ES2800973T3 - Primate embryonic stem cell culture - Google Patents

Abstract

A culture medium comprising albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, at least one insulin or insulin substitute, the culture medium free of animal serum and containing at least 100 ng / ml of a fibroblast growth factor selected from the group consisting of FGF4, FGF9, FGF17 and FGF18, the suitable medium for culturing primate embryonic stem cells in the absence of serum and in the absence of feeder cells and also in the absence of cell-exposed medium feeder, where at least 90% of the stem cells in the culture medium express Oct-4, SSEA4 or Tral-60.

Description

DESCRIPTION

Primate embryonic stem cell culture

BACKGROUND OF THE INVENTION

The present invention relates to culture media and methods for culturing primate embryonic stem cell cultures.

Pluripotent embryonic stem cells from primates (eg, monkey and human) have been derived from embryos prior to implantation. See, for example, US Patent 5,843,780 and J. Thomson et al., 282 Science 1145-1147 (1998). Despite prolonged culture, these cells stably maintain developmental potential to form advanced derivatives of the three embryonic germ layers.

ES cell lines from primates (particularly humans) have widespread utility in relation to human developmental biology, drug discovery, pharmacological testing, and transplantation medicine. For example, current knowledge of the post-implantation human embryo is largely based on a limited number of static histological sections. Due to ethical considerations, the underlying mechanisms that control developmental decisions of the early human embryo remain essentially unexplored.

Although the mouse is the mainstay of experimental mammalian developmental biology, and although many of the fundamental mechanisms that control development are preserved between mice and humans, there are significant differences between early development of the mouse and the human. Therefore, ES cells from primates / humans should provide important new insights into their differentiation and function.

Differentiated derivatives of ES cells from primates could be used to identify genetic targets for new drugs, to test the toxicity or teratogenicity of new compounds, and could be used in transplantation to replace cell populations in disease. Possible conditions that could be treated by ES cell-derived cell transplantation include Parkinson's disease, heart attacks, juvenile-onset diabetes mellitus, and leukemia. See, for example, J. Rossant et al. 17 Nature Biotechnology 23-4 (1999) and J. Gearhart, 282 Science 1061-2 (1998).

Long-term proliferative capacity, developmental potential after prolonged culture, and karyotypic stability are key characteristics regarding the utility of primate embryonic stem cell cultures. Cultures of such cells (especially in fibroblast feeder layers) have typically been supplemented with animal serum (especially fetal bovine serum) to allow the desired proliferation during such culture.

For example, various culture conditions were described in US Patents 5,453,357, 5,670,372, and 5,690,296, including some that use a basic type of fibroblast growth factor in conjunction with animal serum. Unfortunately, serum tends to have variable properties from batch to batch, which affects the characteristics of the culture.

In WO 98/30679 the provision of a serum-free supplement was discussed as a replacement for animal serum to support the growth of certain embryonic stem cells in culture. Serum replacement included albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors and one or more trace elements. It was observed that this replacement could also be supplemented with leukemia inhibitory factor, steel factor or ciliary neurotrophic factor. Unfortunately, in the context of primate embryonic stem cell cultures (especially those grown in fibroblast feeder layers), these culture media were not satisfactory.

In the context of nutrient serum culture media (eg, fetal bovine serum), WO 99/20741 discusses the benefit of using various growth factors such as bFGF in primate stem cell culture. However, culture media without nutrient serum are not described.

Growth media for bone marrow hematopoietic cells and stromal cells are disclosed in US Patent 5,405,772. It is suggested to use fibroblast growth factor in serum deprived medium for this purpose. However, the conditions for the growth of primate embryonic stem cells are not described.

The first human embryonic stem cell cultures were grown using a fibroblast feeder cell layer, which has the property of allowing human embryonic stem cells to proliferate without remaining differentiated. Later, it was discovered that it is sufficient to expose the culture medium to feeder cells, to create what is called conditioned medium, which had the same property as using the feeder cells directly. Without the use of feeder cells or conditioned medium, cultured human embryonic stem cells could not be maintained in an undifferentiated state. Since the use of feeder cells, or even exposure of the medium to feeder cells, risks contamination of the culture with unwanted material, it is desirable to avoid the use of feeder cells and conditioned medium. Medium that has not been exposed to feeder cells is referred to herein as unconditioned medium.

Therefore, it can be seen that there is still a need for techniques to stably grow primate embryonic stem cells without the requirement for the use of animal serum.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention provides a culture medium comprising albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, at least one insulin or insulin substitute, the culture medium free from animal serum and containing al less 100 ng / ml of a fibroblast growth factor selected from the group consisting of FGF4, FGF9, FGF17 and FGF18, the medium suitable for culturing primate embryonic stem cells in the absence of serum and in the absence of feeder cells and also in the absence of medium exposed to feeder cells, where at least 90% of the stem cells in the culture medium express Oct-4, SSEA4 or Tral-60. In another aspect, the invention provides a method for culturing primate embryonic stem cells, comprising: culturing the stem cells in a mammalian fetal serum-free culture and in a stem cell culture medium that includes amino acids, vitamins, salts, minerals, transferrin or a transferrin substitute, insulin or an insulin substitute, albumin, and a minimum concentration of fibroblast growth factor (FGF) of 100 ng / ml, where the FGF is selected from the group consisting of FGF4 , FGF9, FGF17 and FGF18, and where the medium is capable of supporting the culture and proliferation of human undifferentiated proliferative euploid pluripotent stem cells, without feeder cells or exposure of the medium to feeder cells, and where at least 90% of the cells mother in culture express Oct-4, SSEA4 or Tral-60. In a further aspect, the invention provides a method for culturing primate embryonic stem cells in defined medium without serum and without feeder cells, the method comprising: culturing the stem cells in a culture medium containing albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, at least one insulin or an insulin substitute, the culture medium free from fetal mammalian serum and containing at least 100 ng / ml of a fibroblast growth factor selected from the group that consists of FGF4, FGF9, FGF17 and FGF18, supporting the medium that the proliferation of stem cells in an undifferentiated state without feeder cells or conditioned medium, and where at least 90% of the stem cells in the culture express Oct-4, SSEA4 or Tral-60.

Fibroblast growth factors are essential molecules for the development of mammals. There are currently more than twenty known fibroblast growth factor ligands and five fibroblast growth factor receptors signaling for them (and their spliced variants). See generally D. Ornitz et al., 25 J. Biol. Chem. 15292-7 (1996); US Patent 5,453,357. Slight variations in these factors are expected between species and therefore the term fibroblast growth factor is not limited by species. However, we prefer to use human fibroblast growth factors. These compounds are readily available in quantity from Gibco BRL-Life Technologies and others.

It should be noted that, for the purposes of this patent, the culture may still be free of the specified serum even though a discrete component (eg, bovine serum albumin) has been isolated from the serum and is then supplied in a exogenous. The point is that when the serum itself is added, problems of variability arise. However, when one or more well-defined purified components of said serum are added, they do not vary.

Preferably, the primate embryonic stem cells that are cultured are human embryonic stem cells that are true ES cell lines in which: (i) they are capable of indefinite proliferation in vitro in an undifferentiated state; (ii) they are able to differentiate into derivatives of the three embryonic germ layers (endoderm, mesoderm, and ectoderm) even after prolonged cultivation; and (iii) they maintain a normal karyotype (they are euploid) throughout the prolonged culture. Therefore, these cells are called pluripotent.

Culture allows embryonic stem cells to stably proliferate in culture for more than one month (preferably six months; even more preferably twelve months) while maintaining the potential of stem cells to differentiate into endoderm tissue derivatives , mesoderm and ectoderm, and while maintaining the karyotype of the stem cells.

Another method for culturing primate embryonic stem cells is also described herein. The stem cells are cultured in a culture substantially free of fetal mammalian serum (preferably also substantially free of any animal serum) and in the presence of a growth factor capable of activating a fibroblast growth factor signaling receptor, where the factor growth is supplied from a source other than just a fibroblast feeder layer. While the growth factor is preferably a fibroblast growth factor, it could also be other materials such as certain small synthetic peptides (eg, produced by recombinant DNA variants or mutants) designed to activate fibroblast growth factor receptors. See generally T. Yamaguchi et al., 152 Dev. Biol. 75-88 (1992) (signaling receptors).

Also described herein is a culture system for culturing primate embryonic stem cells. It has a human basic fibroblast growth factor supplied by other than just the fibroblast feeder layer. The culture system is substantially free of animal serum.

Also described herein are cell lines (preferably cloned cell lines) derived using the above procedure. "Derivatives" is used in its broadest sense to encompass directly or indirectly derived lines.

In this way, variability in results due to differences in animal serum lots is avoided. Furthermore, it has been found that avoiding the use of animal serum while using fibroblast growth factor can increase the efficiency of cloning.

Therefore, it is an advantage of the present invention to provide culture conditions for primate embryonic stem cell lines where conditions are less variable and allow more efficient cloning. Other advantages of the present invention will be apparent from a study of the specification and claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In some of the following experiments, one of the inventors here used the methods and culture systems described herein to grow human ES cell lines without adding serum to the culture medium. Two clonally derived human ES cell lines proliferated for more than eight months after clonal derivation and maintained the ability to differentiate into advanced derivatives of the three embryonic germ layers when cultured in medium without serum constituent.

In another of the experiments discussed below, the addition of relatively large amounts of a human fibroblast growth factor (FGF) has now been shown to aid in the cultivation and growth of human embryonic stem cells, even in the absence of serum and feeder cells. This allows the cultivation of stem cells that have never been exposed to either animal cells or to media in which animal cells have been cultured. These stem cell culture conditions (ie, no feeder cells and no conditioned medium) are referred to herein as feeder independent. Previous culture conditions have been described, based on the use of feeder cell conditioned medium, which are described as feeder free. However, the use of conditioned medium does not resolve the dependency on the use of feeder cells, which must still be used to condition the medium. The techniques described herein allow indefinite and feeder-independent culture of human embryonic stem cells that have normal karyotype and with respect to which the stem cells remain undifferentiated.

Techniques for the initial derivation, culture, and characterization of the H9 human ES cell line (described herein for reference only) were described in J. Thomson et al., 282 Science 1145-1147 (1998). The experiments described below are for reference only and were performed with this and other cell lines, but the processes and results are independent of the particular ES cell lines.

It is described herein that the addition of fibroblast growth factor (FGF) helps in the cultivation and cloning of human ES cells. The addition of FGF is important in two different ways. First, the addition of FGF at moderate levels (eg, 4 ng / ml) allows the cultivation of undifferentiated human ES cells in serum-free medium. At this level, the rate of differentiation of stem cells slows down, compared to lower levels of FGF, but the cells will eventually differentiate. Second, the addition of FGF at higher levels makes the culture conditions of the feeder medium independent, since feeder cells are not required to indefinitely maintain pluripotency of undifferentiated euploid human ES cells in culture.

This first phenomenon is believed to be activated by the action of FGF interacting with the FGF receptors in the cells. human ES cells. To avoid the use of serum, it is not particularly critical which of the many known FGF variants are used in culture. Basic FGF or bFGF, also known as FGF2, is commonly used here, but that's only because bFGF is one of the readily commercially available members of the FGF family of factors. More than twenty different members of the FGF family have been identified, and are known as FGF-1 through FGF-27. While the FGF concentration here is given in amounts of bFGF, it should be understood that this is intended to quantify the amount of stimulation of FGF receptors and that the FGF concentration may have to be adjusted, up or down, for other members of the FGF. the FGF family. For bFGF, the preferred concentration of FGF in ES cell medium is in the range of about 0.1 to about 1000 ng / ml, with concentrations in a range in excess of about 4 ng / ml being useful to avoid the need for serum. in the middle.

Surprisingly, it has been found that for the second attribute of FGF in a human ES cell medium, the selection of the FGF variant is of some importance. For this purpose, it has been found that when the concentration of bFGF is about 100 ng / ml, this condition is sufficient to avoid the need for both serum and feeder cells, making the culture feeder independent. For this purpose, it has been found that the members of the FGF family FGF2 (bFGF), FGF4, FGF9, FGF17 and FGF18 are sufficient at 100 ng / ml of culture each to make the human ES cell culture feeder independent. . By contrast, it has been found that members of the FGF FGF1 family (acidic FGF), FGF1p, FGF3, FGF - 5, FGF6, FGF7, FGF8, FGF10, FGF16, F ^g F19 and FGF 20 are not sufficient to 100 ng / ml to support the independence of the feeder. We believe, but do not have current data, that results using these forms of FGF are not the result of concentration and that higher concentrations of the particular FGF would also not be successful in supporting feeder independence. For FGF9, our data suggest that at this level (100 ng / ml) FGF9 supports human ES cell culture, but the data have been slightly more equivocal.

The exact minimum amount of effective FGF variants that will be sufficient to support human ES cells as independent feeders in culture is not precisely known at this time, but can be determined by empirical testing. It is known that for FGF2, 4 ng / ml added to the medium alone is insufficient for the indefinite maintenance of undifferentiated euploid human ES cells in culture, while 100 ng / ml of FGF2 alone in the medium is sufficient. While ES cells grown in unconditioned medium containing as little as 4 ng / ml will remain undifferentiated for some time, and perhaps one or two passages, the cells will possibly begin to differentiate. In our hands, the ability of an ES cell culture medium to remain indefinitely undifferentiated and euploid is demonstrated when the cells are grown for at least six passages while remaining proliferating, undifferentiated, euploid and maintaining the characteristic morphology of human ES cells. . As used herein, a maintenance concentration of an FGF is the concentration of that FGF necessary to support the maintenance of human ES cells in an undifferentiated, euploid, and proliferative state for at least six passages. For FGF2, the minimum maintenance concentration is between 4 ng / ml and 100 ng / ml and the exact minimum maintenance concentration can be determined using the following protocols to interpolate those amounts. For each of the other effective FGFs, eg, FGF4, FGF9, FGF17, and FGF18, the corresponding minimum maintenance concentration for each FGF can be determined by similar assays.

Human ES cell cultures in the defined human ES cell media described below in the reference examples can be grown indefinitely in the total absence of fibroblast feeder cells and without conditioned media while remaining euploid. ES cells are thus truly feeder independent. Human ES cells retain all the characteristics of human ES cells, including characteristic morphology (small and compact with indistinct cell membranes), proliferation, and the ability to differentiate into many, if not all, cell types in the human body. . Human ES cells will also retain the characteristic that they can form the three primordial cell layers when injected into immunosuppressed mice. In particular, ES cells retain the ability to differentiate into ectoderm, mesoderm, and endoderm. ES cells still display markers indicative of ES cell status, such as the expression of the nuclear transcription factor Oct4, which is associated with pluripotency. Throughout the process and at the end, human ES cells retain normal karyotypes.

REFERENCE EXAMPLES

In the first experiments described herein, human ES cells were plated on irradiated mouse embryonic fibroblasts (gray gamma irradiation 35). The culture medium for the present work consisted of 80% Dulbeco's modified Eagle's medium "KnockOut" (DMEM) (Gibco BRL, Rockville, MD), 1 mM L-Glutamine, 0.1 mM p-mercap-toethanol and 1% non-essential amino acid stores (Gibco BRL, Rockville, MD), supplemented with 20% fetal bovine serum (HyClone, Logan, UT) or 20% KnockOut SR, a serum-free substitute originally optimized for mouse ES cells (Gibco BRL, Rockville, MD). The components of KnockOut SR are those described for serum substitutes in WO 98/30679.

In alternative experiments, the medium was supplemented with serum or the previously mentioned KnockOut SR serum substitute, and with or without recombinant human basic fibroblast growth factor (bFGF, 4 ng / ml). The preferred concentration range of bFGF in the culture was between 0.1 ng / ml and 500 ng / ml.

To determine cloning efficiency under varying culture conditions, H-9 cultures were dissociated into single cells for 7 minutes with 0.05% trypsin / 0.25% EDTA, washed by centrifugation, and plated. on mitotically inactivated mouse embryonic fibroblasts (105 ES cells per well of a 6-well plate). To confirm growth from individual cells for derivation of clonal ES cell lines, individual cells were selected by direct observation with a stereomicroscope and transferred by micropipette to individual wells of a 96-well plate containing embryonic fibroblast feeders. of mouse with medium containing 20% serum substitute and 4 ng / ml bFGF.

Clones were expanded by routine passage every 5-7 days with 1 mg / ml type IV collagenase (Gibco BRL, Rockville, MD). Six months after shunt, H9 cells showed a normal XX karyotype by standard G-banding techniques (20 chromosomal extensions analyzed). However, seven months after shunt, in a single karyotype preparation, the 16/20 chromosome tracts had a normal XX karyotype, but the 4/20 tracts demonstrated random abnormalities, including one with a translocation to the short arm of chromosome 13 , one with an inverted chromosome 20, one with a translocation to short arm number 4, and another with multiple fragmentation. Subsequently, at 8, 10 and 12.75 months after shunt, the H9 cells presented normal karyotypes in the 20 chromosomal extensions examined.

The cloning efficiency of human ES cells under previously described culture conditions including animal serum was found to be poor (regardless of the presence or absence of bFGF). It was also observed that in the absence of animal serum the cloning efficiency increased, and increased even more with bFGF. It has now been established that the addition of FGF facilitated the cultivation of human ES cells in general and is of particular help in facilitating the cloning of human ES cultures.

The data expressed below are the total number of colonies resulting from 105 individualized ES cells treated, / - standard error of the mean (percent colony cloning efficiency). With 20% fetal serum and without bFGF there was a result of 240 / - 28. With 20% serum and bFGF (at 4 ng / ml) the result was approximately the same, 260 / - 12. In the absence of serum (presence 20% serum substitute) the result without bFGF was 633 / - 43 and the result with bFGF was 826 / - 61. Thus, serum negatively affected cloning efficiency, and the presence of bFGF in the absence of serum had an additional synergistic benefit in terms of cloning efficiency.

Long-term culturing of human ES cells in the presence of serum does not require the addition of exogenously supplied bFGF, and (as noted above) the addition of bFGF to serum-containing medium does not significantly increase the cloning efficiency of ES cells. human. However, in serum-free medium, bFGF increased the initial cloning efficiency of human ES cells.

Furthermore, the supply of exogenous bFGF has been found to be very important for the continued undifferentiated proliferation of primate embryonic stem cells in the absence of animal serum. In serum-free medium lacking exogenous bFGF, human ES cells differentiated uniformly over two weeks of culture. The addition of other factors such as LIF (in the absence of bFGF) did not prevent differentiation.

The perceived results are particularly applicable to clonal lines. In this regard, clones for expansion were selected by placing cells individually in wells of a 96-well plate under direct microscopic observation. Of 192 H-9 cells treated in wells of 96-well plates, two clones (H-9.1 and H-9.2) were successfully expanded. Both clones were subsequently grown continuously in media supplemented with serum substitute and bFGF.

H9.1 and H9.2 cells maintained a normal XX karyotype even after more than 8 months of continuous post-cloning culture. Clones H-9.1 and H-9.2 retained the potential to derive all three embryonic germ layers even after long-term culture in serum-free medium. After 6 months of culture, clones H9.1 and H9.2 were confirmed to have normal karyotypes and were then injected into SCID-beige mice.

Both H9.1 and H9.2 cells formed teratomas containing derivatives of all three embryonic germ layers, including the embryonic kidney of the intestinal epithelium (endoderm), skeletal muscle, smooth muscle, bone, cartilage (mesoderm), and neural tissue (ectoderm). The range of differentiation observed within the teratomas of the high-pass H9.1 and H9.2 cells was comparable to that observed in the teratomas formed by the parental low-pass H9 cells.

It should be appreciated from the above description that, while animal serum is growth-friendly, it is a complex mixture that may contain compounds that are beneficial and detrimental to human ES cell culture. In addition, different batches of serum vary widely in their ability to support vigorous and undifferentiated proliferation of human ES cells. Replacing the serum with a clearly defined component reduces the variability of results associated with this variation of the serum lot, and should allow more carefully defined differentiation studies.

Furthermore, the lower cloning efficiency in serum-containing medium suggests the presence of compounds in conventionally used serum that are detrimental to stem cell survival, particularly when the cells disperse into individual cells. Avoiding the use of these compounds is therefore highly desired.

Feeder independent cultivation

Other investigations later turned to culturing ES cell lines in higher concentrations of FGF, but in the absence of serum and feeder cells. Three different medium formulations have been used in this work, and those medium formulations are referred to herein as UM100, BM +, and DHEM. The UM100 nomenclature refers to unconditioned medium to which 100 ng / ml of bFGF has been added. UM100 medium contains the 'Gibco Knockout' serum substitute product, but does not include or require the use of fibroblast feeder cells of any kind. BM + medium is basal medium (DMEM / F12) plus additives, described below, which also allows the cultivation of cells without feeder cells, but this medium omits the serum replacement product. Finally, the name DHEM refers to a defined human embryonic stem cell medium. This medium, also described below, is sufficient for the culture, cloning and indefinite proliferation of human ES cells, as it is composed entirely of inorganic constituents and only human proteins, unlike BM + medium that contains bovine albumin.

Culture of H1 and H9 human ES cell lines in UM100 / BM + / DHEM media

UM100 was prepared as follows: unconditioned media (UM) consisted of 80% (v / v) DMEM / F12 (Gibco / Invitrogen) and 20% (v / v) 'Knockout' serum substitute (Gibco / Invitrogen) supplemented with 1 mM glutamine (Gibco / Invitrogen), 0.1 mM p-mercaptoethanool (Sigma-St. Louis, MO) and 1% non-essential amino acid pools (Gibco / Invitrogen). To make up the medium, 100ng / ml bFGF was added and the medium was filtered through a 0.22uM nylon filter (Nalgene).

BM + medium was prepared as follows: 16.5 mg / ml BSA (Sigma), 196 mg / ml insulin (Sigma), 108 mg / ml transferrin (Sigma), 100 ng / ml bFGF, 1 mM glutamine (Gibco / Invitrogen), 0.1 mM p-mercaptoethanol (Sigma) and 1% non-essential amino acid pool (Gibco / Invitrogen) were combined in DMEM / F12 (Gibco / Invitrogen) and the osmolality was adjusted to 340 mOsm with 5M NaCl. The medium was then filtered through a 0.22 uM nylon filter (Nalgene).

DHEM medium was prepared as follows: 16.5 mg / ml HSA (Sigma), 196 mg / ml insulin (Sigma), 108 mg / ml transferrin (Sigma), 100 ng / ml bFGF, 1 mM glutamine (Gibco / Invitrogen), 0.1 mM p-mercaptoethanol (Sigma), 1% non-essential amino acid pool (Gibco / Invitrogen), vitamin supplements (Sigma), trace elements (Cell-gro®), and 0.014 mg / L at 0.07 mg / L selenium (Sigma), they were combined in DMEM / F12 (Gibco / Invitrogen) and the osmolarity was adjusted to 340 mOsm with 5M NaCl. It is observed that vitamin supplements in the medium can include thiamine (6.6 g / L), reduced glutathione (2mg / L) and ascorbic acid PO ⁴ . Furthermore, the trace elements used in the medium are a combination of trace elements B (Cell-gro®, Cat #: MT 99-175-Cl and C (Cell-gro®, Cat #: MT 99-176-Cl); each of which is sold as a 1,000 X solution. It is well known in the art that trace elements B and C contain the same composition as Cleveland trace elements I and II, respectively. (See Cleveland, WL, Wood, I. Erlanger, BF, J. Imm. Methods 56: 221-234, 1983.) The medium was then filtered through a 0.22 uM nylon filter (Nalgene) .Finally, defined sterile lipids (Gibco / Invitrogen ) to complete the middle.

H1 or H9 human embryonic stem cells that were previously grown in MEF feeder cells (mouse embryonic fibroblasts) were mechanically passaged with dispase (1 mg / ml) and plated on Matrigel (Becton Dickinson, Bedford, MA). The appropriate medium was changed daily until the cell density was determined to be adequate for cell passage. Cells were then dispase passaged as described and maintained in Matrigel (Becton Dickinson).

Growth rates

To determine the growth rate of human ES cells in the various media, cells were plated at a density of approximately 5 x 10 5 cells / well in triplicate in 6-well tissue culture plates (Nalgene). On days 3, 5, and 7, triplicate wells were trypsin / EDTA (Gibco / Invitrogen), individualized, and cell numbers counted. On day 7, additional wells were trypsinized, counted, and used to reseed a new plate at a cell density of approximately 2 x 105 cells / well. The cultures from day 7, which had been processed with trypsin, were analyzed for ES Oct4, SSEA4 and T ral-60 cell surface markers by FACs analysis. The growth rates were collected during 3 consecutive passes. Growth rate experiments show that human ES cells cultured with UM100 grow as robustly as human ES cells cultured with CM.

Union dynamics

To determine the binding rate of human ES cells in the various media, cells were plated at a density of approximately 2 x 10 5 cells / well in a 6-well tissue culture plate (Nalgene). At times ranging from 30 minutes to 48 hours, the unbound cells were washed and the attached cells were removed with trypsin / EDTA (Gibco / Invitrogen) and counted. These experiments were performed to examine whether the growth rate data with UM100 was due to a combination of better cell attachment and lower growth compared to equivalent growth rates for UM100 and CM. We found that the binding percentages were equivalent for both media at all times. Therefore, they grow at the same rate.

FACS analysis of human ES cells

Human ES cells were removed from a 6-well tissue culture plate (Nalgene) with trypsin / EDTA (Gibco / Invitrogen) 2% chicken serum (ICN Biomedicals, Inc., Aurora, OH) for 10 minutes. at 37 ° C. Cells were diluted in an equal volume of FACS buffer (PBS 2% FBS 0.1% sodium azide) and filtered through an 80 mM cell filter (Nalgaziene). The pellets were collected for 5 minutes at 1000 RPM and resuspended in 1 ml of 0.5% paraformaldehyde. Human ES cells were fixed for 10 minutes at 37 ° C and pellets were collected as described. ES cells were resuspended in 2 ml of FACS buffer and the total number of cells was counted with a hemocytometer. Cells were pelleted as described and permeabilized for 30 minutes on ice in 90% methanol. Human ES cells were pelleted as described and 1 x 105 cells were diluted in 1 ml of 0.1% Triton X-100 FACS buffer (Sigma) in a FACS tube (Becton Dickinson). hESCs were pelleted as described and resuspended in 50 ml of primary antibody diluted in 0.1% Triton X-100 FACS buffer (Sigma). Appropriate control antibody samples were applied in parallel. The hESCs were incubated overnight at 4 ° C. The supernatants were removed and the cells were incubated in the dark for 30 minutes at room temperature in 50 ml of secondary antibody (Molecular Probes / Invitrogen). FACS analysis was performed on a Facscalibur cell sorter (Becton Dickinson) with CellQuest software (Becton Dickinson). This procedure to perform the FACS analysis allows the detection of cell surface markers, in order to demonstrate that ES cells are present. The observed result was that human ES cells cultured in UM100 were 90% positive for Oct-4 as a population. This is comparable to ES cells grown with CM and confirms that the cells are a population of ES cells. For the analysis of SSEA4 and Tral-60, the process was performed as for Oct-4, except that the cells were not treated in paraformaldehyde or methanol. After cell staining, cells were resuspended in FACS buffer (without Triton) and analyzed as described with appropriate antibodies in FACS buffer, again without Triton. Undifferentiated ES cell cultures averaged approximately 90% positive results for these two cell surface markers as well. This was demonstrated by the FACS analysis discussed above.

Results

Cells of the human ES cell line H1 have now been cultured in UM100 medium for more than 33 passages (more than 164 population doublings) while preserving the morphology and characteristics of human ES cells. The H1 cells were cultured in BM + medium for more than 6 passages (70 days) maintaining the morphology and characteristics of human ES cells. H9 cells have been cultured in DHEM medium for more than 5 passages (67 days). Human ES cells H9 and H7 were also cultured in UM100 medium in an undifferentiated state for 22 passages and 21 passages, respectively. Subsequent testing of cells cultured with BM + and UM100 established normal karyotypes.

The study of FGF cell shapes

Human ES of the H1 line were cultured under standard conditions in conditioned medium for three passages before switching to test media. For test conditions, cells were grown in conditioned medium for 24 hours (day 0) and then switched to test media the next day (day 1). Subsequently, the cells were cultured in the respective test media. The H9 human ES cell line was also cultured in Matrigel in conditioned media for five passes before switching to parallel test media. Cells were passaged using the following procedures. Cell cultures were grown to suitable densities (which took approximately 7 days) in 6-well tissue culture plates and then the cultures were treated with 1 ml of dipase (1 mg / ml) (Gibco / Invitrogen) for 5-7 minutes at 37 ° C. The dipase was then removed and replaced with 2 ml of the appropriate growth medium. Using a 5 ml pipet, cells were mechanically removed from the tissue culture plate and then dispersed by pipetting. The cells were then pelleted in a clinical centrifuge for 5 minutes at 1000 rpm. The pellet was resuspended in an appropriate volume of medium and re-plated at the desired dilution. The media formulation was consistent in addition to the selection for added FGF. The base medium was UM100, the FGF being variable depending on the desired test condition. The following FGF variants were tested, each added to the medium at 100 ng / ml: FGF1 (acidic FGF), FGF1 p (acidic FGF isoform), FGF2 (basic FGF), FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, FGF20. All FGFs were commercially purchased or produced in recombinant hosts.

The competence of the particular form of FGF to support human ES cell cultures was assessed after each passage. Conditions that were deemed capable of supporting culture-supported cultures of human ES cells that adequately proliferated in an undifferentiated state in culture, independent of feeder cells, could be effectively overcome and continued to express the Oct4 human ES cell markers. , SSEA4 and Tra1-60. Conditions that were judged to be unable to admit human ES cells in culture resulted in cultures in which significant differentiation of the cells was evident by morphological observation, and the cells were unable to proliferate after colony passage. The FGF variants that supported human ES cell culture were FGF2, FGF4, FGF17, and FGF18. The FGF variants that did not support the maintenance of human ES cells in an undifferentiated state were FGF1, FGF1B, FGF3, FGF5, FGF6, FGF7, FGF8, FGF10, FGF16, FGF19 and FGF20. The results for the medium with FGF9 added were initially in the margin. By repeating the procedure, it seems likely that FGF9 supplemented at 100 ng / ml can also support undifferentiated human ES cells in culture.

Currently, media supplemented with FGF4, FGF17 and FGF20 have supported cultures of undifferentiated human ES cells from H1 cells for 8 passages. Similar replicas with FGF4, FGF9, FGF17 and FGF18 in human ES cell lines H9 and H14 have been extended by 3 and 2 passages respectively.

While recombinantly produced basic human fibroblast growth factor was used in the above experiments, naturally isolated fibroblast growth factor should also be suitable. Industrial application

The present invention provides methods for culturing primate embryonic stem cells and culture media for use therewith.

Claims (6)

1. A culture medium comprising albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, at least one insulin or insulin substitute, the culture medium free of animal serum and containing at least 100 ng / ml of a fibroblast growth factor selected from the group consisting of FGF4, FGF9, FGF17 and FGF18, the suitable medium for culturing primate embryonic stem cells in the absence of serum and in the absence of feeder cells and also in the absence of exposed medium to feeder cells, where at least 90% of the stem cells in the culture medium express Oct-4, SSEA4 or Tral-60. 2. The medium of claim 1, wherein the primate embryonic stem cells are human embryonic stem cells. 3. The medium of claim 1 or 2, wherein the fibroblast growth factor is present at a concentration of 100 ng / ml. 4. A procedure for culturing primate embryonic stem cells, comprising: culturing the stem cells in a fetal mammalian serum-free culture and in a stem cell culture medium that includes amino acids, vitamins, salts, minerals, transferrin or a transferrin substitute, insulin or an insulin substitute, albumin, and a minimum fibroblast growth factor (FGF) concentration of 100 ng / ml, where the FGF is selected from the group consisting of FGF4, FGF9, FGF17 and FGF18, and where the medium is capable of supporting culture and proliferation of human undifferentiated proliferative euploid pluripotent stem cells, without feeder cells or exposure of the medium to feeder cells, and where at least 90% of stem cells in the culture express Oct-4, SSEA4 or Tral-60. 5. A procedure for culturing primate embryonic stem cells in defined serum-free and feeder-free media, the procedure comprising: culturing the stem cells in a culture medium containing albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, at least one insulin or an insulin substitute, the culture medium free from fetal mammalian serum and containing at least 100 ng / ml of a fibroblast growth factor selected from the group consisting of FGF4, FGF9, FGF17 and FGF18, the medium supporting the proliferation of stem cells in an undifferentiated state without feeder cells or conditioned medium, and where at least 90% of stem cells in culture express Oct-4, SSEA4 or Tral-60. 6. The method of claim 5, wherein said culture step includes embryonic stem cells that proliferate in culture for more than one month while maintaining the potential of stem cells to differentiate into tissue derivatives of endoderm, mesoderm, and ectoderm, and while maintaining the karyotype of stem cells.