possible and should be appropriate for the chosen hybridization conditions. For example, hybridization conditions may include a combination of salt and temperature conditions that is about 20-25°C below the calculated _Tm for the specific hybrid, and wash conditions typically include a combination of salt and temperature conditions that is about 12-20°C below the calculated _Tm for the specific hybrid. An example of hybridization conditions suitable for DNA:DNA hybrids includes hybridization in 6X SSC (50% formamide) at about 42°C for 2-24 hours, followed by a wash step comprising one or more washes in about 2X SSC at room temperature, followed by additional washes at higher temperature and lower ionic strength (e.g., at least one wash in about 0.1X-0.5X SSC at about 37°C, followed by at least one wash in about 0.1X-0.5X SSC at about 68°C).

In one embodiment of the present invention, any of the amino acid sequences described herein, including truncated forms (fragments or portions) and homologs thereof, can be produced with from at least one to about 20 additional heterologous amino acids on each side of the C- and/or N-terminus of a given amino acid sequence. The resulting protein or polypeptide can be referred to as "consisting essentially of a given amino acid sequence." According to the present invention, a heterologous amino acid is a sequence that is not naturally found flanking a given amino acid sequence (i.e., not naturally found in vivo) when the nucleotides in the naturally occurring sequence are translated using the standard codons of the organism from which the given amino acid sequence is derived, or, if present in the gene, is not encoded by the nucleotides flanking a naturally occurring nucleic acid sequence encoding the given amino acid sequence. Similarly, "consisting essentially of," when used herein with respect to a nucleic acid sequence, means that the nucleic acid sequence encoding the given amino acid sequence may be flanked by at least one to about 60 additional heterologous nucleotides on the 5' and/or 3' ends of the nucleic acid sequence encoding the given amino acid sequence. As present in the native gene, heterologous nucleotides are not naturally found flanking the nucleic acid sequence encoding a given amino acid sequence (ie, not naturally found in vivo).

According to the present invention, recombinant vector (commonly referred to as recombinant nucleic acid molecule, particularly when it comprises the target nucleic acid sequence according to the present invention) is a kind of engineered (that is, artificially produced) nucleic acid molecule, which is used as the nucleic acid sequence of operation selection and the tool of described nucleic acid sequence being imported into host cell.Therefore recombinant vector is applicable to the cloning, order-checking and/or other operation of the nucleic acid sequence selected, for example, by expressing the nucleic acid sequence selected and/or delivering it into host cell.Described carrier typically comprises heterologous nucleic acid sequence, that is, naturally or usually finds not to be cloned or the nucleic acid sequence adjacent to the nucleic acid sequence of delivery, although carrier also can comprise regulatory nucleic acid sequence (for example, promoter, non-translational region), this regulatory nucleic acid sequence is found adjacent to nucleic acid molecule of the present invention by natural, or can be used for expressing nucleic acid molecule of the present invention (discussed in more detail below).Carrier can be RNA or DNA, prokaryotic or eukaryotic, typically plasmid or viral vector.Carrier can be kept as extrachromosomal element (for example, plasmid) or can be integrated into the chromosome of host cell.Complete carrier can remain in proper position in host cell, or under certain conditions, plasmid DNA can be removed, leaving nucleic acid molecule of the present invention. Under other conditions, the vector is designed to be excised (removed) from the genome of the host cell at a selected time (discussed in more detail below). The integrated nucleic acid molecule can be under the control of a chromosomal promoter, under the control of a natural or plasmid promoter, or under the control of a combination of several promoters. Single or multiple copies of the nucleic acid molecule can be integrated into the chromosome. The recombinant vector of the present invention can contain at least one selectable marker.

According to the present invention, "effectively linked" refers to a way of connecting a nucleic acid molecule to an expression control sequence (e.g., a transcription control sequence and/or a translation control sequence) so that the molecule can be expressed when transfected (e.g., transformed, transduced, transfected, conjugated or caused) into a host cell. A transcription control sequence is a sequence that controls the initiation, extension or termination of transcription. Particularly important transcription control sequences are sequences that control the initiation of transcription, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in the host cell or organism into which the recombinant nucleic acid molecule is to be introduced.

According to the present invention, the term "transfection" refers to any method for inserting an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) into a cell. The term "transduction" is a specific type of transfection in which genetic material is transferred from one source to another, such as by a virus (e.g., a retrovirus) or a transducing phage. When the term is used to refer to the introduction of nucleic acid molecules into microbial cells such as bacteria and yeast, the term "transformation" can be used interchangeably with "transfection". In microbial systems, the term "transformation" is used to describe genetic changes caused by the acquisition of exogenous nucleic acids by microorganisms and is essentially synonymous with "transfection". However, in animal cells, transformation has a second meaning and can refer to changes in the growth properties of cells in culture after (for example) becoming cancerous. Therefore, to avoid confusion, "transfection" is preferably used herein for the introduction of exogenous nucleic acids into animal cells. Therefore, the term "transfection" generally includes the transfection or transduction of animal cells and the transformation or transduction of microbial cells, which terms relate to the introduction of exogenous nucleic acids into cells. Transfection techniques include, but are not limited to, transformation, transduction, particle bombardment, diffusion, active transport, bath sonication, electroporation, microinjection, lipofection, adsorption, infection, and protoplast fusion.

As used herein, the isolated proteins or polypeptides referred to in the present invention include full-length proteins, fusion proteins, chimeric proteins, or any fragments (truncated forms, portions), or homologs of such proteins. More specifically, isolated proteins according to the present invention are proteins (including polypeptides or peptides) that have been separated from their natural environment (i.e., have been subjected to artificial manipulation) and may include, but are not limited to, purified proteins, partially purified proteins, recombinantly produced proteins, membrane-bound proteins, and proteins isolated from other proteins. Therefore, "isolated" does not reflect the degree to which a protein has been purified. Preferably, the isolated proteins of the present invention are recombinantly produced. In addition, as an example of a specific protein (Bcl-2), "human Bcl-2 protein" or a protein "derived from" a human Bcl-2 protein refers to a Bcl-2 protein from Homo sapiens (including homologs or portions of naturally occurring Bcl-2 proteins) or a Bcl-2 protein produced by other means based on knowledge of the structure (e.g., sequence) and (perhaps) function of a naturally occurring human Bcl-2 protein. In other words, human Bcl-2 proteins include any Bcl-2 protein that has a structure and function substantially similar to that of a naturally occurring human Bcl-2 protein or that is a biologically active (i.e., biologically active) homolog of a naturally occurring human Bcl-2 protein as described herein. Thus, human Bcl-2 proteins may include purified, partially purified, recombinant, mutated/modified, and synthetic proteins. According to the present invention, the terms "modification" and "mutation" are used interchangeably, particularly with respect to modifications/mutations of the amino acid sequence (or nucleic acid sequence) of the proteins described herein.

As used herein, the term "homolog" refers to a protein or peptide that differs from a naturally occurring protein or peptide (i.e., a "prototype" or "wild-type" protein) due to modifications (including minor modifications) to the naturally occurring protein or peptide, but retains the basic protein and side chain structure of the naturally occurring form. Such changes include, but are not limited to, changes in one or a few (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid side chains; changes in one or a few amino acids, including deletions (e.g., proteins or truncated forms of proteins or peptides), insertions, and/or substitutions; changes in the stereochemistry of one or a few atoms; and/or minor derivatizations, including, but not limited to, methylation, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation, and/or addition of glycosylphosphatidylinositol. Homologs may have improved, reduced, or substantially similar properties compared to the naturally occurring protein or peptide. Homologs may include agonists of the protein or antagonists of the protein.

Homologues can be the result of natural allelic variation or natural mutation. The naturally occurring allelic variant of a nucleic acid encoding a protein is a gene that is substantially present at the same genomic locus as the gene encoding the protein, but due to natural variation caused by, for example, mutation or recombination, has a similar but not identical sequence. Allelic variants typically encode proteins that have similar activity to the protein encoded by the gene being compared. A group of allelic variants can encode the same protein, but have different nucleic acid sequences due to the degeneracy of the genetic code. Allelic variants can also be included in changes in the 5' or 3' untranslated regions (e.g., in regulatory control regions) of a gene. Allelic variants are well known to those skilled in the art.

Homologs can be produced using protein production techniques known in the art, including but not limited to direct modification of isolated naturally occurring proteins, direct protein synthesis, or modification of the nucleic acid sequence encoding the protein, for example, by random or targeted mutagenesis using classical or recombinant DNA techniques.

In one embodiment, a homolog of a given protein comprises, consists essentially of, or consists of an amino acid sequence that is at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% identical, or at least about 95% identical, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least about 99% identical (or any percent identity in all integer increments between 45% and 99%) to the amino acid sequence of the reference protein. In one embodiment, a homolog comprises, consists essentially of, or consists of an amino acid sequence that is less than 100% identical, less than about 99% identical, less than about 98% identical, less than about 97% identical, less than about 96% identical, less than about 95% identical, etc., in increments of 1%, up to less than about 70% identical to the naturally occurring amino acid sequence of the reference protein.

As used herein, unless otherwise indicated, percent (%) identity refers to an assessment of homology performed by: (1) BLAST 2.0 Basic BLAST homology searches with standard default parameters, using blastp for amino acid searches and blastn for nucleic acid searches, with the query sequence filtered for low complexity regions by default (described in Altschul, S.F., Madden, T.L., Schauffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, DJ. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs." Nucleic Acids Res. 25:3389-3402, which is incorporated herein by reference in its entirety); (2) BLAST 2 alignments (using the parameters described below); (3) and/or PSI-BLAST (site-specific iterative BLAST) with standard default parameters. Should be noted that, because the standard parameters between BLAST 2.0 BASIC BLAST and BLAST 2 have some differences, two kinds of specific sequences may utilize BLAST 2 programs to be defined as having significant homology, and in BLAST 2.0 BASIC BLAST, use a kind of sequence to search as query sequence and may not identify the second sequence of extremely coupling.In addition, PSI-BLAST provides a kind of " profile " search pattern that is automatic, easy to use, and it is a kind of sensitive method for finding sequence homologues.This program first carries out gap BLAST database search.The PSI-BLAST program uses the information from any significant comparison, and said information is returned to make up a kind of site-specific scoring matrix, and it substitutes the query sequence that is used for next round database search.Therefore, can understand that percent identity can utilize any one of these programs to determine.

Two specific sequences can be aligned with each other using the BLAST 2 program as described in Tatusova and Madden, (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174: 247-250, which is hereby incorporated by reference in its entirety. BLAST 2 sequence alignments are performed using blastp or blastn, using the BLAST 2.0 algorithm to perform a gapped BLAST search (BLAST 2.0) between two sequences, allowing for the introduction of gaps (deletions and insertions) in the resulting alignment. For clarity, BLAST 2 sequence alignments are performed using the following standard default parameters:

For blastn, use the 0 BLOSUM62 matrix:

Matching bonus points = 1

Mismatch penalty = -2

Open gap (5) and extended gap (2) penalties

Gap x_drop(50) Expected (10)WordLength(11)Filter(on)

For blastp, use the BLOSUM62 matrix:

Open gap (11) and extended gap (1) penalties

Gap x_drop(50) Expected (10)WordLength(3)Filter(on)

According to the present invention, isolated protein, including its biologically active homologue or fragment, has at least one biological activity characteristic of wild type or native protein. Generally, the biological activity or biological action of a protein refer to any function exhibited or performed by a protein extract owing to the naturally occurring form of the protein, which is measured or observed in vivo (i.e., in the natural physiological environment of the protein) or in vitro (i.e., under laboratory conditions). Modifications, activities or interactions that cause protein expression to decrease or protein activity to decrease can be referred to as inactivation (completely or partially), downward regulation, effect reduction or effect or activity reduction of the protein. Similarly, modifications, activities or interactions that cause protein expression to increase or protein activity to increase can be referred to as amplification, overproduction, activation, enhancement, upward regulation or effect enhancement of the protein.

Conditional immortalization method of the present invention

One embodiment of the present invention relates to a method for producing conditionally immortalized adult stem cells, preferably long-term stem cells. The method generally comprises the following steps: (a) obtaining an expanded adult stem cell population; (b) transfecting (transducing) the stem cells with a vector comprising a proto-oncogene that promotes cell survival and proliferation, wherein the proto-oncogene is regulatable (inducible, controllable); (c) transfecting the stem cells with a vector encoding a protein that inhibits apoptosis; and (d) amplifying the transfected cells in the presence of a combination of stem cell growth factors and under conditions that make the proto-oncogene active. In one embodiment, the vector is an integrating vector. The cells produced by this method can be cultured, amplified, stored, recovered, used in therapeutic methods, used in research and discovery methods, genetically manipulated, induced to differentiate by eliminating conditions that keep the proto-oncogene active, and/or used in any other method described herein or apparent to those skilled in the art based on this disclosure. Steps (b) and (c) can be performed in any order.

According to the present invention, "conditional immortalization" refers to cells that have been immortalized in a reversible manner (e.g., able to grow indefinitely without differentiation in a cytokine-dependent manner while maintaining the ability and potential to differentiate into a large number of different lineages under appropriate conditions). The cells are immortalized under a set of specific conditions. When the conditions are eliminated or changed (or other conditions are added), the cells no longer proliferate indefinitely and can differentiate into other cell types. "Conditional immortalization" can be used interchangeably with "reversible immortalization." For example, with respect to the methods of the present invention, when stem cells are placed under conditions that allow proto-oncogene activation (e.g., tamoxifen or its agonist in the example of MYC-ER), the presence of regulatable proto-oncogenes that promote cell survival and proliferation allows the cells to maintain an immortalized phenotype. In other words, the cells grow and expand indefinitely in culture and maintain an undifferentiated state under these specific conditions. When these conditions are eliminated (e.g., removing tamoxifen in the example of MYC-ER), the stem cells no longer proliferate indefinitely and can differentiate into a variety of cell lineages according to the appropriate environment (e.g., an appropriate combination of growth factors).

As used herein, "stem cell" refers to a term generally understood in the art. For example, stem cells, regardless of their origin, are cells that can self-divide and renew over a long period of time, are non-specialized (non-differentiated), and can produce (differentiate into) specific cell types (i.e., they are progenitors or precursors of a variety of different specific cell types). "Long-term," when used in conjunction with stem cells, refers to the ability of stem cells to self-renew over a long period of time (e.g., several months, such as at least 3 months, to several years) by dividing into the same non-specific cell type, depending on the specific type of stem cell. As described herein, the phenotypic characteristics of various long-term stem cells, such as long-term hematopoietic stem cells (lt-HSCs), derived from different animal species are known in the art. For example, mouse lt-HSCs can be identified based on the presence of the following cell surface marker phenotypes: c-kit+, Sca-1+, CD34-, flk2- (see Examples). Adult stem cells include stem cells that can be obtained from any non-embryonic tissue or source, and typically produce the cell type of the tissue in which they are located. The term "adult stem cell" can be used interchangeably with the term "somatic stem cell." Embryonic stem cells are stem cells obtained from any embryonic tissue or source.

In one embodiment of the invention, the stem cells used in the present invention may include any adult stem cells obtained from any source. In another embodiment of the invention, the stem cells may include embryonic stem cells. Useful stem cells in the present invention include, but are not limited to, hematopoietic stem cells, mesenchymal stem cells (including but not limited to lung mesenchymal stem cells, bone marrow stromal cells), neural stem cells, epithelial stem cells (including but not limited to lung epithelial stem cells, breast epithelial stem cells, vascular epithelial stem cells, and intestinal epithelial stem cells), intestinal stem cells, cardiomyocyte progenitor stem cells, skin stem cells (including but not limited to epidermal stem cells and follicular stem cells (hair follicle stem cells)), skeletal muscle stem cells, osteoblast precursor stem cells, and liver stem cells.

Hematopoietic stem cells give rise to all types of blood cells, including but not limited to erythrocytes (red blood cells), B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, macrophages, and platelets.

Mesenchymal stem cells (including bone marrow stromal cells) give rise to a variety of cell types, including but not limited to bone cells, cartilage cells, adipocytes, lung cells, and other types of connective tissue cells, such as those in tendons.

Neural stem cells in the brain give rise to three main cell types: nerve cells (neurons) and two types of neural cells in the lung: astrocytes and oligodendrocytes.

Epithelial stem cells in the lining of different tissues give rise to the several cell types that form the tissue's epithelium.

Skin stem cells are found in the bottom layer of the epidermis and at the base of hair follicles. Epidermal stem cells give rise to keratinocytes, which migrate to the surface of the skin to form a protective layer, while follicular stem cells can give rise to hair follicles and the epidermis. Other sources of adult stem cells are also well known to those skilled in the art.

Embryonic stem cells can give rise to all tissues and cells of the body.

Methods for obtaining the stem cells and providing initial culture conditions such as liquid culture medium or semi-solid culture medium are known in the art. Initially, cells are expanded in vivo or in vitro by contacting the stem cell source with a suitable reagent that expands or enriches the cells in a tissue source or culture. For example, in the case of hematopoietic stem cells, the donor individual can be treated with reagents such as 5-fluorouracil that enrich hematopoietic stem cells and promote the proliferation of the cells without differentiation. Other reagents suitable for expanding the desired stem cell type are well known in the art. Alternatively, and preferably, adult stem cells are separated from a tissue source and then expanded or enriched in vitro by exposure to suitable reagents. For example, for hematopoietic stem cells, methods for producing expanded cultures of adult hematopoietic progenitor cells are described in Van Parijs et al. (1999; Immunity, 11, 763-70). Cells are obtained from an individual by any suitable method of obtaining a cell sample from an animal, including but not limited to bone marrow collection, collection of umbilical cord blood, tissue aspiration, and tissue dissection of a body fluid (e.g., blood), particularly including but not limited to any biopsy of fine needle aspirates of skin, intestine, cornea, spinal cord, brain tissue, scalp, stomach, breast, lung (e.g., including lavage and bronchial examination), bone marrow, amniotic fluid, placenta, and yolk sac.

In one embodiment, cells for use in the present invention can also be obtained from fresh or cryopreserved (banked) umbilical cord blood, hematopoietic progenitor cell populations that can be derived from in vitro directed differentiation of embryonic stem cells (ES), hematopoietic stem cells (HSCs) obtained from the peripheral blood of normal or granulocyte colony stimulating factor (G-CSF) treated patients who have been induced to mobilize their lt-HSCs into the peripheral circulation.

Once an amplified stem cell population is obtained (can be obtained, provided or produced), the cells are transfected sequentially (in any order) or simultaneously with: (1) a vector comprising a proto-oncogene that promotes cell survival and proliferation, wherein the proto-oncogene is regulatable (inducible, controllable), and (2) a vector encoding a protein that inhibits apoptosis of the cell. Preferably, the vector is an integrative vector, defined herein as any vector (e.g., a retroviral vector) having the ability to integrate into the cell genome. A variety of vectors and transfection methods are described in more detail below. The proto-oncogene is regulatable (inducible or controllable) so that it can be activated and inactivated (i.e., turned on or off) as needed to maintain the stem cell in an infinite proliferation state or to differentiate it into a desired cell type. The proto-oncogene can be selected or designed to be regulated by any appropriate method, including in response to any condition, such as the presence or absence of a compound or reagent, temperature, or any other appropriate condition. As an example, the proto-oncogenes MYC-ER (estrogen receptor (ER)-regulated MYC) and ICN-1-ER (ER-regulated intracellular portion of Notch-1) described herein are both inducible in the presence of tamoxifen. It should be noted that the genes can also be engineered to respond to other dimerizing drugs, such as FK1012, modified forms of rapamycin, or can be expressed from vectors containing tetracycline response elements. The latter case regulates the expression of the protein, not the function of the polypeptide present in the cell. Other similar modifications of this platform technology will be apparent to those skilled in the art.

The proto-oncogene useful in the method of the present invention is any proto-oncogene that promotes cell survival and proliferation. Preferred proto-oncogenes for use in the method of the present invention include, but are not limited to, MYC, ICN-1, hTERT (the reverse transcriptase component of human telomerase), NMYC, S-MYC, L-MYC, Akt (myristylation). In addition, other applicable genes and methods of the present invention or methods of modifying genes to achieve a desired result include, but are not limited to, the use of downstream signaling effectors such as pyruvate dehydrogenase kinase 1 (PDK-1); mammalian target of rapamycin (mTOR); loss of phosphatase and tensin homolog (PTEN) by shRNA; Bcl-3, cyclin D1, cyclin D3, Bcl-10, Bcl-6, BCR-ABL (breakpoint cluster region fused to ABL) and its various mutant forms, constitutively active forms of stat5 and stat3, AML1-ETO (fusion of acute myeloid leukemia 1 and short stature-associated transcription factor 1), MLL-ENL (mixed lineage leukemia and eleven nineteen leukemia) and other leukemias. leukemia)), Hox genes, activated forms of interleukin-3 (IL-3) receptor β chain, and other cytokine receptor chains (epidermal growth factor receptor (EGFR), c-kit, platelet-derived growth factor receptor (PDGFR), etc.), as well as wnt (all mammalian forms), β-catenin, sbh protein (shh-1 and all mammalian forms), bmi-1 and c-jun (all mammalian forms). In addition, the present invention includes shRNA-induced (or inhibition) loss of cell cycle protein kinase inhibitors, including but not limited to pi 6, pi 9, p21 and p27. In one embodiment, the present invention includes the use of any or said proto-oncogenes (e.g., MYC-ER or ICN-1-ER) or regulatable homologs of other genes. The examples describe the successful production of conditionally immortalized lt-HSCs using the methods of the present invention using MYC-ER or ICN-1-ER.

The nucleic acid sequence encoding human MYC is represented herein as SEQ ID NO: 1, which encodes the amino acid sequence represented herein as SEQ ID NO: 2. The nucleic acid sequence encoding hTERT is represented herein as SEQ ID NO: 3, which encodes the amino acid sequence represented herein as SEQ ID NO: 4. The nucleic acid sequence encoding human ICN-1 is represented herein as SEQ ID NO: 11, which encodes the amino acid sequence represented herein as SEQ ID NO: 12. ICN-1 is part of Notch-1, specifically, amino acids 1757-2555 of Notch-1 (see Aster et al., Mol Cell Biol. 2000 Oct; 20(20): 7505-15, which is incorporated herein by reference in its entirety). The nucleotide and amino acid sequences of MYC-ER are known in the art, and the MYC-ER protein is described in Soloman et al., Oncogene. 1995 Nov 2; 11(9): 1893-7, which is incorporated herein by reference in its entirety. ICN-1-ER was created by the present inventors. The nucleic acid sequence encoding this protein is represented herein as SEQ ID NO: 13, which encodes the amino acid sequence represented by SEQ ID NO: 14.

Similarly, a preferred anti-apoptotic gene is Bcl-2, although the present invention also encompasses other genes encoding proteins that inhibit apoptosis and maintain cell survival, particularly when proto-oncogenes are inactivated in stem cells. The nucleic acid sequence encoding Bcl-2α is represented herein as SEQ ID NO:5, which encodes the amino acid sequence of SEQ ID NO:6. Bcl-2β is represented herein as SEQ ID NO:7, which encodes the amino acid sequence of SEQ ID NO:8. An "anti-apoptotic" gene is defined herein as any gene encoding a protein that inhibits (reduces, prevents, reduces) apoptosis-related processes in a cell or promotes (enhances, enhances, stimulates, permits) cell survival, even in the presence of conditions capable of inducing apoptosis. Proteins associated with apoptosis, and genes encoding such proteins, are well known in the art. Such other genes include, but are not limited to, any gene in the Bcl-2 family that may play an important role in the conditional transformation of adult stem cells. These genes include, but are not limited to, other pro-survival members of the Bcl-2 family, such as Bcl-X, Bcl-w, BclXL, Mcl-1, Dad-1, or hTERT (the reverse transcriptase component of human telomerase, which has been shown to inhibit proliferation). These genes are abnormally overexpressed in the presence of regulated oncogenes, such as Bcl-2 described in the Examples herein. In addition, this aspect of the invention includes shRNA-mediated gene knockdown (or disruption or inhibition by any other method) of BH3-only members of the pro-apoptotic Bcl-2 family (e.g., Bim, PUMA, NOXA, Bax, Bak, BclXS, Bad, Bar, etc.), as well as disruption of caspases 3, 9, 10, MLL-1 (and all mammalian forms), Enl-1 (Endospermless-1) and all mammalian forms, Apaf-1, and other components that form part of the apoptotic body.

The nucleic acid sequences of each of the above genes or their coding regions are known in the art and are publicly available, including human genes. Similarly, the amino acid sequences of the proteins encoded by these genes are known in the art and are publicly available.

The present inventors have used the methods of the present invention to generate several different long-term conditionally immortalized stem cells using different combinations of oncogenes and anti-apoptotic genes, including the following combinations: MYC-ER and Bcl-2; MYC-ER and hTERT (the reverse transcriptase component of human telomerase); ICN-1-ER and Bcl-2; ICN-1-ER and hTERT; and MYC-ER and ICN-1-ER.

It should be noted that for proto-oncogenes or genes encoding anti-apoptotic proteins used in the present methods, it is not necessary to use the entire gene in the constructs described herein, as the present invention includes any portion of the gene or nucleic acid sequence (e.g., cDNA) encoding the desired functional protein product, a functional portion thereof, or a functional homolog thereof. Accordingly, references herein to genes or transgenes for transfection of stem cells are to be understood as exemplary and include the use of any nucleic acid molecule encoding the entire gene, the entire coding region of the gene, or a portion of the gene or a homolog thereof, as long as the nucleic acid sequence encodes a functional protein suitable for use in the present invention.

In one embodiment of the invention, the method further comprises the use of shRNA or siRNA directed against RNA encoding pro-apoptotic proteins, such as pro-apoptotic members of the Bcl-2 family, i.e., members of the BH3-only type (Bim, Bax, Bak, Puma, Noxa, etc.). It is expected that the destruction of pro-apoptotic genes in the context of regulated oncogenes will lead to more effective immortalization of certain stem cell populations. RNA interference (RNAi) is a process in which double-stranded RNA and (in mammalian systems) short interfering RNA (siRNA) or short hairpin RNA (shRNA) are used to inhibit the expression of complementary genes or to silence them. In the target cell, the siRNA is unwound and binds to the RNA-induced silencing complex (RISC), which is then guided to the mRNA sequence complementary to the siRNA, whereby the RISC shears the mRNA. The shRNA is transfected into the target cell in a vector, where it is transcribed and then processed by the DICER enzyme to form an siRNA-like molecule, which activates RISC. RISC and the siRNA are then guided to the mRNA sequence complementary to the shRNA, where it cleaves the mRNA.

Any suitable method for transfecting cells, particularly mammalian cells, can be used, including utilizing a combination of various techniques to transfect stem cells with vectors comprising proto-oncogenes and encoding anti-apoptotic proteins. The inventors have discovered that there is a particular synergistic effect between the expression genes (or constructs) that produce the conditionally immortalized long-term stem cells described herein. The examples have demonstrated the use of retroviral vectors, and other methods include, but are not limited to, the use of lentiviral vectors, parvoviruses, vaccinia viruses, coronaviruses, caliciviruses, papillomaviruses, flaviviruses, orthomyxoviruses, togaviruses, picornaviruses, adenoviral vectors, modified and attenuated herpes viruses. Any of the viruses can be further modified with specific surface-expressed molecules that target them to HSCs or other stem cells, such as membrane-bound SCF, or other stem cell-specific growth factor ligands. Other methods for transfecting mammalian cells include, but are not limited to, direct electroporation of mammalian expression vectors, for example, utilizing NUCLEOFECTOR ^™ technology (AMAXA Biosystems). This technology is a highly efficient non-viral gene transfer method for most primary cells and difficult-to-transfect cell lines. It is an improvement on the long-known electroporation method and is based on the cell type-specific combination of electric current and solution to transfer polyanionic macromolecules directly into the cell nucleus. In addition, suitable transfection methods may include any bacteria, yeast or other artificial gene delivery methods known in the art.

The step of expanding the transfected stem cells or culturing the stem cells and exogenous fusion proteins (e.g., Tat-fusion proteins described in variations of the method described below) in the presence of appropriate growth factors may include the use of any appropriate culture conditions, including the conditions specifically described herein. The combination of appropriate stem cell growth factors may include any stem cell factor that allows the transfected (e.g., transduced) cells of the present invention to grow, survive, and proliferate in culture. Although specific combinations are described herein, and although this is an important step in the present method, this step can be simply described as providing any combination of growth factors suitable for stem cell growth, proliferation, and survival, and includes any combination known in the art. Therefore, the present invention is not limited to specific combinations. A preferred combination of growth factors includes interleukin-6 (IL-6), IL-3, and stem cell factor (SCF). Another preferred combination of growth factors includes stem cell factor (SCF), thrombopoietin (TPO), insulin-like growth factor 2 (IGF-2), and fibroblast growth factor 1 (FGF-1) in serum-free medium. This latter combination was recently described by Zhang and Lodich (2005; Murine hematopoietic stem cells change their surface phenotype during ex vivo expansion, Blood 105, 4314-20). It is expected that stem cells transfected with nucleic acid molecules encoding the combination proteins described herein (e.g., MYC-ER and Bcl-2 as described in the Examples) will also be conditionally immortalized in a growth factor cocktail, as described in the examples above (using IL-3, IL-6, and SCF). Other growth factors useful in the present invention include, but are not limited to, angiopoietin-like proteins (e.g., Agptl2, Angptl3, Angptl5, Angptl7, etc.), proliferator-activated protein kinase-2 (PLF2), glycogen synthase kinase-3 inhibitors, inducers of the wnt and Notch signaling pathways, Flt3L and related cytokines, fibroblast growth factor 2 (FGF2) and related cytokines, wnt-1 and other activators of the wnt pathway, shh protein (shh-1) and other activators of the pathway. Other suitable growth factor combinations are suitable for the methods of the present invention and will be apparent to those skilled in the art. Indeed, the cell lines generated using the methods of the present invention can be readily used to screen for other cytokines and growth factors that can be used to expand long-term stem cells or any progenitor cells derived therefrom in vitro under neutral or directed conditions.

According to the present invention, the substratum that is suitable for cultivating animal cells can comprise any obtainable substratum developed for cultivating animal cells, particularly mammalian cells, or the substratum that can prepare in the laboratory with the necessary appropriate composition of available animal cell growth for example assimilable carbon, nitrogen and micronutrients.Described substratum comprises basal medium, and it is any basal medium that is suitable for animal cell growth, includes but not limited to the Eagles substratum (DMEM) (DMEM) (Iscove's improved Dulbecco's medium (IMDM), Dulbecco's improved Eagles substratum (DMEM), αMEM (Gibco), RPMI 1640 or any other applicable commercially available substratum. Assimilable carbon source, nitrogen source and micronutrient source have been added in the basal medium, include but not limited to serum source, somatomedin, amino acid, antibiotic, vitamin, reducing agent and/or sugar source. It should be noted that the complete medium that comprises the many additional ingredients that basal medium and animal cell growth need is commercially available, and some substratums can be used for the cell culture of specific types. In addition, many serum-free mediums are obtainable, and may be particularly suitable for the cultivation of stem cell according to the present invention.

Cells and compositions

Another embodiment of the present invention relates to cells, cell lines, or cell colonies produced according to the methods of the present invention as described herein. The present invention also includes compositions comprising the cells, cell lines, or cell colonies. When used in therapeutic methods, the compositions may comprise a pharmaceutically acceptable carrier, including a pharmaceutically acceptable excipient and/or a delivery vehicle, for delivering the cells, cell lines, or cell colonies to a patient. As used herein, a pharmaceutically acceptable carrier refers to any substance suitable for delivering the therapeutic composition useful in the methods of the present invention to an appropriate site in the body.

Modifications of the methods of the invention for the production of cell lineages at intermediate developmental stages

Another embodiment of the present invention relates to modifying the novel methods described herein to produce cell lines that capture intermediate developmental stages of the hematopoietic lineage. According to the present invention, an "intermediate" stage of development or differentiation refers to a multipotent stage of cell development or differentiation that is downstream of the stem cell development or differentiation stage that produces the "intermediate" cell, but upstream of the endpoint or final point of cell differentiation. For example, a pre-B cell is an intermediate stage of a hematopoietic stem cell that can still differentiate into a mature B cell. Intermediate stages of development or differentiation are understood by those skilled in the art.

More particularly, for many therapeutic and discovery or research applications, as well as the preservation of cell lines, it is desirable that the cell line have a stable phenotype and maintain the ability to further differentiate along its directed pathway when the oncogene of the transfected cell is turned off. Accordingly, the present invention includes the additional step of producing cells that are not fully differentiated (not terminally differentiated), but are in an intermediate stage of differentiation. In a non-limiting example of this embodiment, after removing conditions that maintain the activity of proto-oncogenes or other genes that promote cell survival and proliferation (e.g., 4-OHT in the example of tamoxifen-dependent proto-oncogenes), or by applying appropriate conditions that turn off (inactivate) proto-oncogenes/oncogenes, the long-term stem cells produced using the above method are randomly differentiated in vitro. This step can be performed while maintaining the culture under neutral cytokine growth conditions (e.g., IL-3, IL-6, and SCF), or by replacing cytokines that can specifically guide differentiation toward a certain lineage (e.g., IL-7 and Notch ligands for lymphoid lineages, GM-CSF and IL-4 for dendritic cells, G-CSF for myelomonocytic cells, etc.) with cytokines that are neutral for differentiation (do not guide or drive cell differentiation). Once the culture begins to display differentiation markers consistent with a specific lineage, the culture medium is re-applied to conditions that activate the proto-oncogene (e.g., 4-OHT) or exposed to conditions that otherwise reactivate the proto-oncogene to stabilize the phenotype and generate a cell line with a stable intermediate differentiation phenotype.

Using this method as an example, the present inventors have generated CD4+, αβ+ T cells in vitro from ABM42 cells (lt-HSCs produced by the method of the present invention, see Examples) by removing 4-OHT from the culture medium and adding 4OHT after differentiation. The present inventors have also generated a dendritic cell line by culturing ABM46 cells (see Examples) in the presence of GM-CSF, IL-4, and FLT3L and then placing the culture back in the presence of 4-OHT after differentiation.

Another method for producing the cell line includes introducing ctlt-HSC cells into mice to allow differentiation in vivo after injection of 4-OHT and to block or stabilize the phenotype. The method is described in detail in Example 8. In brief, for example, the lt-HSC produced by this method is injected into an animal without an immune response (e.g., a mouse without an immune response). The oncogenes in the lt-HSC are reactivated by injection of an activator (e.g., 4-OHT), and the cells are then collected and cultured in vitro to differentiate the cells, which are then stored or used as needed. This method, and other methods as described above, can be used for mouse and human ctlt-HSC cell lines, for example, using NOD/SCID mice as recipients, or newborn Rag-1 ^-/- mice, which are injected intrahepatically.

Application of the method of the present invention to embryonic stem cells

Another embodiment of the present invention relates to applying the method of conditionally immortalizing stem cells to embryonic stem (ES) cells. The method can be used to generate cell lines that are more readily derived from ES cells, such as cells of the neuronal lineage, including neuronal stem cells.

In the present embodiment, the method of the present invention, including transducing cells (in this example, ES cells) with proto-oncogenes and genes that inhibit apoptosis (for example, MYC-ER and Bcl-2), can be applied to ES cells to further control the directed differentiation of these cells. In this embodiment, the cells can be used to produce transgenic mice, for example, and in addition, any ES cell and its derived related progenitor cell colony can be activated by being exposed to an activator to carry out the activation of proto-oncogenes, therefore can produce new conditionally converted stem cell lines (different tissue types) or mature cell lines for the tissue type of interest. In addition, the directed differentiation of the ES cells of transduction in vitro can also be used for capturing the intermediate state of differentiation as described above. Using ES cells or cells derived from ES in this way provides a new platform for drug discovery and target identification under different disease backgrounds.

For example, neuronal stem cells can be applicable to this embodiment of the present invention, and utilize method of the present invention to make ES cell directed differentiation into neuronal pathway.Separation and transduction from the neuronal stem cells of hippocampus have been described for mice in the past.The culture conditions of neurosphere (neurosphere) enable these cells to proliferate, causing them to be susceptible to viral-mediated gene (for example, MYC-ER and Bcl-2) transduction of the present invention, thereby producing the neuronal stem cell line of conditional conversion.After transplanting, their differentiation in vitro and in vivo can be monitored according to the reporter gene of virus encoding and the marker of previously determined neuronal differentiation. In addition, after the neural stem cell transplantation of conditional conversion, activator (for example, 4-OHT) is used to cause the development (neuroblastoma, glioblastoma, etc.) of neural malignancies to mice. These tumors will provide a kind of novel method of preclinical research and target identification.

The directed differentiation of ES cells that have been transduced with, for example, MYC-ER and Bcl-2 can be carried out in the presence of previously determined growth medium and cytokines. Adding an activator (e.g., 4-OHT) at any time during the culture process will enable the cells to stabilize in an intermediate phenotype and result in the generation of a cell line that still maintains the ability to further differentiate. For example, the generation of dopaminergic neurons from ES cells is typically achieved by adding retinoic acid and FGF8. This type of neuron is ideal for repairing the brain damage observed in Alzheimer's patients. However, the transplantation of fully differentiated neuronal cells may hinder their successful transplantation and implantation. Cells that are directed to the dopaminergic neuron pathway but still maintain the ability to further differentiate after transplantation, as expected herein, can greatly increase the chances of implantation and successful transplantation. For the generation of motor neurons from ES cells, a similar scheme can be suggested, namely, adding retinoic acid and sbh protein agonists to the culture. These neuronal cells can help repair spinal cord injuries. Again, fully differentiated cells will not be used in this embodiment, but directed progenitor cells (produced by the method of the present invention) that maintain the ability to differentiate will be used.

Variations or modifications of conditional immortalization methods for transgene removal

In one embodiment of the invention, to avoid the risks of introducing stem cells containing transgenes such as those described herein (e.g., MYC-ER) into humans and/or mice, the recombinant constructs are designed so that these DNA segments can be excised. This embodiment can be achieved using any suitable method: first, long-term stem cells are established according to the methods of the invention, and then the cells (or patient) are exposed to conditions where the recombinant DNA is removed, excised, or completely silenced.

For example, in one embodiment of the present invention, a bacterial recombinase method is used. In this embodiment of the present invention, two different recombinases are preferably used to allow control of which of the two genes is excised at any time point. Two examples of the recombinases are Cre and Flp recombinases, which are well known in the art. In brief, a recombinase recognition substrate sequence (RSS's) is introduced into a retroviral construct so that they are located on the side of the open reading frame of an oncogene and a reporter gene (e.g., GFP or Thy1.1). In this example, cells are cultured in a culture medium containing a Tat-Cre fusion protein (i.e., HIV or other retroviral Tat protein fused to Cre). This recombinant protein has been described before and shown to be able to passively enter cells and mediate the recombination of genomic DNA that depends on loxP sites. Other gene (nucleic acid molecule) excision methods are also well known to those skilled in the art and can be easily applied to the present invention. Examples 5 and 13 illustrate this embodiment of the present invention.

In another embodiment of the present invention, to provide an alternative method for avoiding the risks of introducing stem cells containing transgenes such as those described herein into humans and/or other animals (e.g., mice), rather than transfecting stem cells with a combination of recombinant oncogenes or anti-apoptotic protein constructs, the present invention utilizes Tat fusion proteins as a method for allowing proteins to enter cells without having to introduce transgenes into the cells. For example, recombinant constructs encoding tat oncogenes or tat anti-apoptotic genes (e.g., Tat-MYC-ER or Tat-Bcl-2) can be used to conditionally immortalize stem cells. In this embodiment of the invention, the target stem cells are cultured under appropriate culture conditions in a culture medium containing purified recombinant Tat fusion proteins encoded by a selected specific gene combination (e.g., MYC-ER and Bcl-2). In this embodiment of the invention, the product of the oncogene or similar gene can be inducible, as in the above embodiment. Alternatively, or in addition, the effect of the protein can be modulated simply by providing or removing the protein from the culture. Although cell lines generated using this method will continue to rely on the addition of exogenous Tat-fusion proteins, they will not have specific exogenous nucleotide sequences introduced into them. The absence of exogenous oncogene sequences is expected to improve the clinical utility of the methods of the present invention. Human immunodeficiency virus-1 (HIV-1) Tat is an example of a Tat protein, and other retroviral Tat proteins are known in the art. As a non-limiting example, the nucleic acid sequence encoding HIV-1 Tat is shown herein as SEQ ID NO:9, which encodes the amino acid sequence shown herein as SEQ ID NO:10.

In another embodiment, to provide another method for avoiding the risk of introducing stem cells containing transgenes such as those described herein into humans and/or other animals (e.g., mice), rather than transfecting stem cells with a combination of recombinant constructs of proto-oncogenes or anti-apoptotic proteins, the present invention is implemented by introducing proteins (e.g., MYC and Bcl-2) into cells using aptamer technology. Aptamers are short chains of synthetic nucleic acids (usually RNA, but also DNA) selected from randomized combinatorial nucleic acid libraries based on their ability to bind to predetermined specific target molecules with high affinity and specificity. Aptamers have a defined three-dimensional structure and are able to distinguish compounds with minimal structural differences. Accordingly, aptamers can be linked to non-integrating cDNAs encoding proteins or proteins used in the present invention, for example, for delivering proteins or DNA into cells. In addition, aptamers can be easily used to deliver siRNA into cells, for example, when destroying pro-apoptotic proteins according to the present invention. Aptamer technology is described, for example, in Davidson, 2006, Nature Biotechnol. 24(8):951-952; and McNamara et al., 2006, Nature Biotechnol. 24(8):1005-1015. Furthermore, the absence of exogenous oncogene sequences is expected to improve the clinical utility of the methods of the invention.

In another embodiment, to provide an alternative method for avoiding the risks of introducing stem cells containing transgenes such as those described herein into humans and/or other animals (e.g., mice), instead of transfecting stem cells with a combination of recombinant constructs of proto-oncogenes or anti-apoptotic proteins, the present invention is practiced by introducing proto-oncogenes and/or anti-apoptotic proteins into cells using CHARIOT ^™ technology (Krackeler Scientific, Inc., Albany, NY). Using this technology, a non-covalent bond is formed between the CHARIOT ^™ peptide and the target protein. This prevents protein degradation and preserves its native properties during the transfection process. After delivery into the cell, the complex dissociates and the CHARIOT ^™ is transported to the cell nucleus, leaving the delivered protein biologically active and free to enter its cellular target. Effective delivery can occur in the presence or absence of serum and is independent of the endosomal pathway, which can modify macromolecules during cellular internalization. This delivery system also bypasses the transcription-translation process. Thus, proteins useful in the present invention can be delivered into cells and released to immortalize the cells without the need to introduce proto-oncogenes or oncogenes into the cells. As noted above, the absence of exogenous oncogenic sequences is expected to improve the clinical utility of the methods of the invention.

As another alternative (or additional) method for controlling the possibility of proto-oncogene insertion into the host cell genome by the various viral approaches described herein and thereby avoiding transformation events, a drug sensitivity (drug susceptibility) box can be introduced into the viral construct to be used so that it will be expressed in each transduced cell and its differentiated progeny. A drug sensitivity box or drug susceptibility box is a nucleic acid sequence encoding a protein that makes a cell susceptible or sensitive to the presence of a specific drug so that after exposure to the drug, cell activity is inhibited and preferably undergoes apoptosis. For patients in whom the level of a specific blood cell population increases without obvious cause (e.g., infection, trauma, stress, etc.), a drug to which sensitivity has been introduced can be given for a period of time to remove these cells and alleviate any possible additional complications, which involve cells in which genetic insertion may have inadvertently caused oncogenic mutations. Accordingly, as a non-limiting example, a box encoding the cDNA of HPRT can be introduced into the construct used in the method of the present invention to make the transduced cells susceptible to 6-thioguanine. Another non-limiting example is the introduction of thymidine kinase cDNA from a member of the herpes simplex virus family (HSV-TK) to render the transduced cells susceptible to related inhibitors such as ganciclovir, acyclovir, and any related derivatives. In addition, any other such drug sensitivity cassettes and their associated agonists are useful herein.

Other methods of introducing nucleic acids or proteins into cells according to the present invention will be readily apparent to those skilled in the art. Preferred methods of the present invention minimize or eliminate the risk of introducing recombinant DNA into the host cell genome, many of which are described above.

Methods of using the conditionally immortalized cells of the present invention

Another embodiment of the invention includes any stem cell population produced by the methods of the invention, including mixed populations and clonal populations, and the use of the stem cells of the invention in any of the methods described herein, including differentiation into desired cell types, and any method of transplantation, cell replacement, disease treatment, genetic engineering, drug discovery, and cell development and differentiation research described herein.

Since it is now possible to produce homogeneous stem cells that are easy to preserve, recover, amplify and operate in virtually unlimited supply, the stem cells can be used as stem cells or differentiated into different cell lineages and used in experiments to detect the effects of different compounds on cell differentiation, gene expression and/or cell processes. Therefore, one embodiment of the present method relates to a method for identifying a compound that affects cell differentiation, gene expression and/or cell processes. The method generally comprises the following steps: contacting the stem cells produced by the method of the present invention with a test compound and measuring a specific result, particularly a desired result, such as gene expression, biological activity, cell differentiation, cell growth, cell proliferation, etc. (see below), comparing the result without the compound to determine whether the test compound has a desired effect on the stem cell. The method can be used to test virtually any aspect of cell differentiation, cell activity or gene expression. In one aspect, stem cells are manipulated before contacting the compound, for example, genetic manipulation is performed. For example, in order to identify therapeutic compounds (e.g., cancer therapeutic agents) and evaluate gene replacement therapy, the test evaluation can be used to evaluate stem cells from individuals with genetic defects. In fact, the technology of the present invention provides opportunities for identifying drug candidates and treatment candidates and strategies for "fitting" individual cells for cells of specific individuals. Furthermore, as described above, the assay can also be used to identify other growth factors or culture conditions suitable for maintaining the stem cells of the present invention in culture. An example of such an assay is described in detail in Example 7, although the present invention is not limited to this assay.

Another embodiment of the present invention relates to a method for studying cell lineage commitment and/or cell differentiation and development from stem cells, which generally comprises culturing conditionally immortalized stem cells of the present invention and evaluating the cells for genetic and biomarkers related to cell development and differentiation under various conditions and in the presence and absence of compounds or agents that can affect cell lineage commitment or differentiation. As described above, prior to the present invention, such research was severely hampered by the inability to obtain and generate sufficient numbers of desired cell populations to conduct the desired experiments. For example, to identify or screen for intermediates in the differentiation of a particular set of cell lines, sufficient numbers of cells must be obtained to provide meaningful and reproducible results. This was not possible using the technology available prior to the present invention. However, the present invention solves this problem by providing an expandable and essentially unlimited supply of homogenous stem cells that can be used in a variety of experiments. This technology will greatly enhance research capabilities in the field of cell differentiation and discovery. In one approach, the conditionally immortalized stem cells of the present invention are expanded, and then a portion of them are cultured in the absence of conditions that maintain the cells in a conditionally immortalized state (e.g., in the absence of tamoxifen, according to the exemplary methods described herein). Cells can be evaluated for changes in gene expression, cell surface markers, biomolecule secretion, or any other genotype or phenotypic markers to study the process of cell differentiation and lineage orientation. Growth factors or other factors can be added to the culture, for example, to drive differentiation along a specific cell lineage pathway, and the changes in the cells can be evaluated in the presence or absence of these factors. In addition, cells can be used to evaluate the culture conditions, in vivo conditions, factors, and reagents that affect (regulate) cell differentiation and development.

A variety of methods for detecting changes in genotypic or phenotypic characteristics of cells in any of the assays of the present invention are known in the art. Examples of methods that can be used to determine or detect gene sequence or expression include, but are not limited to, polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR), in situ PCR, quantitative PCR (q-PCR), in situ hybridization, Southern blotting, Northern blotting, sequence analysis, microarray analysis, reporter gene detection, or other DNA/RNA hybridization platforms. Methods for determining protein levels include, but are not limited to, Western blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescence polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, microcell analysis, microarrays, microscopy, fluorescence activated cell sorting (FACS), flow cytometry, and assays based on protein properties, including, but not limited to, DNA binding, ligand binding, interaction with other protein partners, cell signaling, enzyme activity, and secretion of soluble factors or proteins.

In drug screening assays, the terms "test compound," "putative inhibitory compound," or "putative modulatory compound" refer to compounds that have unknown or previously unrecognized modulatory activity in a particular process. Similarly, the term "identification" in the method of identifying a compound is intended to include all compounds whose availability for a particular purpose (e.g., regulating cell differentiation) is determined by the method of the present invention, preferably in the presence and absence of the compound. Compounds screened using the methods of the present invention include known organic compounds, such as antibodies, products of peptide libraries, and products of chemical combinatorial libraries. Compounds can also be identified using rational drug design. Such methods are known to those skilled in the art and may include the use of three-dimensional imaging software programs. For example, a variety of drug design methods that can be used to design or select mimetics or other therapeutic compounds useful in the present invention are disclosed in Maulik et al., 1997, Molecular Biotechnology: Therapeutic Applications and Strategies, Wiley-Liss, Inc., the entire text of which is incorporated herein by reference.

In any of the above-described assays, the conditions under which the cells, cell lysates, nucleic acid molecules, or proteins of the present invention are exposed to or contacted with the putative regulatory compound (e.g., by mixing) are any suitable culture or assay conditions, which may include the use of an effective culture medium in which the cells can be cultured (e.g., as described above) or the evaluation of cell lysates in the presence and absence of the putative regulatory compound. The cells of the present invention can be cultured in a variety of containers, including, but not limited to, tissue culture flasks, test tubes, microtiter dishes, and petri dishes. The culture is carried out at a temperature, pH, and carbon dioxide content suitable for the cells. The culture conditions are also within the skill of those skilled in the art, and conditions particularly suitable for culturing the immortalized stem cells of the present invention are described in detail elsewhere herein. The cells are contacted with the putative regulatory compound under conditions that take into account the number of cells in each contact container, the concentration of the putative regulatory compound applied to the cells, the incubation time of the putative regulatory compound with the cells, and the concentration of the compound applied to the cells. Determination of an effective protocol can be achieved by those skilled in the art based on variables such as the size of the container, the volume of liquid in the container, conditions known to be suitable for culturing the specific cell type used in the assay, and the chemical composition (i.e., size, charge, etc.) of the putative regulatory compound being tested.

In one embodiment of the present invention, the cells and methods of the present invention are useful for methods designed to evaluate the multipotency of ctlt-HSCs, CD34+ cells derived from human umbilical cord blood, or adult CD34+ cells isolated from peripheral blood. The methods are described in Example 11.

Another embodiment of the present invention relates to the use of ctlt-HSC cell lines as a platform for generating new models of acute myeloid leukemia (AML). More particularly, the inventors have used ctlt-HSCs of the present invention to generate a mouse model of acute myeloid leukemia. This is a leukemia composed of cells similar to HSCs (based on their surface marker expression). To generate ctlt-HSCs that promote leukemia in mice, 10 ³ -10 ⁵ ctlt-HSCs were transferred into lethally irradiated recipient mice along with 10 ⁵ Rag-1 ^-/- whole bone marrow cells. The mice were given 4-OHT weekly to maintain oncogene activity and monitored for clinical signs associated with leukemia, which are known in the art. Tumors were recovered from these animals and could be propagated in cultures lacking 4-OHT. These cells maintained their HSC-like phenotype, indicating that their proliferation, survival, and blocked differentiation were no longer strongly dependent on the high activity of MYC. The leukemic cell line also caused disease after being re-transplanted into irradiated recipient mice. These tools provide a new platform for studying the biology of AML and related diseases and exploring new therapeutic avenues. Furthermore, introducing ctlt-HSC lines into mice treated with 4-OHT will provide a good internal positive control for treatment: withdrawal of 4-OHT. Secondary cell lines generated after in vivo tumor establishment can also be used to understand relevant therapeutic targets for drug-resistant forms of AML.

Other embodiments of the present invention relate to the use of stem cells produced by the methods of the present invention and cells differentiated from these stem cells in a variety of therapeutic and health-related methods. These methods generally include the steps of obtaining a population, culture, or cell line of conditionally immortalized stem cells produced by the methods of the present invention, removing the conditions that caused the cells to conditionally immortalize, and then using the cells in a therapeutic regimen. For example, the cells can be directly administered to an individual in need of the cells, or the cells can be differentiated into a desired cell type in vitro and then administered to the individual. In addition, before or immediately after removing the conditions that caused the cells to immortalize, the cells can be genetically modified in vitro to express or silence one or more genes as a new method of gene therapy in a controlled environment. The cells can then be administered to the individual as stem cells, or first differentiated into the desired cell lineage in vitro.

To obtain stem cells, in one embodiment, stem cells are obtained from the individual to be treated and then conditionally immortalized according to the methods of the present invention. These cells can be extensively expanded and stored (e.g., frozen or refrigerated), then recovered and re-expanded, manipulated, and/or reused as needed. In another embodiment, stem cells are obtained by obtaining a previously preserved source of conditionally immortalized stem cells from the individual to be treated. In another embodiment, stem cells are obtained from a previously generated group of human stem cell lines that cover a high percentage of the population according to current criteria for identifying "matched" donors. In one embodiment, the cells are obtained from fresh or refrigerated umbilical cord blood, a population of hematopoietic progenitor cells that can be generated by directed differentiation of ES cells in vitro, or HSCs obtained from the peripheral blood of normal patients or patients treated with G-CSF, where the 1-HSCs have been induced to mobilize into the peripheral circulation. Other sources of stem cells will be apparent to those skilled in the art. The cells are cultured according to the methods described above, and the conditions controlling immortalization can be removed at the appropriate time. Additionally, prior to administration to an individual, the cells can be manipulated to excise the gene or construct responsible for conditional immortalization (i.e., a proto-oncogene and/or an anti-apoptotic encoding gene), or if soluble fusion proteins are utilized to maintain the cells in culture, as described above for Tat-fusion proteins, these fusion proteins can be gradually or immediately removed from the culture.

Therefore, the present invention includes delivering the stem cells (including the compositions comprising the stem cells) produced by the methods of the present invention, or the cells differentiated by these cells, to an individual (which may include any animal). Since the stem cells used in these methods are produced in vitro, even if stem cells are initially separated from the patient, the entire cell administration process is essentially an ex vivo administration scheme. Ex vivo administration refers to performing a partial adjustment step outside the patient to produce conditional immortalized stem cells (except for substantially normal stem cells, which may include the stem cells that produce genetic modification) from the individual, and returning the cells or the cells differentiated by the cells to the patient. The stem cells produced according to the present invention or the cells differentiated by these cells can be returned to the individual, or applied to the individual, by any appropriate method of administration. The administration can be systemic administration, mucosal administration and/or administration adjacent to the target site. Preferred routes of administration are apparent to those skilled in the art and depend on the type or reason of administration of the disease to be prevented or treated. Preferred methods of administration include, but are not limited to, intravenous administration, intraperitoneal administration, intramuscular administration, intranodal administration, intracoronary administration, intraarterial administration (e.g., administration into the carotid artery), subcutaneous administration, transdermal delivery, intratracheal administration, subcutaneous administration, intraarticular administration, intraventricular administration, intraspinal administration, pulmonary administration, catheter infiltration, and direct injection into tissue (e.g., liver cannulation).

The cells can be administered with a carrier or pharmaceutically acceptable excipient. The carrier is typically a compound that increases the half-life of the therapeutic composition in the individual being treated. Suitable carriers include, but are not limited to, polymeric controlled-release formulations, biodegradable implants, liposomes, oils, esters, and ethylene glycol. As used herein, a pharmaceutically acceptable excipient refers to any substance suitable for delivering the cells produced by the methods of the present invention to an appropriate site in the body. Preferred pharmaceutically acceptable excipients are those that maintain the cells in a form that allows them to function in a manner that is beneficial to the individual after they reach the target tissue or site in the body.

According to the present invention, effective administration regimen includes appropriate dosage parameters and mode of administration, which result in an effective number of functional cells being delivered to the patient to provide short-term or long-term benefits for the patient. Effective dosage parameters can be determined using method standards for specific conditions or diseases in the art. The method includes, for example, determining survival rate, side effects (i.e., toxicity) and development or degradation of the disease.

An appropriate single dose of stem cells or their differentiated cells according to the present invention is a dose that can provide a beneficial number of cells to a patient when administered once or multiple times over an appropriate period of time. For example, a preferred single dose of stem cells of the present invention is one in which each individual administers approximately ^0.5x10 to approximately ^5.5x10 , or from approximately ^0.5x10 to approximately ^5.5x10 , or from approximately ^0.5x10 to approximately ^5.5x10, stem cells. Doses from approximately ^1x10 to approximately ^5.5x10 are more preferred. The present invention includes any dose increased in increments of ¹⁰ cells between ^0.5x10 and approximately ^5.5x10 . Higher or lower doses are known to those skilled in the art and depend on the type of stem cell or differentiated cell to be administered, as well as on the route of administration. It is understood by those skilled in the art that the dose administered to an animal depends on the extent of the disease or condition and the individual patient's response to treatment. Therefore, within the scope of the present invention, an appropriate dose includes any dose required for the treatment of a particular disease.

As used herein, the phrase "preventing a disease" refers to alleviating the symptoms of a disease, reducing the occurrence of a disease, and/or reducing the severity of a disease. Protecting an animal (individual, subject) refers to the ability of the cells produced according to the present invention to prevent the occurrence of a disease and/or treat or alleviate the symptoms, signs, or causes of a disease when administered to an animal. Similarly, preventing an animal's disease includes preventing the occurrence of a disease (prophylactic treatment) and treating an animal suffering from a disease or experiencing the initial symptoms of a disease (therapeutic treatment). The term "disease" refers to any deviation from the normal health state of a mammal, including a state in which symptoms of a disease appear, as well as a situation in which deviations occur (e.g., infection, gene mutation, genetic defect, etc.) but no symptoms have yet been manifested.

As described above, the stem cells of the present invention can be administered to an individual to treat or prevent a variety of diseases. For example, the stem cell lines of the present invention provide a unique source of expandable stem cells that can be used in a variety of transplantation and treatment strategies, including the treatment of cancer, particularly, radiation-treated cancer. In addition, various immunodeficiency disorders and anemic disorders (e.g., aplastic anemia or hemolytic anemia) will also greatly benefit from this technology, as the present invention provides the ability to regenerate an individual's hematopoietic cells according to individual needs. Another application of the present invention involves the generation of continuously expandable and renewable hair follicle stem cells for use, for example, in plastic surgery on burn victims, in any patient who has experienced irreversible loss of hair growth due to chemotherapy and/or radiation therapy, and in patients who have undergone any surgical procedure affecting the skull or in selective treatments to induce hair growth in individuals affected by hereditary forms of baldness. Similarly, the application of the stem cells of the present invention to the skin is invaluable for the treatment of wound healing and treatment in burn victims, as well as plastic surgery on wounds and other patients, and selective surgery, including but not limited to cosmetic surgery. The cells can also be genetically manipulated to correct congenital or acquired genetic defects in young and elderly individuals. Those skilled in the art will appreciate, based on this disclosure, that the present invention may be used to advantage with a variety of other stem cell populations, including but not limited to stem cells derived from lung, breast, and intestinal epithelium and stem cells derived from neural and cardiac tissue, to name a few.

In addition, as described above, the present invention provides individuals with a unique opportunity to obtain an expandable supply of autologous stem cells and their differentiated cells as needed throughout their lifetime. The stem cells produced by the present method can be stored and used as part of a treatment regimen throughout the individual's lifetime as needed (e.g., if the individual develops cancer or an immunodeficiency disease).

It is now possible to correct genetic defects or introduce beneficial genetic modifications into somatic cells by manipulating autologous stem cells obtained from an individual that have been immortalized and expanded using the conditions of the present invention. The stem cells can then be reintroduced into the individual from whom they originated.

Other applications of the invention include using stem cell lines to repair lung damage caused by COPD, IPE, emphysema, asthma, and smoking. Additionally, the cells can be used to treat vascular damage in the heart and help with autoimmune diseases (e.g., SLE, diabetes, RA) after lethal irradiation.

In the methods of the present invention, cells produced according to the methods of the present invention and compositions comprising these cells can be administered to any animal, including any member of the Vertebrata class, the Mammalia class, including but not limited to primates, rodents, livestock, and domesticated pets. The preferred mammal to be treated is a human.

The various aspects of the present invention are described in more detail in the following examples and drawings. However, the present invention is not limited to these examples and illustrations of the invention.

Example

Example 1

The following examples describe the development of a method for reversibly immortalizing long-term hematopoietic stem cells (lt-HSCs).

Historically, low frequencies of related cell populations have made it difficult to elucidate the molecular basis of impaired hematopoietic lineage development, which has prevented biochemical analysis of signal transduction and downstream responses. In fact, this is a major limiting factor in all hematopoietic studies. In addition, the limited availability of LT-HSCs is also a major obstacle in treating many types of cancer and several immunodeficiencies in humans.

In an effort to overcome this limitation, the inventors developed a method for generating conditionally transformed cell lines representing early hematopoietic stem cell progenitors. The initial strategy involved retroviral transduction of bone marrow stem cells from young and immunologically aged 3-83 mice treated with 5FU. The inventors used a pMSCV bicistronic retroviral vector [Van Parijs, L., Y. Refaeli, A.K. Abbas, and D. Baltimore. (1999) Autoimmunity as a consequence of retrovirus-mediated expression of C-FLIP in lymphocytes. Immunity, 11, 763-70] with inserts encoding Bcl-2 and GFP, as well as MYC-ER and GFP. These genes were chosen because the inventors knew that MYC has the ability to replace cytokine-derived survival and proliferation signals in lymphocytes. By restricting target cells, the inventors hypothesized that stem cell tumors could be formed. Importantly, in this case, MYC-ER function was dependent on tamoxifen, and MYC function and transformation were terminated by removing tamoxifen from the animal or culture. In cells transduced with MYC-ER, the fusion protein is produced but remains in the cytoplasm until exposure to tamoxifen.

More specifically, stem cell populations from mice treated with 5FU were transduced with two retroviruses (encoding MYC-ER and Bcl-2) and transferred into recipient mice subjected to lethal irradiation (1200 rads). Ten days later, weekly intraperitoneal injections of 1 mg/mouse of 4-hydroxytamoxifen (40HT) emulsified in oil were started to activate MYC function (Figure 1). Within four weeks, the recipients of young (rather than old) transduced stem cells developed tumors. Tumors were harvested from bone marrow, spleen, and lymph nodes and cultured in vitro with tamoxifen without the addition of cytokines. These cells grew for about 10 days, then stopped growing, and the cells eventually died. The inventors suspect that the cells are differentiating and believe that this may be due to the need for cytokines for cell growth. Referring to Figure 1, the curve represents the kinetics of mortality after transplantation and the activation of MYC function in vivo. All mice died of leukemia. Although overexpression of MYC can replace cytokine-dependent proliferation and survival functions, it does not seem to involve differentiation signals derived from cytokines.

When sick, mice were euthanized, spleen and lymph node cells were harvested and placed in a culture with tamoxifen and a stem cell growth factor mixture (IL-6, IL-3 and stem cell factor (SCF)). In parallel, cells were analyzed by flow cytometry (Fig. 2). Referring to Fig. 2, the dot plots represent flow cytometric analysis data of forward (FSC) and side (SSC) scatter characteristics of HSC after three days of cultivation with IL-3, IL-6 and SCF. These two criteria are related to cell size (FSC) and granularity (SSC). The two colonies have similar distributions. The histogram represents the GFP level expressed in each cell colony. This reflects the efficiency of in vitro retroviral transduction using a retrovirus encoding the cDNA of MYC-ER and Bcl-2.

In all cases, the ex vivo GFP+ cells were >90% Sca-1 ⁺ and negative for lineage markers. After a few days of culture, the cells began to grow and approximately 400 lines were frozen for subsequent studies. After proliferation, these cells maintained expression of EGFP and were consistently positive for SCA1 and negative for CD34, Flk2, and lineage markers (Figure 3). The only difference in marker expression between markers derived from young mice and markers derived from old mice was increased expression of c-kit in young mice. Without being bound by theory, the inventors believe this may be due to the longer culture time of the old cell lines in the c-kit ligand before the markers were analyzed (3 months versus 3 weeks). Ultimately, the inventors found that these cell lines could be easily recovered and maintained their original phenotype after freezing. Importantly, these cell lines are phenotypically homogeneous and appear to provide the phenotype of all long-term reconstituting lt-HSCs in mice (Reya, T., Duncan, AW, Ailles, L., Domen, J., Scherer, DC, Willert, K., Hintz, L., Nusse, R., and Weissman, LL. (2003). A role for Wnt signaling in self-renewal of hematopoietic stem cells. Nature 423, 409-14).

Recently, the inventors melted 10 bone marrow-derived cell lines produced as described above and were able to easily recover 9 of the 10 cell lines by culturing in a cytokine mixture and 4OHT. The inventors determined the phenotype of these tumors and the results were very promising. Specifically, based on forward and 90° light scattering, each cell line contained two different cell populations. These 9 cell lines differed only in the proportions of these populations. The larger cells in these populations were all GFP-luminescent and Scal, endoglin, and ckit-positive, but negative for Flt3, B220, CD19, and mIgM. ^CD34- was also shown to be negative, although this needs to be confirmed (Figure 3). This phenotype corresponds exactly to the characteristics disclosed by long-term regenerative pluripotent stem cells (Reya et al., supra). The inventors observed the same initial phenotype on cell lines recently obtained from leukemias that developed from transduced HSCs obtained from young donor mice (Figure 3).

To test the ability of these cells to differentiate, representative cell lines were cultured with and without tamoxifen in the presence of IL-3, IL-6, and SCF to inhibit MYC-ER function for 7 days before analysis of phenotypic markers. As shown in Figure 4, a significant proportion of cells acquired B lineage markers, including B220 (~12%), CD19 (~10%), and mIgM (-10%). Furthermore, the inventors have been able to generate the following lineages in vitro by removing 4OHT from the culture: CD4+ab T-cells, myeloid cells (Mac-1+), ter-119+ erythroid progenitors, NK1.1-expressing cells, and neutrophils (Gr-1+ cells). Further experiments will evaluate the ability of these cells to generate additional lineages, as well as the effects of varying cytokine regimens on differentiation. Although no comparisons have been performed, the inventors anticipate that differentiation from young animals will be more efficient in producing B cells compared to old animals. To the best of the inventors' knowledge, this is the first example of conditionally immortalized hematopoietic stem cells that can be induced to differentiate in vitro.

Example 2

The following examples describe the results of adoptive transfer of LT-HSC cell lines into lethally irradiated recipients.

If the HSC cell lines described in Example 1 are suitable subjects for analyzing the basis of defective B cell lymphopoiesis in aged animals, they should be able to reproduce the defect in vivo. The inventors have begun to address this issue by adoptively transferring LT-HSC cell lines into lethally irradiated recipients. In initial experiments, cell lines from aged animals (>60% ^ID- ) were transferred along with RAG2 ^-/- bone marrow, and the recipients were not treated with tamoxifen to silence MYC-ER. Six weeks later, the recipients' bone marrow and spleen cells were harvested and analyzed for the recovery and phenotype of GFP+ cells (GFP-labeled cells derived from the HSC line) (Figure 5).

In the data from three mice shown in Figure 5, one mouse received the aging HSC line ABM42 and two mice received the aging HSC line ABM46. Depending on the cell line transferred, 30 to 70% of the cells in the lymphoid scatter gate were GFP ⁺ . As shown in Figure 5, both cell lines tested (ABM46 and ABM42) produced B (CD19 ⁺ ) and T (TCR ⁺ , CD4 ⁺ , CD8 ⁺ ) cells, macrophages (CDl1b ⁺ ) and granulocytes (GR1 ⁺ ). There was some inter-receptor variation in the proportions of these lineages. However, importantly, both test cell lines produced mature CD4 and CD8 single-positive T cells (Figure 7), but B cell development did not exceed the progenitor stage (Figure 6). Although B220 ⁺ , CD19 ⁺ cells developed, they did not develop to the mIg ⁺ stage. This is entirely predictable based on experimental results involving both autologous and adoptive reconstitution of BM HSCs from immunologically aged mice (Johnson, SA, SJ. Rozzo and JC Ampier, Aging-dependent exclusion of antigen-inexperienced cells from the peripheral B cell repertoire. J Immunol, 2002. 168(10): p. 5014-23). In other words, the same developmental arrest was observed when intact bone marrow from immunologically aged mice was used for transplantation.

The present inventors have discovered that this system can be taken a step further and that LT-HSC lines can be successfully reconstituted from the bone marrow of adoptive recipients of the original HSC line (data not shown). This was accomplished simply by culturing the bone marrow cells in the presence of stem cell cytokines plus tamoxifen to reactivate MYC. These cells are growing and displaying a primitive phenotype.

Example 3

The following example describes a method for reversibly immortalizing HSCs using a procedure performed entirely in vitro.

In addition to the previously described methods for generating conditionally immortalized long-term HSC cell lines, the present inventors have been able to perform this procedure entirely in vitro. The above method relies on introducing transduced HSCs into mice and inducing their transformation in vivo. The advantage of performing this procedure in vitro is that every aspect of the method is carried out under a controlled environment.

The method first includes treating donor mice with 5-fluorouracil (5-FU) to enrich HSCs and induce the proliferation of these cells. 5FU-enriched hematopoietic stem cells from mouse tibias and femurs are collected and then placed in DMEM culture medium on 24-well tissue culture plates at a density of 1.8-2.0x10 ⁶ cells per well. The culture medium contains 15% heat-inactivated fetal bovine serum and IL-3, IL-6, and SCF. The cells are subjected to three rounds of spin infection to transduce the cells with retroviral vectors encoding MYC-ER and Bcl-2. In brief, cells are transfected with pMIG-MYC.ER or pMIT-Bcl2. The supernatant containing the virus is collected and supplemented with 4 μg/ml of 1,5-dimethyl-1,5-diazoundecamethylene polybrene and 10mM HEPES and passed through a 0.45 μm filter. Two different viral supernatants were mixed in a 1:1 ratio and added to the wells. The cells were then centrifuged at 2000 rpm for 1 hour. The viral supernatant was replaced at the end of each spin infection. 24 hours after the last round of infection, the transduction level was determined by flow cytometry to determine transduction efficiency. The transduced cells were then incubated in DMEM medium containing IL-3, IL-6, SCF, and 10 nM 4OHT. The medium was changed every 3 days, with particular emphasis on ensuring a fresh supply of cytokines and 4OHT. Cells were slowly passaged as needed.

Using this in vitro approach, the inventors have been able to generate conditionally immortalized cell lines harboring the following gene combinations: MYC-ER and Bcl-2; MYC-ER and hTERT (the reverse transcriptase component of human telomerase); ICN-1-ER (the ER-regulated active component of the intracellular portion of Notch-1) and Bcl-2; ICN-1-ER and hTERT; and MYC-ER and ICN-1-ER. The data presented in Figures 8-11 demonstrate the starting characteristics of most of these cell lines. They generated cell lines composed of c-kit+, Sca-1+, CD34-, flk2- cells, a phenotype consistent with that exhibited by normal, long-term hematopoietic stem cells. The data presented in Figures 8-11 are derived from flow cytometric analysis of retrovirally encoded reporter genes (GFP and thy1.1) and four stem cell markers: c-kit, sca-1, CD34, and flk-2. The cell lines shown in Figures 8-11 were cultured for 5 weeks before phenotype determination. These cells have now been expanded and divided in continuous culture for over 35 days.

See Figure 8, which shows a phenotypic comparison of cell lines derived from HSCs obtained from young C57/BL6 mice, which were retrovirally transduced with BCL-2 and MYC-ER and maintained in continuous culture in vitro for more than 90 days. Shown is the phenotype of a representative clone at 3 (young) months after 90 days of continuous culture.

See Figure 9, which shows a phenotypic comparison of cell lines derived from HSCs obtained from young C57/BL6 mice, which were retrovirally transduced with different oncogene combinations and maintained in continuous culture in vitro for more than 90 days. 5FU-enriched HSCs were retrovirally transduced with pMIG-MYC and pMIT-Bcl-2 (top panel), pMIG-MYC.ER and pMIG-hTERT (center panel), or pMIG-ICN.1.ER and pMIT-Bcl-2. Cells were maintained in DMEM supplemented with 15% fetal bovine serum and a mixture of IL-6, IL-3, and SCF. Shown is the phenotype of a representative clone at 3 (young) months after 90 days of continuous culture. The figure shows flow cytometric analysis of the expression of virally expressed markers (GFP and Thy1.1) and four markers required to define long-term HSCs in mice (Sca-1, c-kit, CD34, and Flk-2). These four cell lines contain a subpopulation that maintains the phenotype of lt-HSC (Sca-1+, c-kit+, CD34-, flk-2-).

See Figure 10, which shows a phenotypic comparison of cell lines derived from HSCs obtained from young C57/BL6 mice that were retrovirally transduced with different combinations of oncogenes and maintained in continuous culture in vitro for more than 90 days. 5FU-enriched HSCs were retrovirally transduced with pMIG-ICN.1.ER and pMIT-Bcl-2 (top panel), pMIG-ICN.1 and pMIT-Bcl-2 (second row of panels), or pMIG-ICN.1 and pMIG-Bcl-2 (third row of panels), or pMIG-hTERT and pMIT-Bcl-2 (bottom panel). These cells were maintained in DMEM supplemented with 15% fetal bovine serum and a mixture of IL-6, IL-3, and SCF. Shown is the phenotype of a representative clone at 3 (young) months after 90 days of continuous culture. The figure shows the results of flow cytometric analysis of the expression of virally expressed markers (GFP and Thy1.1) and four markers required to define long-term HSCs in mice (Sca-1, c-kit, CD34, and Flk-2). These four cell lines contain subpopulations that maintain the phenotype of lt-HSCs (Sca-1+, c-kit+, CD34-, flk-2-).

Referring to Figure 11, this figure shows the phenotypic comparison of cell lines derived from HSC obtained from young C57/BL6 mice, which were retrovirally transduced with different combinations of oncogenes and maintained for more than 90 days in in vitro continuous culture. 5FU-enriched HSC were retrovirally transduced with pMIG-MYC and pMIG-ICN.1 (upper figure), pMIG-MYC.ER and pMIG-ICN.1 (middle figure) or pMIG-ICN.1.ER and pMIG-MYC. These cells were maintained in DMEM supplemented with 15% fetal bovine serum and a mixture of IL-6, IL-3 and SCF. What is shown is the phenotype of a typical clone at 3 (young) months after 90 days of continuous culture. The figure shows the flow cytometry analysis results for the expression of virally expressed markers (GFP and Thy1.1) and four markers (Sca-1, c-kit, CD34 and Flk-2) required for the long-term HSC of the definition mouse. These four cell lines contain a subpopulation that maintains the phenotype of lt-HSC (Sca-1+, c-kit+, CD34-, flk-2-).

These cell lines have also been used to reconstitute cellular compartments in vivo. See Figure 12, which shows the results of in vivo reconstitution of T and B cell compartments by cell lines derived from HSCs obtained from young C57/BL6 mice, which were retrovirally transduced with different combinations of oncogenes and maintained in continuous culture in vitro for more than 90 days. Briefly, 5FU-enriched HSCs were retrovirally transduced with pMIG-ICN.1-ER and pMIG-hTERT (top panel), pMIG-MYC.ER and pMIG-hTERT (middle panel), or pMIG-MYC-ER and pMIT-Bcl-2 (bottom panel). These cell lines were maintained in DMEM supplemented with 15% fetal bovine serum and a mixture of IL-6, IL-3, and SCF. Bone marrow stem cells from Rag2-/- mice and the in vitro generated LT-HSC line were used to reconstitute young C57/BL16 mice that had been subjected to lethal irradiation. Six weeks later, bone marrow was harvested and stained with a panel of specific lineage markers. The development of mature CD4 and B220 positive/GFP positive cells can be easily observed. Data from four representative mice are shown in this figure. In each group, approximately 30% of the mice maintained the GFP marker.

Example 4

The following examples describe an extension of the method for reversibly immortalizing HCS derived from human umbilical cord blood and bone marrow in vitro.

Another application of this technology is the ability to expand human long-term hematopoietic stem cells in vitro through their conditional immortalization. The inventors therefore adapted the in vitro methods described in the previous examples for use in human cells with some modifications. First, the retrovirus is preferably packaged with an amphotropic envelope to ensure efficient transduction of human cells. In addition, the cell source is human umbilical cord blood, obtained anonymously from an umbilical cord blood bank in accordance with all regulations and systems of the inventors' institutional review boards. The resulting cells will express a reporter gene, which can ultimately be used to isolate pure populations via high-speed cell sorting. The inventors have noted that many mature cells generated from the murine lt-HSC cell line lose expression of surface markers, likely due to methylation of the retroviral genome after lineage commitment and differentiation. The inventors expect to see similar behavior in human cells. In this case, the presence of the reporter gene in these cells, combined with cell surface markers in the cell population, can be used to monitor lt-HSCs and their distribution in transplant recipients.

Example 5

The following example describes a method for serial excision of DNA fragments encoding MYC-ER and Bcl-2 from conditionally immortalized HSC cells.

In order to avoid the risk of introducing transgenic HSCs encoding MYC.ER and Bcl-2 into humans and/or mice, the two DNA fragments will be excised using a bacterial recombinase method. In order to allow control of which of the two genes is excised at any time point, two different recombinases are used. Two examples of the recombinases are Cre and Flp recombinases. In brief, a recombinase recognition substrate sequence (RSS) is imported into a retroviral construct so that they are located on the side of the open reading frame of the oncogene, and a reporter gene (GFP or Thy1.1) is imported. In this example, cells are incubated in a culture medium comprising Tat-Cre fusion protein. Recombinant proteins have been previously described and shown to be able to passively enter cells and mediate the recombination of genomic DNA that depends on loxP sites.

This method will allow to obtain a large number of conditions that can produce many HSCs for in vitro and in vivo differentiation. First, the cells are gradually stopped from the high level of proliferation and survival signals that they have adapted to during the conditional conversion process. Second, the cells are adapted to rely on normal cytokines to exercise their own stable functions and differentiation. Third, the continuous loss of reporter gene expression will allow to determine the deletion state and degree of each gene studied. Accordingly, cells expressing two reporter genes (GFP and Thy1.1) contain two sequences (MYC and Bcl-2, respectively). Cells expressing Thy1.1 but not expressing GFP have successfully deleted the MYC coding sequence, but still contain the Bcl-2 gene. Finally, cells not expressing GFP or Thy1.1 have deleted these two alleles. Figure 13 shows this method in the form of a schematic table.

In addition, the method was tested in mice by obtaining 5FU-enriched BM-HSC from a mouse strain expressing a human MYC transgene, which can be induced by removing tetracycline and containing a bacterial protein called tTA (tetracycline trans-acting protein). Human MYC cDNA was cloned downstream of the tetracycline-regulated transcription element (TRE). TRE-MYC mice were treated with 5FU and used to harvest BM-HSC. These cells were transduced in vitro with retroviruses expressing Bcl-2 and tTA (pMIT-Bcl2 and pMIG-tTA). The cells were cultured in the continuous presence of doxycycline to keep the MYC transgene silent. Once the cells were analyzed by flow cytometry, they were transplanted back into mice that were not given a diet containing doxycycline (a more stable form of tetracycline commonly used in vivo).

Once lt-HSC cell lines were generated, the effects of culturing them in vitro in the presence of doxycycline were examined in parallel with a MYC.ER-containing cell line cultured in the absence of 4OHT. MYC protein levels were monitored throughout the culture by western blotting and intracellular staining.

Example 6

The following examples describe the in vitro generation of numerous hematopoietic lineages following removal of 4OHT from liquid tissue culture medium.

Traditional methods for determining the potential of HSCs involve using semi-solid culture media (methylcellulose) with defined cytokines to enhance the differentiation of HSCs into specific lineages. The inventors were interested in determining the pluripotency of cell colonies generated in vitro using the methods of the present invention. To examine this tissue, the ABM42 and ABM46 cell lines described herein were maintained in a culture medium containing IL-3, IL-6, and SCF, but without 4OHT. In addition to the lineages (i.e., lymphoid, myeloid, and granulocyte lineages) that the inventors were able to detect in reconstructed mice, GFP+ cells expressing NK1.1 or ter-119 were also detected (Figure 14). NK1.1 cells can be NK-cells or NK-T cells. Ter-119-expressing cells belong to the erythroid lineage. These findings show that these cell lines are capable of producing all components of the normal hematopoietic system and that these cells are useful for producing large quantities of specific components for passive therapy. In addition, they have great application and importance for studying early events in hematopoiesis and identifying new therapies for therapeutic intervention in genetic disorders or complications of normal chemotherapy or even infectious diseases.

Example 7

The following examples describe a method for high-throughput screening of small molecules or biologics that induce or inhibit conditionally transformed long-term HSC differentiation.

The following is a general method for screening small molecules or biological agents that induce or inhibit HSC differentiation. Previously, the fact that large numbers of stem cells could not be obtained hindered these types of large-scale screening. Due to the current ability of the inventors to immortalize long-term HSC, such technology is now possible.

For example, one such method is a myeloid differentiation read-out that has been modified by Schneider et al. (Schneider, T. and Issekutz, AC (1996) Quantitation of eosinophil and neutrophil infiltration into rat lung by basic assays for eosinophil peroxidase and myeloperoxidase. Application in a Brown Norway rat model of allergic pulmonary inflammation. J Immunol Methods 198, 1-14). Briefly, conditionally transformed long-term HSCs are plated in 96-well flat-bottom plates at varying cell numbers (typically 2×10 ⁴ -5×10 ⁴ cells/well). Screening is performed in complete medium (DMEM + 15% heat-inactivated fetal calf serum, 1× penicillin/streptomycin, 1× L-glutamine, and 1× non-essential amino acids, supplemented with IL-3, IL-6, and SCF) with or without the addition of 4OHT to maintain the cells in an undifferentiated state. These conditions have been shown to produce Mac-1+ cells, consistent with a myeloid differentiation pattern. Additional cytokines can be added to direct differentiation along a specific pathway, although this system can also be used to screen for specific functions of a panel of cytokines. In this example, complete medium is added without IL-3, IL-6, and SCF, but with the given cytokine to be tested or used to direct differentiation (e.g., CSF-1, G-CSF, GM-CSF, EPO, TEPO, etc.).

If necessary and according to the reagent to be tested or molecule and determine, use 96-well plates to titrate small molecules, biological agents or positive control substances (for example, arsenic trioxide) and incubate with lt-HSC for 24 to 72 hours or longer. After incubation, cells are washed with PBS and resuspended in PBS and stored overnight at -80 ° C to lyse cells. The cells are then thawed at room temperature and the culture plate is centrifuged at 3,000 rpm for 10 minutes. The supernatant is then transferred into a new 96-well plate and mixed with tetramethylbenzidine (TMB) for 40 minutes. The reaction is terminated with 4N H ₂ SO ₄ and OD is read at 450 nm. This type of high-throughput test can be used to test the ability of long-term HSC to differentiate into a wide range of cell types that small molecules or biological agents induce or prevent conditional conversion. The results of these screenings can then further test the ability to induce or inhibit HSC differentiation in vivo. The variation of this test format is obvious to those skilled in the art and is included in the present invention.

Example 8

The following examples describe the use of the methods of the present invention to generate cell lines of intermediate hematopoietic lineages.

The following protocol can be used to induce the development of cell lines representing intermediate stages of hematopoietic lineage development following transplantation of a conditionally immortalized lt-HSC line into lethally irradiated mice. First, 10 ³ -10 ⁵ conditionally transformed lt-HSC lines generated according to the methods of the present invention are transferred into groups of lethally irradiated recipient mice. The transplants may also include 10 ⁵ Rag-1 ^-/- as a carrier to ensure initial survival of the irradiated mice. The mice are given weekly intraperitoneal injections of 1 mg of tamoxifen to immortalize partially differentiated cells derived from the conditionally transformed lt-HSC line. Injections are initiated 3 days to 1 week after the initial transplantation or 8 days after transplantation until the mice have been fully reconstituted with the conditionally transformed lt-HSC line. After 3 days of tamoxifen treatment, or when they display clinical symptoms associated with leukemia, cells are harvested from the spleen and bone marrow of the mice. Cells were cultured under standard bone marrow culture conditions (DMEM, 15% fetal bovine serum, penicillin/streptomycin, L-glutamine, non-essential amino acids, IL-3, IL-6, and SCF) with 4-OHT, or in the presence of other cytokines and media for other hematopoietic cell types. Cell lines were frozen and/or expanded, and cell lines were also cloned by limiting dilution and single cells confirmed by PCR amplification of proviral integration, frozen, and then characterized by flow cytometry for expression of surface markers. These types of methods were used for murine and human ctlt-HSC cell lines, using NOD/SCID mice as recipients or using newborn Rag-1 ^-/- mice, which were injected intrahepatically.

Example 9

The following examples describe the development of cell lines representing intermediate stages of T cell development using the methods of the present invention and a protocol for the in vitro generation of mature D4+αβ T cells.

In this experiment, the conditionally immortalized lt-HSC cell line produced according to the inventive method was inoculated into a culture plate in the presence of a normal cytokine mixture, wherein IL-7 was supplemented and tamoxifen-free. Parallel culture was established on an OP-9 stromal cell layer expressing jagged (a Notch-1 ligand). After starting to cultivate for the indication of monitoring T cell development, cells were stained for T cell lineage markers every 48 hours. The wells showing T-lineage orientation and developmental indications were converted to a culture medium comprising tamoxifen to stabilize the phenotype and establish a cell line. The resulting cell line was amplified, cloned, and characterized as described in Example 8. In order to determine the use of their T cell receptor repertoire, the T cell line was specifically stained for each TCR-V β allele. When appropriate, some mature T-cell lines or cell lines representing progenitor cell colonies were transplanted into Rag-1 ^-/- mice to evaluate their ability to follow the normal tolerance and self-stabilization mechanisms in vivo, as well as their ability to further differentiate in vivo. Finally, their ability to respond to antigenic stimulation was evaluated in vitro and in vivo.

Example 10

The following examples describe the development of intermediate developmental cell lines and myeloid leukemia models using the methods of the present invention and employing protocols for directed differentiation of HSCs into myeloid lineages.

In this experiment, the lt-HSC cell line of conditional immortalization produced according to the inventive method was inoculated in culture plates under the presence of a normal cytokine mixture, wherein supplemented with G-CSF and not containing tamoxifen. After starting to cultivate for the indication of monitoring bone marrow development, within every 48 hours, bone marrow lineage markers were subjected to cell staining. The holes showing bone marrow lineage orientation and development indication were converted to the culture medium comprising tamoxifen, with stable phenotype and set up cell line. As described in Example 8, the cell line obtained was amplified, cloned and characterized. Some cell lines obtained were transplanted back into mice to monitor the ability of their regeneration Op/Op mice (natural mutant mice lacking macrophages). These cell lines were also transplanted into the wild-type mice that will remain under the tamoxifen condition, to determine whether these cell lines will also produce myeloid leukemias that are similar to people AML, CML and APL. These new tumors provide new models for preclinical treatment.

Example 11

The following examples describe the generation of human adult ctlt-HSC cell lines and the testing of their pluripotency in vivo using NOD/SCID or RAG ^<-> xenograft models.

In this assay, CD34+ cells (from mobilized blood or umbilical cord blood) are transduced in vitro with a retroviral vector encoding MYC-ER, Bcl-2, and GFP (for later detection of transplanted cells) that is coated with an amphotropic envelope (according to the methods of the present invention). lt-HSCs are selected by in vitro proliferation in the presence of 4OHT and growth factors, as described above for murine HSCs. The pluripotency of the selected cells is assessed by transplanting the lt-HSC line into sublethally irradiated NOD/SCID or NOD/SCID/β-2M ^-/- or Rag-1 ^-/- or Rag-2 ^-/- mice and analyzing all blood cell lineages by immunofluorescence flow cytometry after 6-12 weeks. More specifically, after generating ctlt-HSC lines using the methods of the present invention, their pluripotency can be examined using two different and complementary methods. In the first approach, varying numbers of cloned ctlt-HSC lines were introduced into sublethally irradiated NOD/SCID mice or NOD/SCID/B-2M ^-/- mice. In this case, 10 ³ -10 ⁵ cells derived from the human ctlt-HSC line were intravenously transferred after the mice were subjected to a sublethally irradiated regimen (0.3 Gy). The mice were analyzed for reconstitution 12 weeks after transplantation. Second, 10 ³ -10 ⁵ cells derived from the human ctlt-HSC line were introduced into the livers of newborn Rag-1 ^-/- or Rag-2 ^-/- mice by direct injection. These xenografts were also analyzed for adequate reconstitution 6-12 weeks after transplantation.

Example 12

The following examples describe the conditional ablation of MYC and Bcl-2 expression in ctlt-HSCs following transplantation.

In this experiment, the virus used to transform stem cells (viral vector) was reengineered to contain two loxP sites flanking the MYC-ER, Bcl-2, and GFP open reading frames (ORFs). When the cells were transplanted, a regulated form of Cre or Cre-TAT fusion protein was used to delete the oncogene coding sequence, thereby eliminating the risk of the inserted fragment causing malignancy in the recipient. This approach was first developed in mice and then applied to human lt-HSCs.

In the second approach, lt-HSCs from TRE-MYC mice are used to generate cell lines harboring retroviruses encoding Bcl-2 or rtTA. These are then transplanted into mice. The mice are fed doxycycline to eliminate MYC expression. lt-HSCs derived from TRE-MYC x TRE-Bcl-2 bigenic mice can be used, which can be transduced with the pMIG-rtTA retrovirus to eliminate MYC and Bcl-2 expression.

Example 13

The following examples describe the use of HIV-1 Tat fusion proteins with MYC and/or Bcl-2 to achieve conditional transformation without genetic modification of lt-HSCs.

MYC-Tat and Bcl-2 fusion proteins were produced and purified using established protocols. The fusion proteins were tested by treating cells where the effects of overexpressing Bcl-2 (e.g., activated T cells, B-cell lymphoma cell lines resistant to BCMA-Fc, etc.) or MYC (e.g., anergic B cells, naive T cells, activated T cells) could be readily determined. A combination of MYC-Tat and Bcl-2-Tat proteins was used to propagate lt-HSCs prior to transplantation. This approach was readily developed and tested in a mouse system and subsequently applied to humans.

The entire disclosures of U.S. Provisional Patent Application No. 60/728,131 and U.S. Provisional Patent Application No. 60/765,993 are incorporated herein by reference.

Although several embodiments of the present invention have been described in detail, modifications and variations of these embodiments will be apparent to those skilled in the art. However, it should be clearly understood that such modifications and variations are within the scope of the present invention, as set forth in the following claims.

Claims (3)

1. A composition comprising: A fusion protein comprising protein transduction domains of MYC and Tat proteins capable of promoting cell proliferation and survival of one or more hematopoietic stem cells in vitro; and A fusion protein comprising a member of the Bcl-2 family that inhibits apoptosis and the protein transduction domain of the Tat protein. 2. A method for promoting the proliferation or survival of one or more hematopoietic stem cells cultured in vitro, comprising: Use one or more hematopoietic stem cells to transduce in the following ways: (i) a first vector containing a nucleic acid sequence encoding the hormone-binding domain (MYC-ER) of the MYC molecule and the human estrogen receptor, and a second vector containing a nucleic acid sequence encoding Bcl-2; (ii) a first vector containing a nucleic acid sequence encoding MYC-ER; and a second vector containing a nucleic acid sequence encoding hTERT; (iii) a first vector containing a nucleic acid sequence encoding an ER-regulated active element of the intracellular portion of Notch-1 (ICN-1-ER), and a second vector containing a nucleic acid sequence encoding hTERT; or (iv) a first vector containing a nucleic acid sequence encoding MYC-ER, and a second vector containing a nucleic acid sequence encoding ICN-1-ER; and Culture the one or more hematopoietic stem cells in a culture medium; One or more of the hematopoietic stem cells have increased survival or proliferation compared to the corresponding non-transduced hematopoietic stem cells. 3. A method for promoting the proliferation or survival of one or more hematopoietic stem cells cultured in vitro, comprising: Provide the composition of claim 1 to one or more hematopoietic stem cells. The MYC protein is MYC-ER and the Bcl-2 family member is Bcl-2; and Culture the one or more hematopoietic stem cells in a culture medium; One or more of the hematopoietic stem cells have increased survival or proliferation compared to the corresponding hematopoietic stem cells not exposed to the composition.