CN101018857B - The extended culture of embryonic stem cell without feeder cells - Google Patents

Abstract

The method of culture human embryo stem cell needed fibroblast feeder cells or once contacted the culture medium of fibroblast feeder cells so that stem cell is maintained undifferentiated state in the past.It has now been found that if bone morphogenetic protein antagonist and fibroblast growth factor are added in the culture medium of culture stem cell, even without feeder cells or conditioned medium, stem cell also will indefinitely remain undifferentiated.

Description

Long-term cultures of embryonic stem cells without feeder cells

Cross References to Related Applications

This application claims priority to US Provisional Patent Application Serial No. 60/573,545, filed May 21,2004.

Statement Regarding Federal Funding for Research or Development

To be determined.

Background of the invention

Stem cells are defined as cells capable of differentiating into many other differentiated cell types. Embryonic stem cells are stem cells derived from embryos that are capable of differentiating into most, if not all, of the differentiated cell types of the mature organism. A stem cell is said to be pluripotent, which means it can differentiate into many cell types. One type of pluripotent stem cells that has received high attention from research institutions is human embryonic stem cells, referred to here as hES cells, which are embryonic stem cells derived from human embryos. Human embryonic stem cells have received great scientific attention due to their ability to proliferate indefinitely in culture and thus be able, at least in principle, to provide cells and tissues to replace defective or damaged human tissues. The presence of human embryonic stem cells in culture has the potential to provide unlimited quantities of human cells and tissues for use in a variety of therapeutic regimens that contribute to human health. It is conceivable that, in the future, human embryonic stem cells will be able to be propagated and directed to differentiate into specific lineages to produce differentiated cells or tissues that can be transplanted into the human body for therapeutic purposes.

One of the most important features of human embryonic stem cells is their ability to self-renew. This means that hES cells can proliferate into a variety of progeny stem cells, each of which appears to have the full potential of its progenitor. In other words, the offspring are renewed to have all the developmental and proliferative capabilities of the parental cells. This property, together with pluripotency, makes hES cells useful for a variety of potential uses, since, in theory, hES cells can replicate in unlimited numbers and can then be induced to become any cell type in the human body. According to the existing knowledge, only undifferentiated hES cells can self-renew indefinitely, and once the cells differentiate, they lose the ability of self-renewal. At least from this perspective, the characteristic of self-renewal seems to be closely related to the characteristic of maintaining undifferentiation relevant. Since human embryonic stem cells will differentiate spontaneously, care must be taken in culture to maintain the cells in an undifferentiated state.

Basic techniques for generating and culturing human embryonic stem cells to maintain the cells in an undifferentiated state have been described. Existing techniques do work, but some of the methods currently used to grow human embryonic stem cells have limitations and drawbacks. One limitation is particularly significant. Most existing human embryonic stem cell lines have been exposed to some degree or another directly to mouse cells or to media in which mouse cells have been cultured. The original technique for generating and culturing hESCs used mouse embryonic fibroblast (MEF) feeder cells as a feeder layer on which hESCs could be cultured. Fibroblast feeder layers enable stem cells to remain in an undifferentiated state through mechanisms that are not yet fully cleared. It was later discovered that the same phenomenon could be obtained if the stem cells were exposed to "conditioned medium". A conditioned medium is nothing more than a stem cell medium in which feeder cells such as MEFs have been cultured. Regardless of whether the feeder cells can impart certain factors to the medium or remove certain factors from the medium, the result is that the conditioned medium can be used to culture stem cells so that they do not undergo differentiation. Whether the culture conditions, growth directly on feeder cells, or use of conditioned media involves one or more agents, such as viruses, that can be delivered from mouse cells to human ES cells. If one purpose of culturing human embryonic stem cells is to generate tissue that can eventually be implanted into a human body, it is highly desirable for the stem cells to be exposed to other types of cells or to media that have been used to culture other types of cells. At the same time, the use of feeder cells of any kind introduces unwanted biological variability when culturing stem cells, which should be avoided as much as possible. Therefore, of greatest interest in the continued development of technologies utilizing hESCs is to find culture conditions capable of proliferating and culturing hESCs without fibroblast feeder layers and without conditioned medium.

Some media formulations can maintain human ES cells in an undifferentiated state for a period of time, but will not maintain this state in long-term culture. Specifically, we define a "passage" in which human ES cells from an initial seed culture are grown to confluence on culture plates. We have found that some media formulations allow the culture of human ES cells for one or two passages without severe differentiation, but with gradual or rapid differentiation in subsequent passages. We have come to believe that in order to have a medium that does indeed support the immortalization of human ES cells without differentiation and without feeder cells or feeder-conditioned medium, the medium must support the culture of human ES cells in a substantially homogeneous and undifferentiated state for at least 5 generations.

Summary of the invention

The present invention can be summarized as a method of culturing human embryonic stem cells that does not require any type of feeder cells or conditioned media, the method comprising the addition of salts, vitamins, amino acids, glucose, fibroblastic Procedure for culturing human embryonic stem cells in culture medium with cell growth factors and bone morphogenetic protein antagonists.

The present invention also relates to in vitro cell cultures of human embryonic stem cells cultured in a medium containing a bone morphogenetic protein antagonist, thereby permitting the unlimited cultivation of stem cells in an undifferentiated state without the need for fibroblast feeder cells or conditioned medium.

It is an object of the present invention to define a long-term culture condition of human embryonic stem cells without the use of feeder cells, including culture without feeder cells and culture without culture on feeder cell-conditioned medium.

Another object of the present invention is to define a culture condition of human embryonic stem cells that is as defined as possible and at the same time completely independent of feeder cells.

Other objects, features and advantages of the present invention will be understood from the following description.

Brief description of the drawings

none.

Detailed description of the invention

The previous observation that cultures of human embryonic stem (ES) cells could be maintained in an undifferentiated state only in the presence of fibroblast feeder cells or in conditioned medium resulted in the release of fibroblasts in the medium capable of inhibiting ES Inference of factors of cell differentiation. This speculation is also based on parallel observations of murine ES cell lines, which when cultured with fibroblast feeder cells remain undifferentiated in response to leukemia inhibitory factor (LIF) secreted by fibroblasts. LIF activates signaling pathways that trigger self-renewal in murine ES cells. However, human ES cells do not respond to LIF and appear to have virtually no LIF receptors on their cell surface. Since no single factor that appears to prevent differentiation of human ES cells has been isolated from the conditioned medium, we developed a new hypothesis. We hypothesized that fibroblasts inactivate differentiation factors present in non-conditioned media.

Various research groups have investigated the factors that initiate the differentiation of human ES cells into progeny cell cultures enriched in cells of one or more specific lineages. One of these differentiation factors is a class of protein factors known as bone morphogenetic proteins (BMPs). BMPs are members of the transforming growth factor beta (TGFβ) superfamily of secreted signaling molecules. They play a broad role in almost all aspects of embryonic development. BMP4 and other members of the BMP family such as BMP2, -5 and -7 bind the type II BMP receptor BRII, which recruits the type I receptors BR1A (ALK3) or BR1B. After ligand activation, the intracellular kinase domain of type I receptors phosphorylates Smad1, -5, and -8, which then enter the nucleus under the protection of Smads and activate target genes. The relative expression levels of BMPs, receptors and Smads in cells are important determinants of BMP-induced responses. Costimulation of other signal transduction pathways can also alter the properties of BMP effects. A classic example is the altered action of BMPs caused by coactivation of LIF signaling in mouse ES cells: BMP signaling alone induces non-neuroepithelial differentiation, whereas BMP and LIF signaling together inhibit differentiation into any lineage. Extracellular BMP antagonists such as noggin, gremlin, chordrin, inhibin, follistatin, twisted gastrulation and members of the DAN family can be modified , weaken BMP activity or make it completely ineffective. On the other hand, some signaling pathways can interrupt intracellular BMP signaling. For example, fibroblast growth factor (FGF)-activated MAPK signaling inhibits BMP signaling by phosphorylating the linker domain of Smads and preventing nuclear translocation of Smads. Activation of transforming growth factor beta (TGFβ), Nodal or Activin signaling pathways can antagonize BMP signaling through intracellular communication such as competition with Smad4 for entry into the nucleus. All of these molecules are expected to be used to antagonize BMP signaling to achieve the effects described here.

It was also observed that the level of bone morphogenetic protein (BMP)-stimulated intracellular signaling was lower in human ES cells grown in conditioned medium, whereas the level of the same signal was lower in human ES cells grown in non-conditioned Fibroblast feeder cells) were higher in human ES cells. The effect produced by the conditioned medium may be due to the suppression of the effects of BMP-induced signals present in the non-conditioned medium. We therefore investigated the possibility of culturing human ES cells with antagonists of BMP activity and maintaining them in an undifferentiated state without the use of feeder cells or conditioned medium. We find, and report here, that this possibility is true. Human ES cells can be cultured indefinitely by antagonizing BMP activity while retaining all known properties of embryonic stem cells.

A wide variety of BMP antagonists are useful in the present invention. The most potent such antagonist known is noggin. Other proteins known to function as BMP antagonists include gremlin, chordrin, inhibin, follistatin, twisted gastrulation, and members of the DAN family. As noted above, other proteins include TFG[beta] and activins and other molecules that activate the MAPK signaling pathway. It is not required that the antagonist protein is the human form of the protein. It is only required that it can make the non-conditioned medium maintain ES cells from differentiation. Antagonist antibodies specific for all BMPs or specific BMPs may also be used. Which protein is chosen as a BMP antagonist is not as important as the result to be achieved, which is that BMP signaling activity is inhibited by the molecule added to the medium. The simplest and most direct way to achieve this is to add BMP antagonists to the culture medium of human ES cells.

The most potent BMP inhibitor identified to date is Noggin, which was originally cloned for its dorsalizing activity in Xenopus embryos. The mouse noggin cDNA encodes a precursor protein of 232 amino acid (aa) residues, cleaved at 19 amino acid residues of what is assumed to be the signal peptide to generate a mature protein of 213 amino acids, which is Secreted as a homodimeric glycoprotein. Noggin is a highly conserved molecule. Mature mouse noggin shares 99% and 83% amino acid sequence identity with human and Xenopus noggin, respectively. Noggin has a complex expression pattern during embryogenesis. In adults, noggin is expressed in the central nervous system and, in some adults, peripheral tissues such as lung, skeletal muscle and skin. Noggin has been shown to be a high-affinity BMP-binding protein that antagonizes almost all BMP biological activities.

It has also been found that high levels of fibroblast growth factor (FGF) can also be used to culture stem cells with or without conditioned medium. Although it has been reported that FGF is a beneficial additive to stem cell culture conditions, such as WO 01/66697, in this document, the FGF added to the culture medium is basic FGF (bFGF or FGF2) at a concentration of 4 ng/ml, but this paper reports that, with Better results were obtained with approximately 10-fold higher concentrations of FGF cultured. Herein, the preferred concentration of bFGF is 40 ng/ml. While various other FGF variants can also be used for this purpose, the concentration of FGF needs to be adjusted to be consistent with the efficacy of this level of bFGF, which we have shown also inhibits intracellular BMP activity.

There appears to be a synergistic relationship between the efficacy of BMP antagonists and the efficacy of high levels of FGF in culture. In other words, using high levels of bFGF (e.g. 100ng/ml) helps to culture hES cells in an undifferentiated state without feeder cells or conditioned media, but simultaneously using lower levels of bFGF (e.g. 40ng/ml) and BMP Antagonists such as noggin can also achieve this effect. Either combination enables culture not only "feeder-free" but also completely "feeder-independent", the term "feeder-free" is used for culture with conditioned medium (conditioned with feeder cells), "independent "Feeder cells" means completely independent of all types of feeder cells.

The data below will prove this hypothesis to be correct. With the addition of Noggin or other BMP signaling inhibitors and stimulation of fibroblast growth factor (FGF) signaling, human ES cells can grow indefinitely in an undifferentiated state without feeder cells or conditioned media. This enables the initiation and maintenance of culture of human ES cells without contact with feeder cells or media exposed to feeder cells, thus enabling the propagation of human ES cell lines in well-defined media in the absence of animal cells.

A related issue in culturing human ES cells is to remove as much as possible undefined components and components of animal origin from ES cell culture conditions. This is done for two reasons. One is to standardize culture conditions to minimize the normal variation of biological material as much as possible. Another aim is to avoid the use of materials, cells, exudates or components of animal origin to avoid any possible cross-species virus transmission through the culture system. Therefore, one purpose of defining culture conditions is to avoid the use of products of animal origin.

Thus, the defined culture medium for human ES cells starts with a basal medium containing salts, vitamins, glucose and amino acids. The basal medium can be any of a number of commercially available media. We recommend the combination of Dulbecco's Modified Eagle Medium and Hams F12 Medium sold as a combination (DMEM/F12). Glutamine, β-mercaptoethanol, and non-essential amino acids are added to this basal medium. Other possible additives include antioxidants and lipids. The protein component of the medium is serum in place. Albumin or purified albumin products such as the commercial product AlbuMax ^™ can be used, but we recommend a defined protein product consisting of albumin, insulin and transferrin. Human proteins are preferred, but not required, as long as uncharacterized animal products are excluded.

Notably, the observations here point to fundamental differences in the mechanisms of stem cell self-renewal among mammalian species. Conditions that support the maintenance of murine stem cells, including the addition of LIF and BMP to the medium, are not sufficient to maintain hES cells in an undifferentiated state. We were unable to maintain undifferentiated hES cells in serum-free medium supplemented with LIF and BMP4. BMPs differentiate human ES cells into trophoblasts. Indeed, the data presented here indicate that BMP signaling must be antagonized to promote human stem cell self-renewal. It is thus clear that, at least at a minimum, the cellular self-renewal mechanisms of mouse and human cell systems are very different.

Example

Methods and materials

Media and cell cultures. Unconditioned medium (UM) containing 80% DMEM/F12 and 20% KNOCKOUT serum replacement supplemented with 1 mM L-glutamine, 1% non-essential amino acids (both from Invitrogen) and 0.1 mM β-mercaptoethanol (Sigma) . To prepare conditioned medium (CM), incubate non-conditioned medium with mouse embryonic fibroblasts overnight, collect the medium, add 4ng/ml bFGF to it, freeze, and use within 2 weeks. hESCs were incubated on Matrigel (BDScientific) coated plates with CM or UM supplemented with or without 0.5 μg/ml mouse Noggin (R&D Systems) or 40 ng/ml human bFGF (Invitrogen) or both, and treated with 2 mg /ml Dispase (Invitrogen) was propagated to disperse cell colonies. To assess Oct4 ⁺ cell numbers, suspension colonies containing 35,000 cells were added to the medium of each well and incubated for 7 days. Cells were harvested and counted on days 1 and 7, and Oct4 ⁺ cells on day 7 were detected by fluorescence activated cell sorting (FACS, see below). To form embryoid bodies (Eb), hESCs that had been cultured in CM or UM/bFGF/Noggin (UMFN) were suspended as cell clumps in UM on uncoated plates and cultured on a shaker for 7 sky. The EB cells were then replated on gelatin-coated plates in DMEM supplemented with 10% fetal calf serum, incubated for 5 days, and then harvested for reverse transcription-PCR (RT-PCR) analysis. Experiments were repeated multiple times and ANOVA statistical analysis was performed for the entire study.

Immunoprecipitation and western blotting. Condition 15 ml of DMEM/F12 medium overnight in a T75 flask with 2.12 x 10 ⁵ /ml irradiated mouse embryonic fibroblasts. The medium was collected, concentrated to approximately 0.7 ml with a 5 kD cut-off filter (Millipore), and immunoprecipitated with goat anti-mouse noggin and gremlin antibodies (R&D Systems) (5 μg each) or 10 μg goat IgG as a negative control. Precipitated proteins or cell lysates were electrophoresed on a 4%-20% linear gradient polyacrylamide gel Tris-HCl precast gel (BioRad) for western blotting (Fig. 2A). Antibodies against mouse noggin and gremlin were used to immunoprecipitate proteins, anti-human Smad1/5/8, phosphorylated Smad1/5/8 (CellSignaling Technology), BMP2/4 (R&D Systems) and β-actin ( Abcam) antibody was used in cell lysates. Blots were processed with ECL surrogate solutions 1 and 2 (Amersham Biosciences) and exposed to a Fuji imager for analysis of chemiluminescence.

BMP/Smad-Luciferase reporter assay. hESCs cultured in CM were transfected with the BMP/Smad-responsive firefly luciferase reporter plasmid pID120-Lux and the pRL-tk plasmid (Promega) expressing a trace amount of flowerworm luciferase as an internal control. Cells were treated with different methods for 24 hours 1 day after transfection. Cell lysates were extracted and firefly and flowerworm luciferase activities were detected on a 3010 Luminometer (BD Biosciences) with the Dual Luciferase Reporter Assay System (Promega). Results are expressed as firefly luciferase activity normalized to flower insect luciferase activity.

Quantitative-PCR and RT-PCR. Total cellular RNA was extracted with the RNeasy kit (Qiagen) and treated with RNase-free DNase following the manufacturer's instructions. 1 μg of RNA was reverse transcribed into cDNA using the Improm-II reverse transcription system (Promega). Quantitative-PCR was performed on the AB 7500 real-time PCR system (Applied Biosystems) with SYBR greenQ-PCR Mastermix (Stratagene), and the quantitative-PCR conditions were as follows: 95°C for 10 minutes, 40 rounds of 95°C for 30 seconds, 60°C for 1 minute, 72 °C for 1 min and an extension at 72 °C for 3 min. Following the instructions of Applied Biosystems, GAPDH transcripts were detected as an endogenous reference to calculate the relative expression levels of target genes. The RT-PCR conditions were as follows: 94°C for 3 minutes, cycled through different rounds (see below) of 94°C for 20 seconds, 55°C for 30 seconds, and 72°C for 1 minute. The PCR reaction products were separated on a gel by electrophoresis and the DNA bands were photographed under UV light. Primer sequences and PCR round numbers are as follows.

Table 1. Primers and number of rounds for Q-PCR and RT-PCR

FACS and immunocytochemistry. FACS analysis was performed on hESCs cultured in various media to detect Oct4 ⁺ cells. Mouse anti-human Oct4 antibody (Santa Cruz Biotechnology) at 2 μg/ml and 1:1000 dilution of fluorescent isothiocyanate-labeled rabbit anti-mouse secondary antibody (Molecular Probes) were used. Statistical analysis was performed on the arcsine numbers transformed by the percentage of Oct4 ⁺ cells. For immunocytochemistry, a mouse anti-Oct4 antibody (0.2 μg/ml) and a 1:1000 dilution of Alexa Fluor 488-labeled anti-mouse IgG secondary antibody (Molecular Probes) were used.

Immunoassay of HCG in media. hESCs cultured in UMFN (non-conditioned medium with bFGF and Noggin) for multiple passages were cultured in CM supplemented with 100 ng/ml BMP4 for up to 7 days with daily refreshment of medium and BMP4. Spent medium was collected on days 3, 5 and 7 and assayed for HCG as described.

G banding and fluorescence in situ hybridization. hESCs cultured in UMFN for multiple passages were processed for G-banding and fluorescence in situ hybridization. In all scattered and fixed cells, 20 metaphase cells were selected for G-banding analysis, and 100-200 nuclei were used for fluorescent in situ hybridization to detect marker genes in chromosomes of interest. observe through Individual images captured by a digital imaging system (Applied Imaging).

result

UM has BMP-like differentiation-inducing activity. UM contains 20% KNOCKOUT ^™ Replacement Serum (Invitrogen), which includes ALBUMAX ^™ , a lipid-rich proprietary bovine albumin fraction. UM was conditioned overnight with fibroblasts, then 4 ng/ml human bFGF was added to obtain CM. We cultured hESC(H1) in CM, UM, a 1:1 mixture of CM and UM or a 1:1 mixture of CM and DMEM/F12. Cells in CM or CM-DMEM/F12 1:1 mixture remained undifferentiated and had typical hESC morphology. However, cells differentiated rapidly within 48 hours in either UM or CM-UM 1:1 mixture. We then replaced the surrogate serum with purified fetal bovine serum albumin (16.6 g/L, Fisher Scientific) to determine whether albumin was responsible for the differentiation. This medium was able to maintain hESCs in an undifferentiated form for approximately 7 days; however, the cells proliferated slowly and gradually differentiated into a mixed cell population. These results demonstrate that replacement of components in serum other than albumin resulted in rapid differentiation of UM-cultured cells. CM reduced this differentiation-inducing activity but also provided favorable factors to maintain hESC self-renewal. In addition to albumin, surrogate serum also contains other components required for culturing hESCs, so all subsequent studies used surrogate serum instead of albumin.

To test whether the differentiation-inducing activity of UM stimulates BMP signaling in hESCs, we used western blotting to assess the level of phosphorylated Smad1, an immediate downstream effector of the BMP receptor. Smad1 phosphorylation (antibodies used here also detect phosphorylation of other BMP effectors Smad5 and Smad8) was lower in H1 cells cultured in CM but higher in cells cultured in UM or CM+BMP4 for 24 hours . The addition of Noggin to UM decreased the phosphorylation level of Smad1, but the addition of 40 ng/ml bFGF to UM did not change the phosphorylation level of Smad1. BMP signaling can induce the expression of BMP ligands, forming a positive feedback loop in various cell types including hESCs. Indeed, UM-cultured hESCs had elevated BMP2/4 protein levels compared to cells cultured in CM or UM plus Noggin. It is unclear whether BMPs in UM directly stimulate BMP signaling in hESCs or whether other differentiation-inducing molecules stimulate BMP signaling indirectly by inducing BMP secretion. Both noggin and gremlin, another BMP antagonist, were detected in media conditioned with fibroblasts. These data demonstrate elevated but repressible BMP signaling activity in UM-cultured hESCs and the presence of both BMP agonists and antagonists in hESCs' fibroblast-supported media.

We also assessed BMP signaling in hESCs (H14) cultured in different media in the presence or absence of protein factors using a luciferase reporter plasmid specifically responsive to BMP/Smad. Reporter activity increased with surrogate serum or BMP4 concentrations and decreased with noggin or bFGF concentrations. Noggin at 500 ng/ml and bFGF at 40 ng/ml had a synergistic effect on reducing reporter activity to levels similar to those obtained with CM. Somewhat oddly, higher concentrations of bFGF (100 ng/ml) reduced BMP signaling to levels comparable to those obtained with CM when noggin was added. These results suggest that the surrogate serum actually has BMP-like activity that can be reduced by Noggin and/or bFGF.

The Id1 promoter contains a BMP-responsive element, and Id1 has been shown to be a target of BMP signaling in human and mouse ESCs. We therefore examined the expression of the Id gene, a secondary indicator of BMP signaling activity, in hESCs cultured in various media. Id1-4 transcripts were higher in hESCs (H9) cultured in UM or CM+BMP4 for 24 hours than in cells cultured in CM, and the addition of noggin to UM reduced the expression of Id genes.

UM/bFGF/Noggin maintains undifferentiated proliferation of hESCs. UM supplemented with 0.5 μg/ml Noggin and 40 ng/ml bFGF maintained the undifferentiated proliferation of hESCs. Equal numbers of H1 cells were plated and incubated for 7 days in CM, UM, UM+bFGF, UM+Noggin or UM+bFGF+Noggin. After 7 days, the number of Oct4 ⁺ cells was significantly higher in CM and UM/bFGF/Noggin than in UM, UM/bFGF or UM/Noggin. The number of Oct4 ⁺ cells measured in UM/bFGF and UM/Noggin was intermediate, indicating that Noggin and bFGF have a synergistic effect. hESCs cultured in UM/bFGF or UM/Noggin were able to propagate for multiple passages, but differentiated cells aggregated in the middle (UM/bFGF) or edges (UM/Noggin) of hESC colonies. Cells cultured in UM/bFGF/Noggin also showed increased differentiation if the concentration of albumin was reduced to 0.1 μg/ml while the concentration of bFGF was reduced to 10 ng/ml. Noggin in UM/bFGF/Noggin could be replaced by gremlin (5 μg/ml) or soluble BMP receptor IA (0.5 μg/ml) (data not shown), thus supporting that the action of noggin is actually interrupted by BMP Activity of BMP receptors.

Three different hESC lines (H1, H9, and H14) expanded over 40 days in UM/bFGF/Noggin (7, 6, and 6 passages, respectively) were still Oct4-positive, but if switched to lacking bFGF and Noggin After UM of the protein differentiation subsequently occurs. UM/bFGF/Noggin cultured hESCs continued to express other ES cell markers (including Nanog and Rexl) and cell surface markers SSEA4 and TRA-1-60 (data not shown). Even in the best cultures hESCs are mixed with small numbers of spontaneously differentiating cells. For example, low levels of the trophoblast marker chorionic gonadotropin β-subunit (CGβ) can be detected in CM-cultured ES cells, suggesting the presence of a minor trophoblast population. However, this labeling was not detected in UM/bFGF/Noggin cultured cells. Neural progenitor markers Pax6 and NeuroD1, mesoderm marker brachyury, and endoderm marker HNF3α were all negative in hESCs cultured in CM and UM/bFGF/Noggin. Thus, ES cells propagated in UM/bFGF/Noggin maintained characteristic ES cell markers after long-term culture.

We also examined hESCs after long-term culture in UM/bFGF/Noggin. H9 cells were continuously cultured in UM/bFGF/Noggin for 32 passages. H1 and H14 cells were cultured in UM/bFGF/Noggin for 20 and 16 passages, respectively, and then frozen. H14 cells were then directly thawed into UM/bFGF/Noggin and cultured to passage 18. The doubling time and ratio of Oct4 ⁺ cells of H9 and H14 cells cultured in UM/bFGF/Noggin for passages 27 and 18, respectively, were similar to control hESCs cultured in CM.

UM/bFGF/Noggin maintained the developmental potential of hESCs. When treated with BMP4 in CM for 3-7 days, hESCs that have been cultured for 10 passages in UM/bFGF/Noggin differentiate into squamous epithelium and secrete human chorionic gonadotropin (HCG) in the medium, which is a sign of trophoblastoma stratification. Embryoid bodies (EBs) from H1 cells cultured for 5 passages in UM/bFGF/Noggin and from control cells cultured in CM expressed the trophoblast marker CGβ and markers of the three germ layers, including Pax6, NeuroD1, brachyury, and HNF3α. EB cells also had reduced expression of ES cell markers Oct4, Nanog and Rexl. H1 and H9 cells cultured in UM/bFGF/Noggin for passage 7 and 6, respectively, were injected into SCID-beige mice. Mice developed teratomas showing complex differentiation 5-6 weeks after inoculation.

The karyotype of ES cells cultured with UM/bFGF/Noggin was normal. The karyotypes of H1 cells cultured in UM/bFGF/Noggin for 5 passages, H9 for 33 passages and H14 for 19 passages were analyzed by standard G-banding, and chromosomes 12 and 17 were detected by fluorescence in situ hybridization. The cells maintained a normal karyotype.

ES cells cultured in defined humanized systems remain undifferentiated. Although substituting UMFN for CM can eliminate the use of mouse-derived feeder cells, UM still contains fetal bovine serum-derived albumin extract (a component that is not fully defined), and the material used to coat plates, Matrigel, is a A soluble basement membrane matrix extracted from mouse tumors. Therefore, it is appropriate to further remove these animal materials to define a humanized culture system for human ES cells. We first looked for a defined humanized replacement serum to replace the previously used KNOCKOUT SR product, considering Sigma's 50x SeralReplacement 3 (SR3) composed of three human proteins: albumin, insulin and transferrin. It is known that laminin can be used instead of Matrigel to coat plates for culturing human ES cells with CM. We then established a system in which human ES cells were cultured on laminin-coated plates with UM containing 5XSR3 instead of KNOCKOUT SR and supplemented with 40 ng/ml FGF2 and 0.5 μg/ml Noggin. The ES cells in this system also maintained the consistency of ES cells after several weeks of passage. Therefore, this combination of defined humanized culture systems is suitable for human ES cells.

Together these data sets demonstrate that the use of feeder cells and conditioned media can be avoided by using culture conditions that include BMP antagonists such as Noggin as well as FGF. Other components of the medium can then be selected to avoid the use of animal products. The resulting highly defined medium enables the long-term culture and proliferation of human embryonic stem cells while retaining the full potential of those cells.

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R.-H. Xu (XU, Ren-He)

<120> Long-term cultures of embryonic stem cells without feeder cells

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<223> Synthetic PCR primers

<400>19

cgtccatctt tgcttgggaa atc 23

<210>20

<211>23

<212>DNA

<213> Artificial

<220>

<223> Synthetic PCR primers

<400>20

gagcctcatc tgaatcttct ccg 23

<210>21

<211>24

<212>DNA

<213> Artificial

<220>

<223> Synthetic PCR primers

<400>21

aagccatgaa cgcagaggag gact 24

<210>22

<211>20

<212>DNA

<213> Artificial

<220>

<223> Synthetic PCR primers

<400>22

agctgtccat ggtaccgtaa 20

<210>23

<211>24

<212>DNA

<213> Artificial

<220>

<223> Synthetic PCR primers

<400>23

aacccaactg tggagatgat gcag 24

<210>24

<211>24

<212>DNA

<213> Artificial

<220>

<223> Synthetic PCR primers

<400>24

aggggcttca ctaata actg gacg 24

<210>25

<211>21

<212>DNA

<213> Artificial

<220>

<223> Synthetic PCR primers

<400>25

ccaagccgcc ttactcctac a 21

<210>26

<211>21

<212>DNA

<213> Artificial

<220>

<223> Synthetic PCR primers

<400>26

cgcagatgaa gacgctggag a 21

<210>27

<211>25

<212>DNA

<213> Artificial

<220>

<223> Synthetic PCR primers

<400>27

tggcaccaca ccttctacaa tgagc 25

<210>28

<211>25

<212>DNA

<213> Artificial

<220>

<223> Synthetic PCR primers

<400>28

gcacagcttc tccttaatgt cacgc 25

Claims (11)

1. cultivating human embryo stem cell in a kind of culture independent of feeder cells and maintaining the human embryo stem cell Method in undifferentiated state, the method includes containing salt, vitamin, amino acid, glucose, fibroblastic growth The step of human embryo stem cell is cultivated in the culture medium of the factor and bone morphogenetic protein antagonist, the human embryo stem cell comes From human embryonic stem cell has been established, the human embryonic stem cell that has been established is selected from human embryonic stem cell H1, H9 and H14；

Wherein, the bone morphogenetic protein antagonist is selected from the group：Noggin, Ge Lemulin elements, notochord occur element, promote folliculus Plain inhibin, the embryogenesis of torsion type primitive gut, DAN families member and be specific to the antagonist antibodies of bone morphogenetic protein；And its In, the fibroblast growth factor and bone morphogenetic protein antagonist are to be enough to maintain the human embryo stem cell to be in The amount of undifferentiated state provides.

2. the method as described in claim 1, which is characterized in that the culture medium contain concentration be at least 4ng/ml at fiber Porcine HGF.

3. method as claimed in claim 1 or 2, which is characterized in that the bone morphogenetic protein antagonist is noggin.

4. method as claimed in claim 1 or 2, which is characterized in that the fibroblast growth factor is bFGF, concentration For 40ng/ml.

5. method as claimed in claim 3, which is characterized in that the fibroblast growth factor is bFGF, a concentration of 40ng/ml。

6. a kind of culture medium, the culture medium contains salt, vitamin, amino acid, glucose, fibroblast growth factor and bone Morphogenic protein antagonist,

Wherein, the bone morphogenetic protein antagonist is selected from the group：Noggin, Ge Lemulin elements, notochord occur element, promote folliculus Plain inhibin, the embryogenesis of torsion type primitive gut, DAN families member and be specific to the antagonist antibodies of all bone morphogenetic proteins； And wherein, the offer amount of the fibroblast growth factor and bone morphogenetic protein antagonist is enough to maintain embryonic stem cell In undifferentiated state the culture medium of feeder cells is exposed to without contacting feeder cells or contact.

7. culture medium as claimed in claim 6, which is characterized in that the bone morphogenetic protein antagonist is noggin.

8. culture medium as claimed in claims 6 or 7, which is characterized in that the culture medium also contains protein selected from the group below： Albumin, insulin and transferrins.

9. culture medium as claimed in claims 6 or 7, which is characterized in that the fibroblast growth factor is bFGF, Concentration in the culture medium is at least 4ng/ml.

10. culture medium as claimed in claim 8, which is characterized in that the fibroblast growth factor is bFGF, at this Concentration in culture medium is at least 4ng/ml.

11. culture medium as claimed in claim 6, which is characterized in that the embryonic stem cell is human embryo stem cell.