HK1261077A1 - Simplified basic media for human pluripotent cell culture - Google Patents

Description

Simplified basal medium for human pluripotent cell culture

Cross Reference to Related Applications

Not applicable.

Statement regarding federally sponsored research or development

The present invention was made with government support under ES017166 awarded by the national institutes of health. The government has certain rights in this invention.

Background

Pluripotent cells, such as Embryonic Stem (ES) cells and Induced Pluripotent Stem (iPS) cells, have the potential to differentiate into all three primitive germ layer cells (Thomson et al, Science 282,1145-1147 (1998)). The great potential for development of pluripotent cells has been demonstrated for both basic research and clinical applications. Many basic methods of human pluripotent cell culture, such as growth media, plating and other conditions, have been developed and improved (Ludwig et al, nat. Biotechnol 24, 185-. For example, when human ES cells are initially cultured on Murine Embryonic Fibroblast (MEF) feeder cells in a medium containing Fetal Bovine Serum (FBS), a fully defined medium and a defined protein matrix are now available (Ludwig et al, Nat. Biotechnol 24, 185-.

During the past decade, pluripotent cell culture methods have advanced significantly. Several growth media have been developed that provide basic nutrients and growth factors for the survival and expansion of pluripotent cells and directly determine how the cells grow and differentiate. TeSR^TMIs one of the first defined media that supports pluripotent cells in an undifferentiated state over multiple culture passages in the absence of feeder cells or conditioned media (Ludwig et al, nat. methods 3,637-646 (2006); U.S. patent No. 7,449,334, each incorporated herein by reference and as if fully set forth). TeSR^TMIn addition to the basal medium DMEM/F12, which itself contained 52 components, 18 components were included (Table 1).

The diversity of different growth media available for pluripotent cell culture has led to inconsistent results. The media currently used for the derivation and growth of pluripotent cells, including fully defined media, contain components that affect pluripotent cells in different ways. Prior to the invention described herein, it was not known how each medium component (alone or in combination with other components) affected the function of different pluripotent cells in cell culture, such as viability, pluripotency or differentiation.

For example, albumin, the most abundant protein in most media, is a lipid carrier and can also influence differentiation or maintenance of pluripotency by its associated lipids. The quality of albumin and its associated lipids determines whether it can be used in human pluripotent cell culture. However, the quality of albumin varies widely depending on its source, even when produced from recombinant genetic material, resulting in differences between experiments performed under different equivalent experimental conditions. Also, although cloned human serum albumin is available, it is rarely used in routine experimentation because of its relatively expensive price.

Efforts to remove albumin from the culture medium have proven unsuccessful. Removal of albumin or any other growth factor present in TeSR results in a significant reduction in human ESC culture performance, such as a reduction in cell viability, proliferation and pluripotency (Ludwig et al, nat. biotechnol 24,185-187 (2006)).

To fully exploit the potential of pluripotent cells for drug discovery, testing and transplantation therapy, it is desirable to derive and grow these cells under well-defined and ideally xeno-free conditions. Thus, there is an unmet need in the art for a culture medium that is free of components that cause inconsistencies to maintain control over pluripotent cell culture conditions. There is a particular need in the art for pluripotent cell culture media that contain only those components that support pluripotent cell functions important for a particular culture objective.

Brief description of the drawings

The present invention relates generally to media, compositions and methods for deriving and culturing pluripotent cells, and more particularly to fully defined media for pluripotent cells.

A first aspect of the invention is summarized as an albumin-free medium that supports the viability, growth and pluripotency of pluripotent cells.

In some embodiments of the first aspect, the medium comprises selenium.

In some embodiments of the first aspect, the medium comprises NODAL.

In some embodiments of the first aspect, the medium comprises transferrin.

In some embodiments of the first aspect, the medium comprises transforming growth factor- β (TGF- β).

In some embodiments of the first aspect, the medium comprises only water, salts, amino acids, vitamins, a carbon source, insulin, and Fibroblast Growth Factor (FGF), each in an amount sufficient to support the viability of the pluripotent stem cells.

In some embodiments of the first aspect, the medium comprises only water, salts, amino acids, vitamins, a carbon source, insulin, FGF, selenium, transferrin, and one of TGF- β and NODAL, each in an amount sufficient to support proliferation of pluripotent stem cells.

In some embodiments of the first aspect, the culture medium supports the survival of pluripotent cells after passaging, freezing, proliferating, pluripotency, derivation, and cloning.

In some embodiments of the first aspect, the medium is xeno-free.

In some embodiments of the second aspect, the medium used to culture pluripotent cells comprises only water, salts, amino acids, vitamins, a carbon source, insulin, and FGF, each in an amount sufficient to support the viability of the pluripotent cells.

In some embodiments of the third aspect, the culture composition is free of fibroblast feeder cells, conditioned medium, and xeno contaminants.

A fourth aspect of the invention is summarized as a method for deriving iPS cells from mature individuals under well-defined conditions. The method comprises the following steps: somatic cells from a mature individual are cultured in a medium comprising water, salts, amino acids, vitamins, a carbon source, insulin, and FGF, all in amounts sufficient to maintain viability, and the cells are reprogrammed in defined conditions (e.g., the conditions used to derive iPS cells).

In some embodiments of the fourth aspect, the medium comprises TGF- β in part or all of the reprogramming steps.

In some embodiments of the fourth aspect, the medium comprises butyrate.

In some embodiments of the fourth aspect, the medium comprises hydrocortisone.

In some embodiments of the fourth aspect, the medium is xeno-free.

A fifth aspect of the invention is summarized as a method for cloning pluripotent stem cells in an albumin-free medium. The method comprises the following steps: pluripotent stem cells are plated at clonal density in albumin-free medium that supports the cloning of pluripotent stem cells.

In some embodiments of the fifth aspect, the medium comprises a ROCK inhibitor.

In some embodiments of the fifth aspect, the medium comprises myostatin (blebbistatin).

In some embodiments of the fifth aspect, the medium comprises only water, salts, amino acids, vitamins, a carbon source, insulin, FGF, selenium, transferrin, and one of TGF- β and NODAL, each in an amount sufficient to support the cloning of pluripotent stem cells.

A sixth aspect of the invention is summarized as a method for cryopreservation of pluripotent stem cells in albumin-free medium. The method comprises the following steps: the pluripotent stem cells were frozen in albumin-free medium.

In some embodiments of the sixth aspect, the medium comprises only water, salts, amino acids, vitamins, a carbon source, insulin, FGF, selenium, transferrin, one of TGF- β and NODAL, and dimethyl sulfoxide (DMSO).

A seventh aspect of the invention is summarized as iPS cells derived in the absence of albumin. iPS cells derived in the absence of albumin were not contaminated with endogenous albumin.

The methods and compositions described herein can be used in a variety of applications to derive, culture, and use pluripotent cells. It is an object of the present invention to define short and long term culture conditions for pluripotent cells that are restricted to factors that support the desired culture goal.

It is another object of the present invention to provide culture conditions for pluripotent cells under which the percentage of cultured cells in an undifferentiated state is maximized.

It is another object of the invention to provide a culture medium that can serve as a necessary platform to test how various conditions affect pluripotent cells and to compare previously reported experiments in different media contexts.

These and other features, objects and advantages of the present invention will be better understood from the following description. In this description, reference is made to the accompanying drawings, which form a part hereof, and in which is shown by way of illustration only, and not by way of limitation, or embodiments of the invention. The description of the preferred embodiments is not intended to limit the invention to the precise forms disclosed, and all modifications, equivalents, and alternatives may be apparent. Accordingly, reference should be made to the claims herein for interpreting the scope of the invention.

Brief description of the drawings

The present invention will be better understood and features, aspects and advantages other than those described above will become more apparent in view of the following detailed description. The detailed description makes reference to the following drawings, in which:

FIGS. 1A-E show the media components that allow human ES cells to survive and self-renew in culture, FIG. 1A shows the 24 hour survival index of individualized cells plated in different media, the media abbreviations are listed in Table 1 the presence of insulin and fibroblast growth factor (IF), Bovine Serum Albumin (BSA), β -mercaptoethanol (BME) is indicated by "+" and the absence is indicated by "-", FIG. 1B shows the 24 hour or 96 hour survival index of individualized cells plated in different media, the addition of insulin and Fibroblast Growth Factor (FGF) is indicated by "+" and the removal is indicated by "-", FIG. 1C shows the TeSR cultured in vitamin C^TMMedium (TeSR), vitamin C free TeSR^TMMedium (TeSR)^TM-survival index at 24 hours or 129 hours for individualized cells in LAA) or DF5 medium. FIG. 1D shows cell proliferation after three passages each in DF5, DF5 with selenium added (DF5+ selenium), DF12 or DF12 with selenium removed (DF 12-selenium). FIG. 1E shows comparative analysis of twelve different basal media.

Figures 2A-F show optimization of culture conditions for human ES cells and iPS cells using DF 5S. FIG. 2A is shown at DF5S (lower panel) or TeSR^TM(upper panel) at low density (about 1,500 cells/cm)²) Inoculating in different O₂And CO₂Concentration (O15C 5: 15% O₂And 5% CO₂；O15C10：15％O₂And 10% CO₂；O5C10：5％O₂And 10% CO₂) Survival index of cultured individualized cells. Cell survival was measured at 24 hours and 124 hours. FIG. 2B shows the culture in the presence of small molecule HA100(+ HA100) or in the absence of small molecule HA100(CM 100: 100 n-containing)g/ml conditioned medium of FGF) cloning efficiency of H1 cells in different media (cloning efficiency). Fig. 2C shows the cloning efficiency of H1 cells and iPS cells derived from foreskin fibroblasts in different media. Fig. 2D shows the cloning efficiency of iPS cells derived from foreskin fibroblasts in different media. DF5S trFe represents DF5S medium to which holotransferrin was added. FIG. 2E shows the cloning efficiency of H1 cells cultured in different media in the presence of HA100 (10. mu.M, 24 hours), myosistatin (10. mu.M, 4 hours), or Y27632 (10. mu.M, 24 hours), compared to the cloning efficiency in the absence of these factors (control). Asterisks denote p<0.05. FIG. 2F shows the cloning efficiency of H1 cells in both normoxic (dark gray column) and hypoxic (light gray column) conditions in Conditioned Medium (CM), CM containing a ROCK inhibitor (HA100), TeSR containing a ROCK inhibitor, and E8 containing a ROCK inhibitor. Error bars represent standard error of the mean; asterisks denote p<0.05。

FIGS. 3A-B show pluripotent cell growth and gene expression in DMEM/F12 (referred to herein as "E8 (TGF- β)" and "E8 (NODAL)", respectively) supplemented with insulin, transferrin, selenium, L-ascorbic acid, FGF2 and TGF- β or NODAL FIG. 3A shows growth and gene expression in TeSR^TMFold expansion of H1ES cells (top panel) and iPS cells (bottom panel) maintained in (dark gray line) or E8(TGF- β) (light gray line FIG. 3B shows H1ES cells grown in E8(TGF- β) and in TeSR^TMRNA of H1 cells maintained in TeSR or E8(TGF β) medium for three generations was analyzed by RNA-sequencing with an eluminar Genome Analyzer (Illumina Genome Analyzer) GAIIX (whole gene expression Correlation coefficient R ═ 0.954 (Spearman Correlation)).

Fig. 4A-F show iPS cell-derived under various defined conditions fig. 4A shows foreskin fibroblast proliferation in DF5 SFe-based medium supplemented with various Fibroblast Growth Factors (FGFs), compared to proliferation in FBS-containing medium fig. 4B shows fibroblast growth in different media supplemented with hydrocortisone fig. 4C shows expression of pluripotency markers OCT4 (left panel) and SSEA4 (right panel) fig. 4D shows selected gene expression of iPS cells derived from foreskin fibroblasts, hES cells, feeder cells (iPS cells (feeder cells)) and iPS cells derived in E8 medium (iPS cells (E8)) prior to RNA analysis, all cells except fibroblasts are maintained in E8 containing hydrocortisone, all cells are maintained in E8(β) medium fig. 4E shows whole human iPS cell-derived in E8(TGF 2) medium and TGF s cells derived in TGF 3 medium (TGF 730. TGF t) and TGF t 3 is whole human iPS cell-derived gene expression in E8 medium (TGF 8).

FIGS. 5A-C show the improvement for iPS cell-derived media, FIG. 5A shows foreskin (dark gray column) and PRPF8-2 mature fibroblast (light gray column) proliferation in DF5SFe media supplemented with TGF- β, hydrocortisone, TGF- β and hydrocortisone, or TGF- β and hydrocortisone but without FGF, FIG. 5B shows the effect of TGF- β and butyrate on foreskin fibroblast reprogramming.

FIGS. 6A-B show iPS cells derived from mature fibroblasts without secondary passaging FIG. 6A shows an example of a reprogramming protocol FIG. 6B shows expression of pluripotency markers OCT4 and SSEA4 as determined by flow cytometry analysis on an iPSC cell line maintained 20 passages in DMEM/F12 supplemented with insulin, transferrin, selenium, L-ascorbic acid, FGF2, and TGF- β or NODAL ("E8"), shaded peaks stained with OCT4 (left) and SSEA4 (right) specific antibodies, non-shaded peaks mouse IgG control antibodies.

FIGS. 7A-C show the human fibroblast reprogramming efficiency in different media. Figure 7A shows iPS cell clone number per 80,000 fibroblasts processed using mouse fibroblast feeder cells (MEFs) or reprogrammed in E8-based medium. To improve efficiency, 100 μ M sodium butyrate was added to both conditions. FIG. 7B is shown in TeSR^TMIn or on E8-based mediumFIG. 7C shows the effect of contact time with TGF- β and butyrate on the efficiency of reprogramming of foreskin fibroblasts under well-defined conditions FIG. 7C shows the reprogramming of fibroblasts in DMEM/F12 supplemented with insulin, transferrin, selenium, L-ascorbic acid and FGF 2(E8 without TGF- β) or in E8 (with or without 100. mu.M butyrate), the reprogramming efficiency under all conditions was examined 30 days after reprogramming<0.05。

Since the invention is susceptible to various modifications and alternative forms, exemplary embodiments are shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the description herein of exemplary embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

Exemplary description of embodiments

The present invention relates to the following observations of the inventors: certain media components that were thought necessary to culture pluripotent cells can be removed from the pluripotent cell culture media formulation for certain culturing purposes.

The term "pluripotent cell" as used herein refers to a cell that has the ability to differentiate into all three germ layer cells. Examples of pluripotent cells include embryonic stem cells and Induced Pluripotent Stem (iPS) cells. The term "iPS cell" as used herein refers to a cell that is genetically substantially identical to its respective differentiated, derived somatic cell and exhibits similar characteristics as higher potential cells, such as the ES cells described herein. The cells may be obtained by reprogramming of non-pluripotent cells such as multipotent cells or somatic cells.

The present invention relates to novel media that do not contain factors that are not essential to a particular culture target. Examples of culture targets include, but are not limited to: cell survival, passage, proliferation, pluripotency, cloning, and iPS cell derivation. The invention relates in particular to albumin-free media.

And (5) clarifying a little: "passaging" and "cloning" are different methods. "passaging" describes the process of dividing cells that have been cultured to a certain density in a culture vessel into clumps, which are then placed in a new culture vessel. These clumps may contain any number of cells, typically between 100 and 1,000 cells, which are susceptible to initiating growth in culture. In contrast, "cloning" refers to the creation of a clonal colony by growing a clone of human ES cells from a single individual ES cell. The term "cloning efficiency" as used herein refers to the number of individualized cells forming new cell colonies divided by the number of individualized cells plated in culture. The cloning efficiency varies greatly depending on the culture conditions. For example, under defined and non-heterologous conditions,the cloning efficiency of human ES cells above is low (i.e., less than about 0.1%), while the cloning efficiency of these cells cultured in fibroblast-conditioned media, while still low (i.e., less than about 2%), is high enough for the creation of a pure line ES cell colony.

The media described herein are substantially free of damaging, differentiating, and indeterminate factors found in most conventional pluripotent cell media.the disclosed media have been successfully used for various culture purposes, such as to support short-term pluripotent cell viability (e.g., 24 hours), short-term proliferation (e.g., 4-5 days), to maintain pluripotent cells over extended culture cycles (e.g., more than 25 passages over 3 months), and to derive iPS cells from adult fibroblasts with lentiviruses and episomal vectors.

A new simplest medium specifically tailored for certain cell culture purposes was developed. Various media components, such as salts, vitamins, glucose sources, minerals and amino acids, alone or in combination, were tested to determine their individual effects on viability, proliferation or pluripotency. A new survival assay was developed and used to determine which components are essential for the survival of pluripotent cells after dissociation (dissociation). The ability of the novel media to support proliferation and maintain pluripotency was tested. These media were also used for cloning analysis to determine how each medium affected individual cells and their cloning efficiency. Table 1 lists a complete list of the components of each of the novel media described herein (light and dark shaded areas indicate the presence of the component in the media, striated areas indicate interchangeable components, and colorless areas indicate the absence of the component in the media).

TABLE 1 Medium composition

Various media described herein can be prepared from the base component. Alternatively, one skilled in the art understands the advantageous efficacy of using a basal medium as a starting material to prepare the disclosed novel media. The term "basal medium" as used herein refers to a medium that supports the growth of certain unicellular organisms and cells without special media additives. The composition of typical basal media is known in the art and includes: salts, amino acids, vitamins and carbon sources (e.g. glucose). Other ingredients that do not alter the basic characteristics of the medium, but are otherwise desirable, such as the pH indicator phenol red, may also be included. For example, Dulbecco's Modified Eagle Medium (Dulbecco's Modified Eagle Medium): Nutrient Mixture (Nutrient Mixture) F-12(DMEM/F12) is a basal medium for preparing a suitable growth medium for mammalian cell culture. The complete composition of DMEM/F12 is tabulated in Table 2.

TABLE 2 DMEM F-12 Medium formulation (ATCC catalog No. 30-2006).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the methods and materials described herein are preferred.

In describing the embodiments and in the claims which follow, the following terminology will be used in accordance with the definitions set forth below.

The term "about" as used herein means within 5% of the stated concentration range or within 5% of the stated time interval.

The term "substantially serum-free" as used herein means that the culture medium does not contain serum or serum substitutes, or that the culture medium does not contain serum or serum substitutes substantially. For example, a substantially serum-free medium may comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% serum, wherein the culture capacity of the medium is still observed.

The term "defined medium" or "defined medium" as used herein means that the identity and amount of each medium component is known.

The term "culture medium consisting essentially of … …" as used herein means that the culture medium contains the specified ingredients and optionally other ingredients that do not materially affect its basic characteristics.

The term "effective amount" as used herein refers to an amount of an agent sufficient to elicit the effect of a particular cell as described herein.

The term "viability" as used herein refers to being in a viable state. Viable pluripotent cells adhere to the surface of the cell plate and are not stained with propidium iodide dye because the cell membrane is intact. Short term viability correlates with the first 24 hours after plating of cells in culture. Cells do not usually proliferate during this period.

The term "short term growth" as used herein refers to the proliferation of cells in culture for 4-5 days.

The term "prolonged growth" as used herein refers to growth for at least five generations. Typically, the test medium supports the ability of pluripotent cells to grow for more than twenty passages (approximately 2-3 months).

The term "long-term culture" as used herein refers to more than 15 generations (about two months in culture).

The term "pluripotency" as used herein refers to the ability of a cell to differentiate into all three germ layer cells.

The term "cloning" as used herein refers to the process of cell culture starting from a starting culture, which is ideally a single pluripotent cell or at least very few cells. The culture conditions that allow clonal culture of undifferentiated pluripotent cells may be the most severe of all those required in normal pluripotent cell culture and propagation.

The term "iPS cell-derived" as used herein refers to reprogramming a non-pluripotent cell to make it pluripotent.

The term "xeno-free" as used herein means that the cell culture conditions do not contain any cells or cell products other than the cultured cell species.

As used herein, the term "normoxic conditions" refers to conditions having about 20% oxygen.

The term "hypoxic conditions" as used herein refers to conditions having less than about 20% oxygen (e.g., about 5% oxygen).

The invention will be more fully understood by consideration of the following non-limiting examples.

Examples

Example 1: pluripotent cell survival assay.

Prior to cell addition, each well of a 12-well plate was filled with 500. mu.L of each test medium. Adherent pluripotent cells were dissociated with TrypLE (Invitrogen) for 5 minutes or until they were completely detached from the plate. An equal volume of medium was added to the culture to neutralize TrypLE. Cells were counted, washed and resuspended in fresh medium at a concentration of 300,000-1,000,000 cells/mL. About 100. mu.L of the cell solution was added to each well of the 12-well plate with 5% O at 37 ℃₂And 10% CO₂Incubating the cells under conditions (a). Cells were dissociated again with 0.4mL of TrypLE at different time points, and then TrypLE was neutralized with an equal volume of DMEM containing 10% FBS. The cells were counted by flow cytometry. 5000 pieces of Comtebrett beads (count bright beads) were added to each sample as an internal reference (each sample counted about 200 beads). All experiments were performed in triplicate.

Example 2: growth factors for survival and short term growth.

In addition to the growth factors present in the basal medium, TeSR medium also contains six growth factors, Fibroblast Growth Factor (FGF), transforming growth factor β (TGF- β), gamma-aminobutyric acid (GABA), pipecolic acid, lithium chloride (LiCl), and insulin (Table)1). Formulation containing all TeSR^TMBasal Nutrient Medium (NM) containing none of these six growth factors. About 2X 10 dissociation⁵H1ES cells were plated on Matrigel (Matrigel). Survival index was determined after 24 h. After dissociation, single NM failed to support cell survival. Cell survival by addition of insulin to NM and use of TeSR^TMSimilar, but not supported, cell growth was observed (fig. 1A). Addition of insulin (20. mu.g/ml) and FGF2(100ng/ml) supported cell survival and also caused cell growth within 96h, which is in contrast to the use of TeSR^TMThe results observed for the medium were comparable (FIG. 1B). Therefore, FGF and insulin supplemented NM support human ES cell culture. Twelve different basal nutrient media supplemented as described above were able to support cell survival and growth (fig. 1E).

Example 3: l-ascorbic acid supports short-term proliferation.

NM contains 11 nutrients, i.e. DMEM/F12, trace element B, trace element C, L-ascorbic acid, thiamine, selenium, L-glutamine, BSA, BME, sodium bicarbonate (NaHCO)₃) And transferrin (table 1). DMEM/F12 as basal medium and NaHCO₃For adjusting the pH. To test which other nutrients are necessary in the presence of insulin and FGF, DMEM/F12, NaHCO₃Insulin and FGF were added separately for each factor. None of the trophic factors was essential for survival after passaging, but L-ascorbic acid (64mg/L) was essential for cell proliferation after passaging (FIG. 1C). L-ascorbic acid, known as vitamin C, is the main antioxidant and a cofactor for several enzymes. Hydroxyproline can partially replace L-ascorbic acid. In DMEM/F12, NaHCO₃Human ES cells plated in L-ascorbic acid, insulin and FGF (restriction factor 5, "DF 5" table 1) maintained a morphology similar to human ES cells plated in TeSR.

Example 4: media components for extended passage.

DF5 supported only one passage growth of the cells. After the second passage, the cells attached poorly and eventually died (fig. 1C). Cells can be passaged in NM + insulin + FGF (data not shown) and DF12 (fig. 1D, table 1), indicating that the presence of one or more factors in NM + insulin + FGF and DF12 is important for prolonged passage. Various trophic factors present in NM were added individually to DF5 to determine their ability to support cell expansion after multiple passages. Addition of selenium alone was sufficient to support cell proliferation after multiple passages (fig. 1D, DF5+ selenium, "DF 5S", table 1).

DF5S was used to expand H1 cells. And in TeSR^TMHowever, H1 cells can grow for weeks (more than 15 passages) during which they retain the morphology of human ES cells and high levels of OCT4 expression (fig. 1E). H1 cells grown in DF5S supplemented with NODAL (100ng/mL) or TGF- β (2ng/mL) express significantly higher levels of mrna than H1 cells cultured in DF 5S. the pluripotency of the two tested human iPS cell lines was also supported by DF5S + NODAL as determined by the high expression of the pluripotency marker OCT 4. all cells grown in DF5S containing NODAL or TGF- β (hES cells and iPS cells) retain normal karyotype after long-term passage.

Example 5: hypoxia improves cell growth and cloning.

And in TeSR^TMH1 cells grew faster in DF5S medium than in the same cells (fig. 1C and 2A). To optimize the growth conditions of pluripotent cells, the cells are grown under conditions of varying osmotic pressure, pH, oxygen levels and CO₂Horizontal DF 5S. To increase the sensitivity of the assay, only 5,000 cells were seeded in each well and analyzed for survival (24h) and proliferation (124 h). With O₂And CO₂The greatest improvement was observed with varying levels. Common culture conditions use about 15% oxygen and 5% CO₂(O15C 5). Higher CO₂Usually resulting in a slight improvement in survival after 24 hours. Lower oxygen levels increased DF5S and TeSR^TMThe cell in (1) is grown. 15% oxygen and 10% CO₂(O15C10), and 5% oxygen and 10% CO₂(O5C10) increased cell survival (FIG. 2A). At a higher O₂Cells at levels (O15C5 and O15C10) did not grow well, but proliferated at lower oxygen levels (O5C10) (fig. 2A). And in TeSR^TMCells in DF5S grew faster than those in DF5S, but at 5% oxygen and 10% CO₂The growth was fastest (fig. 2A). With 5% O₂In contrast, further reduction of oxygen levels to 2% reduced cell growth.

For measuring different oxygen and CO₂Cloning efficiency at concentration 500 cells were seeded per well. Even at low oxygen, the cloning efficiency was too low: (<2%) so that the effect of different conditions on cloning cannot be determined. Increasing the cloning efficiency for oxygen and CO using HA100, a ROCK inhibitor known to increase the cloning efficiency₂And (5) testing the concentration. The best medium known for cloning, Conditioned Medium (CM), was used as a control. The addition of HA100 significantly promoted the cloning efficiency in CM under O5C10 and O15C5 conditions, and the cloning efficiency under O5C10 was higher than O15C5 (fig. 2B). Under both conditions, the cloning efficiency of cells in DF5S was comparable to that of cells in CM (fig. 2B).

Some of the subsequent examples used hypoxic conditions while the cells were kept at low density due to the positive effect of hypoxia on cell survival. However, when the cells were not cultured at low cell density, the experiment was performed under normoxic or hypoxic conditions (FIG. 2B).

Example 6: improved cloning efficiency of iPS cells.

To determine how DF5S affected the cloning efficiency, two iPS cell lines were grown in DF5S and plated at clonal density (approximately 500 cells per well of a 12-well plate) in the presence of HA 100. The cloning efficiency of iPS cells grown in DF5S was lower than that in DF5STeSR^TMOr cloning efficiency of iPS cells grown in CM (fig. 2C), indicating that factors increasing cloning efficiency are present in TeSR^TMMedium, but not DF 5S. To identify this factor, a single TeSR was added to DF5S alone^TMIngredients and their effect on cloning efficiency was tested. Addition of holotransferrin (DF5SFe) to DF5S caused and used TeSR^TMComparable cloning efficiency (fig. 2D). Transferrin also caused a significant improvement in the cloning efficiency of H1 cells in DF5S medium.

In DMEM/F12 supplemented with insulin, transferrin, selenium, L-ascorbic acid, FGF and TGF- β (or NODAL; "E8"), the ROCK inhibitors HA100 and Y27632, and myostatin, increased the cloning efficiency of H1 cells (fig. 2E), which was further increased by the addition of transferrin and by culture under hypoxic conditions (fig. 2F).

Example 7 NODAL and TGF- β support Long term preservation of H1 and iPS cell pluripotency in Albumin-free Medium And (4) maintaining.

As described in example 3, human pluripotent cells, such as H1, H9 and iPS cells, can be grown and passaged more than 15 times in DF5S, but are prone to differentiation, thus requiring additional care to maintain pluripotency in DF 5S. Due to the fact that in TeSR^TMCan maintain pluripotency more easily, and is added into DF5SFe for H1 cell growth^TMWherein said H1 cells were pre-cultured in DF5S and undifferentiated, thereby identifying factors that support long-term pluripotency. The cells were passaged approximately one day after they reached fusion, to promote cell differentiation, and Oct4 expression evaluated by flow cytometry was used as an indicator of pluripotency.

Human pluripotent cells grown in DF5SFe elongate and align with each other just prior to differentiation, resembling a "spindle" shape this phenotype is typically observed at the onset of neural differentiation, and is typically inhibited by the TGF- β/BMP pathwayRecombinant proteins of the road support the ability for long-term pluripotency. With TeSR^TMNODAL supplemented DF5SFe ("E8 (NODAL)") maintained high Oct4 expression at concentrations used DF5SFe ("E8 (TGF- β)") supplemented with TGF- β was used as TeSR^TMOct4 expression was maintained at low levels when used at concentrations (0.6ng/mL), but high Oct4 expression was maintained when used at higher concentrations (1 ng/mL).

The culturing experience of human ES cell lines (e.g. H1 and H9) includes exposure to different complex culture components, such as FBS, feeder cells and knockout serum replacement (knockouts serum replacers) exposure to these components can be confidently made dependent on these components, thus altering the cellular response to the simplified medium since the conditions of derivation are less complex, the culturing experience may play a minor role in iPS cells derived from reprogrammed body cells, therefore, the two original lentiviral iPS cell lines grown in DF5SFe (Yu et al, Science 318:1917(2007)) were tested for different factors, cells were transferred from MEF plates directly to the 5SFe medium for one passage and then transferred to different growth factor conditions, addition of TGF- β (2ng/mL) or NODAL (100ng/mL) (E8 (E- β) and "E2 (NODAL)" support TGF- β (2ng/mL) or addition of NODAL (100ng/mL) (these cells, respectively) to the pluripotent cells of mice injected with serial pluripotent markers such as E8 (E- β) and Tra 2 (NODAL) "support cells to develop a combined pluripotent stem cells expressing a pluripotent stem cell line expressing a pluripotent marker(s) that can also enter the mouse nucleus for a long term, i-expressing a pluripotent cell line, i-10-expressing a pluripotent marker (sci-64).

E8(TGF- β) and E8(NODAL) supported the pluripotency of each pluripotent cell line tested, i.e., two human ES cell lines (H1 and H9) and five iPSC cell lines had no evidence of differentiation at more than 25 passages (about 3 months) (FIG. 3.) H1ES cells grown in E8 medium and in TeSR^TMH1ES cells grown in the culture showed similar gene expression profiles compared to the cells (fig. 3B).

Example 8: iPS cells differentiated in albumin-free medium.

Available reprogramming schemes include: the cells were incubated in FBS for the first few days after viral transduction or electroporation, and then transferred to UM100 (U.S. patent No. 7,439,064, incorporated herein by reference in its entirety) or CM. The ability of the simplified medium as described in the examples above to support reprogramming was tested. Somatic cells from ES can be efficiently reprogrammed in DF5S medium using either lentiviral or episomal vectors and cultured with or without FBS-containing medium for the first two days. However, DF5S does not support reprogramming of primary foreskin cells using Nanog, Oct4, Sox2 and Lin 28. DF5SFe supports reprogramming of foreskin and mature cells on Matrigel (Matrigel) or MEF using modified lentivirus treatment when the cells were initially incubated in FBS-containing medium (Ebert et al, Nature 457(7227): 277-. However, DF5SFe is as effective as CM in supporting reprogramming, initial exposure to FBS indicating the importance of reprogramming.

Foreskin cells grew significantly slower in DF5SFe than in FBS medium. To determine the growth factors that can help the growth of primary foreskin cells, a single growth factor contained in FBS was tested. There are several members of the FGF family of growth factors, one or more of which are commonly used in fibroblast culture. DF5SFe contained 100ng/mL zebrafish recombinant FGF 2. Each FGF family member was tested for its ability to support the growth of foreskin cells. Foreskin cells were aliquoted into wells of culture plates and incubated for 24 hours in DF5SFe without FGF. A single FGF-type was added at a concentration of 100ng/mL and incubated for 96 h. FGF1, zFGF2, FGF4, FGF6, and FGF9 were most effective at supporting foreskin cell growth, but none supported cell growth as well as FBS-containing media (fig. 4A). To identify whether the growth of foreskin cells promoted by growth factors that are members of the non-FGF family was comparable to that seen with FBS, several known fibroblast growth promoting factors were tested. The addition of hydrocortisone (fig. 4B), its derivatives and dexamethasone to DF5SFe to replace FBS significantly promoted cell growth. DF5SFe + hydrocortisone ("DF 5 SFeC") also improved the cloning efficiency of iPS cells.

To determine whether DF 5S-based media could be used for virus-free iPS cell derivation, foreskin cells were reprogrammed under hypoxic conditions (O5C10) using virus-free episomal vectors (as described in Yu et al, Science 324:797(2009), which is incorporated herein by reference in its entirety). Plasmid compositions No. 4 (pEP4EP2SCK2MEN2L and pEP4EO2SET2K, table 3), No. 6 (pEP4EO2SEN2L, pEP4EO2SET2K and pEP4EO2SEM2K, table 3) and No. 19 (pEP4EO2SEN2K, pEP4EO2SET2K and pCEP4-M2L, table 3) were used and passed from 10 after the second passage⁶2 clones were isolated per cell.

TABLE 3 reprogramming vector Components and vector compositions

Plasmid compositions No. 6 and No. 19 were used for the reprogramming. To facilitate entry of the plasmid into the nucleus, ENBAmRNA was electroporated together with the plasmid DNA. Approximately one million cells were transferred to DF5SFeC in two 6-well plates and left for 5 days. Then, the medium was changed to DF5SFe, and left for another 18 to 25 days. At different time points, cells in some of the wells were passaged a second time at a 1:6 ratio. Plasmid composition No. 19 produced more colonies than plasmid composition No. 6, but most of them were not similar to the typical human ES cell morphology. Approximately 25 days later, human ES cell-like colonies appeared on the initial plates of both plasmid compositions, an estimated 24 reprogrammed cells per million foreskin cells using plasmid composition No. 19, and an estimated 8 reprogrammed cells per million foreskin cells using plasmid composition No. 6. After the second passage, the number of human ES cell-like colonies on the plates increased significantly, with an estimated > 500/one million foreskin cells for each plasmid composition. The increase in iPS cell colonies on the second passage plate was probably due to iPS cell division on the initial culture plate. In some cases, the initial plates did not have any colonies with morphology similar to typical human ES cells, but many iPS cells appeared after the second passage, indicating that some iPS cells may not be recognized because of being mixed with somatic cells.

Differentiation began only after two passages of cells in iPS cell colonies derived in DF5 SFe. Six iPS cell colonies were picked from the initial plates and transferred directly into DF5SFeN containing Nodal (E8 (Nodal)). These cells were able to remain for more than 15 passages in E8(Nodal), retain their ES cell-like morphology and use TeSR^TMThe observed results were similar. The cells had a normal karyotype, expressed Oct4 and SSEA4 (fig. 4C), and formed teratomas 5-7 weeks after injection into SCID mice.

Foreskin fibroblasts were also reprogrammed in E8 medium. The whole gene expression of iPS cells derived in E8 medium was similar to that of H1 cells (fig. 4D and E) or iPS cells derived on feeder cells (fig. 4D and F). Both pluripotency markers in ES and iPS cells were highly expressed, while fibroblast-specific marker genes were not expressed (fig. 4D). iPS cells can also be derived in E8 medium using various strategies, for example, using lentiviruses or episomal vectors.

Example 9: iPS cells were derived from patient cells in albumin-free medium.

To determine whether cells from an adult human donor could be reprogrammed in simplified media using virus-free episomal vectors, two million cells of the patient cell line OAT or PRPT8 were electroporated with plasmid composition No. 4 or No. 6 along with EBNA mRNA and transferred to two 10cm plates. To maximize reprogramming, FBS-containing medium was used on the first 6 days. Cells were maintained at O15C5 to meet the maintenance of normal mature cells. Then, the medium was replaced with DF5SFe, and the medium was left for 14 to 21 days. Cells on one plate were passaged at different time points at a ratio of 1: 2. Plasmid composition No. 6 produced more colonies (about 5 per million cells) than plasmid composition No. 4, but the morphology of most of the cells was not similar to that of typical human ES cells. After a period of about 22 days, the composition,human ES cell-like colonies appeared on the initial plate of plasmid composition No. 4. More human ES cell-like colonies appeared on the second passage plate using plasmid composition No. 6, with an estimated approximately 40 colonies per million cells. No iPS cells were produced using plasmid composition No. 4. On the initial culture plate iPS cell colonies appeared among other densely living cells, but were unable to grow beyond their boundaries. However, colonies on the secondary culture plate are enlarged to a large size suitable for colony isolation. Picking the colonies and transferring them directly to the TeSR^TM32 picked colonies survived and showed ES cell morphology. Genetic analysis confirmed that these colonies were derived from the OAT cell line and showed normal karyotype.

To improve the reprogramming efficiency of mature cells, TGF- β was added to the reprogramming media iPS clones did not increase significantly, but the total number of colonies increased significantly when TGF- β was removed from the media when hydrocortisone was removed, indicating that TGF- β maintained reprogramming within the first few days of the process.

Many seemingly non-iPS clones were able to produce iPS clones after secondary passage, indicating that iPS cell derivation may be inhibited by surrounding cells.several agents were tested for their ability to overcome this effect.

Using TGF- β and butyrate, successful reprogramming of somatic cells from mature individuals using an episomal vector system under fully defined conditions the efficiency of deriving iPS cells from three independent mature somatic cell lines (OAT, GRC M1-29, and PRPF8-2) is as followsFrom 1X 10⁶1-100 iPS cells are derived from one PRPF8-2 cell, and 1 iPS cell is derived from 100,000 GRC 1-29 cells.

Example 10: iPS cells were derived from mature individuals under well-defined conditions.

Biopsies were taken from the skin of male adult donors, washed several times with Hank's Balanced Salt Solution (HBSS) containing antibiotics and antifungal agents, and incubated overnight at 4 ℃ in 2mL of 0.25% trypsin/EDTA (table 4) or trypte selection (tryptleselect). The samples were rinsed three times, using trypsin inhibitors after the second rinse (table 4). The dermis and epidermis were separated using sterile forceps. The dermis was cut into small pieces and incubated in 0.75 mL enzyme solution containing the defined enzyme (table 4) at room temperature (12-well or 24-well plates) for 3 hours. After about 35 minutes, the tissue structure begins to disintegrate. An equal volume of medium containing 10 μ g/mL polyvinylpyrrolidone (PVP) was added and the tissue mechanically disintegrated by pipetting about 10 times. The sample was centrifuged at 400g for 10 min at room temperature and washed twice with fresh medium/PVP. The supernatant was discarded, the pellet was resuspended in 3mL of complete medium, and 1mL of cell suspension was transferred to the wells of a 6-well plate coated with 3. mu.g/well vitronectin. At 5% CO₂The plates were incubated at 37 ℃ and the medium was changed daily. When the medium is changed, fibroblasts attach to the culture plate, while non-adherent cells and debris are removed.

TABLE 4 reagents and methods for sample digestion

After 20 days, the reprogramming plasmid was mediated into the fibroblasts by electroporation. Within the next 25 days, multiple iPS colonies appeared, which were picked for further analysis. Reprogramming efficiency was about 10 for 1 million electroporated fibroblasts without secondary passage. iPS cells were further passaged to isolate vector-free cell lines.

Example 11: derivation from non-secondarily passaged adult individuals to iPS cells in albumin-free medium.

Mature fibroblasts were reprogrammed in E8 (DMEM/F12 supplemented with insulin, transferrin, selenium, L-ascorbic acid, FGF2 and TGF- β (or NODAL)) following the overall method shown in figure 6A. the reprogrammed iPS cell line that maintained more than 20 passages in E8 consistently expressed the pluripotency markers OCT4 and SSEA4 (figure 6B).

With mouse fibroblast feeder cells (MEF) (FIG. 7A) or TeSR^TMButyrate (100 μ M) further increased reprogramming efficiency in the presence of TGF- β (E8) or in the absence of TGF- β (E8 without TGF- β, i.e., DF5SFe) (fig. 7C).

Example 12: cryopreservation of pluripotent stem cells in albumin-free medium.

Pluripotent cells were cultured in E8 medium in 6-well plates essentially as described above. The medium was aspirated from each well and the cells were washed twice with 1.0mL EDTA/PBS (0.5mM EDTA in PBS, osmolality 340). The cells were then incubated in EDTA/PBS for 5 minutes at 37 ℃. PBS/EDTA was removed and the cells were rinsed rapidly with 1mL E8 medium. The cells were then resuspended in equal volumes of 20% Dimethylsulfoxide (DMSO) and E8 medium (final concentration: 10% DMSO in E8 medium), aliquoted into cryotubes, and plated with CRYOBOX^TMFreezing at-80 deg.C. The cells were then transferred to a liquid nitrogen tank.

The invention has been described in connection with what is presently considered to be the most practical and preferred embodiment. However, the present invention is provided by way of illustration and is not meant to be limited to the disclosed embodiments. Accordingly, those skilled in the art will recognize that the invention is intended to cover all modifications and alternative arrangements included within the spirit and scope of the invention as set forth in the appended claims.

Claims (26)

1. A fully defined xeno-free albumin-free medium consisting of water, salts, amino acids, vitamins, glucose, insulin, FGF, selenium, transferrin, and one of TGF- β and NODAL, each in an amount sufficient to support proliferation of human pluripotent stem cells.

2. A method for culturing human induced pluripotent stem cells, the method comprising the steps of:

placing the human induced pluripotent stem cells on a substrate; and

contacting said cells with a well-defined heterologous-free albumin-free medium consisting of:

water, salt, amino acids, vitamins, glucose, insulin, FGF, selenium, transferrin, and one of TGF- β and NODAL, each in an amount sufficient to support proliferation of human pluripotent stem cells.

3. The method of claim 2, wherein the substrate comprises laminin.

4. The method of claim 2, wherein the matrix comprises vitronectin.

5. The method of claim 2, wherein the cells are contacted with the culture medium under hypoxic conditions.

6. The method of claim 2, wherein the human induced pluripotent stem cells are produced from adult somatic cells.

7. A method for cloning human pluripotent stem cells, the method comprising the steps of:

human pluripotent stem cells produced from human somatic cells are plated at clonal density in a well-defined xeno-free albumin-free medium consisting of water, salts, amino acids, vitamins, glucose, insulin, FGF, selenium, transferrin, and either TGF- β or NODAL, each in an amount sufficient to support human pluripotent stem cell cloning.

8. The method of claim 8, wherein the medium further comprises a ROCK inhibitor.

9. The method of claim 9, wherein said ROCK inhibitor is selected from the group consisting of HA100 and Y27632.

10. The method of claim 8, wherein the culture medium further comprises myosin statin.

11. A method of cryopreserving human induced pluripotent stem cells, the method comprising the steps of:

freezing human induced pluripotent stem cells in a well-defined xeno-free albumin-free medium consisting of water, salts, amino acids, vitamins, glucose, insulin, FGF, selenium, transferrin, and one of TGF- β and NODAL, each in an amount sufficient to support human pluripotent stem cell proliferation.

12. A method for deriving human Induced Pluripotent Stem (iPS) cells under defined conditions, the method comprising the steps of:

reprogramming human cells in a fully defined xeno-free albumin-free medium consisting of water, salts, amino acids, vitamins, glucose, insulin, FGF, selenium, transferrin, and one of TGF- β and NODAL, each in an amount sufficient to support reprogramming of cells to derive human iPS cells.

13. The method of claim 13, wherein the reprogramming step comprises exposing the cells to TGF- β 5 for 5-10 days.

14. The method of claim 13, further comprising the steps of:

removing TGF- β after 5-10 days, and

contacting the cell with a culture medium consisting essentially of:

water, salt, amino acids, vitamins, a carbon source, insulin, FGF, selenium and transferrin.

15. The method of claim 13, wherein the reprogramming step comprises contacting the cell with hydrocortisone.

16. The method of claim 13, wherein the reprogramming step comprises contacting the cells with butyrate.

17. The method of claim 13, wherein the iPS cells are derived under xeno-free conditions.

18. The method of claim 13, wherein the somatic cell is a mature somatic cell.

19. The method of claim 13, wherein the reprogramming step comprises the use of a viral vector.

20. The method of claim 13, wherein the reprogramming step comprises using an episomal vector.

21. A method for culturing human pluripotent stem cells, the method comprising the steps of:

placing human pluripotent stem cells on a substrate; and

contacting said cells with a well-defined heterologous-free albumin-free medium consisting of:

water, salt, amino acids, vitamins, a carbon source, insulin, FGF, selenium, transferrin, and one of TGF- β and NODAL, each in an amount sufficient to support long-term culture of human pluripotent stem cells.

22. The fully defined, heterologous-free albumin-free growth medium of claim 1, wherein the medium comprises TGF- β.

23. The fully defined, xeno-free, albumin-free growth medium of claim 1, wherein the medium comprises NODAL.

24. The fully defined, heterologous-free, albumin-free growth medium of claim 1, further comprising a ROCK inhibitor.

25. The fully defined heterologous-free albumin-free growth medium of claim 24, wherein said ROCK inhibitor is selected from the group consisting of: HA100 and Y27632.

26. The fully defined, heterologous-free, albumin-free growth medium of claim 1, wherein the medium further comprises myosin statin.