IL182143A - Cultivation of cultured primate embryonic stem cells expressing oct4, ssea4 or tra1-60 - Google Patents

Description

453562 IN SSEA4 TRA1 CULTIVATION OF CULTURED PRIMATE EMBRYONIC STEM CELLS EXPRESSING SSEA4 OR TRA1 WICELL RESEARCH WISCONSIN ALUMNI RESEARCH FOUNDATION BACKGROUND OF THE INVENTION The present invention relates to methods for culturing primate embryonic stem cell cultures and culture media useful Primate monkey and pluripotent embryonic stem cells have been derived from preimplantation for patent and Thomson et 282 Science 1 147 The disclosure of these publications and of all other publications referred to herein are incorporated by reference as if fully set forth Notwithstanding prolonged these cells stably maintain a developmental potential to form advanced derivatives of all three embryonic germ Primate ES cell lines have widespread utility in connection with human developmental drug drug and transplantation For current knowledge of the human embryo is largely based on a limited number of static histological Because of ethical considerations the underlying mechanisms that control the developmental decisions of the early human embryo remain essentially Although the mouse is the mainstay of experimental mammalian developmental and although many of the fundamental mechanisms that control development are conserved between mice and there are significant differences between early mouse and human ES cells should therefore provide important new insights into their differentiation and Differentiated derivatives of primate ES cells could be used to identify gene targets for new used to test toxicity or teratogenicity of new and used for transplantation to replace cell populations in Potential conditions that might be treated by the of ES cells include cardiac diabetes and See Rossant et 17 Nature Biotechnology and 282 Science Long term proliferative developmental potential after prolonged and karyotypic stability are key features with respect to the utility of primate embryonic stem cell Cultures of such cells on fibroblast feeder have typically been supplemented with animal serum fetal bovine to permit the desired proliferation during such For in patents and various culture conditions were including some using a type of basic fibroblast growth factor together with animal serum tends to have variable properties from batch to thus affecting culture In WO there was a discussion of providing a supplement in replacement for animal serum to support the growth of certain embryonic stem cells in The serum replacement included albumins or albumin one or more amino one or more one or more transferrins or transferrin one or more one or more insulins or insulin one or more collagen and one or more trace It was noted that this replacement could be further supplemented with leukemia inhibitory steel or ciliary neurotrophic in the context of primate embryonic stem cell cultures those grown on fibroblast feeder these culture media did not prove In the context of nutrient serum culture media fetal bovine WO discusses the benefit of use of various growth factors such as bFGF in culturing primate stem culture media without nutrient serum are not In Patent growth media for hematopoietic cells and bone marrow stromal cells are There is a suggestion to use fibroblast growth factor in a deprived media for this conditions for growth of primate embryonic stem cells are not IL 151270 discloses a method of culturing primate embryonic stem cells in a defined medium without but IL 151270 does not disclose that at least of the cultured cells express SSEA4 or Tral WO discloses methods for culturing primate ES cells on a prolonged and stable basis the presence of exogenously supplied FGF and in the absence of animal but does not di sclose that at least of the cultured cells express SSEA4 or Tral WO discloses a method for obtaining undifferentiated cells to when the hESCs are cultured at high concentrations of bFGF in conditioned ES cell Amit et Biology of Society for the Study of November pages a culture medium that is lacking a feeder cell layer and that is supplemented with a combination of the following growth and bFGF to support undifferentiated human ES cell The first human embryonic stem cell cultures were grown using a layer of fibroblast feeder which has the property of enabling the human embryonic stem cells to be proliferated while remaining it was discovered that it is sufficient to expose the culture medium to feeder to create what is called conditioned which had the same property as using feeder cells Without the use of either feeder cells or conditioned human embryonic stem cells in culture could not be maintained in an undifferentiated Since the use of feeder or the exposure of the medium to feeder risks contamination of the culture with unwanted avoiding the use of feeder cells and conditioned medium is Medium which has not been exposed to feeder cells is referred to here as unconditioned It can therefore be seen that a need still exists for techniques to stably culture primate embryonic stem cells without the requirement for use of animal BRIEF SUMMARY OF THE INVENTION The invention provides a method of culturing human embryonic stem culturing the stem cell s a culture essentially free of mammalian fetal serum and in a stem cell culture medium including amino transferrin or a transferrin insulin or an insulin and a fibroblast growth factor that is supplied from a source other than just a feeder layer and is present in a concentration that is at least as high as the maintenance the medium capable of supporting the culture and proliferation of human undifferentiated proliferating euploid embryonic stem cells for at least six without the need for feeder cells or for exposure of the to feeder and wherein at least of the stem cells in the culture express SSEA4 or Notice Under Circular dated April 1992 Inasmuch as the invention is defined in the appended it will be apparent that the portions of the present which fall outside the scope of the do not relate directly to the claimed This Notice is not meant to disclaim any legitimate rights to which the Patentee is legally especially any rights in accordance with Section 49 of the Israel Patent ADDITIONAL ASPECTS OF THE APPLICATION In one aspect the invention provides a method of culturing primate embryonic stem One cultures the stem cells in a culture essentially free of mammalian fetal serum also essentially free of any animal and in the presence of fibroblast growth factor that is supplied from a source other than just a fibroblast feeder hi a preferred the fibroblast feeder previously required to sustain a stem cell is rendered unnecessary by the addition of sufficient fibroblast growth Fibroblast growth factors are essential molecules for There are currently more then twenty known fibroblast growth factor ligands and five signaling fibroblast growtli factor receptors therefor their spliced See generally Omitz et aL 25 patent Slight variations in these factors are expected to exist between and thus the term fibroblast growth factor is not species we prefer to use human fibroblast growth more preferably human basic fibroblast growth factor produced from a recombinant This compound is readily available in quantity Gibco Technologies and It should be noted that for purposes of this patent the culture may still be essentially free of the specified serum even though a discrete component bovine serum has been isolated from serum and then is exogenously The point is that when serum itself is added the variability concerns when one or more well defined purified of such serum is they do Preferably the primate embryonic stem cells that are cultured using this method are human embryonic stem cells that are true ES cell lines in that are capable of indefinite proliferation vitro in an undifferentiated are capable of differentiation to derivatives of all three embryonic layers and even after prolonged and maintain a normal karyotype throughout prolonged These cells are therefore referred to as being The culturing permits the embryonic stem cells to stably proliferate in culture for over one month over six even more preferably over twelve while 3a WO the of the cells to differentiate into derivatives of and ectoderm In aspect the invention provides method of primate embryonic stem One stem cells in a cult essentially free of fetal also essentially free of any and the presence a growth factor capable of activating a fibroblast grovrth factor ng rece wherein the growth factor is supplied from a source other just a fibroblast feeder While certa n small peptides variants or designed to activate generally Yamaguchi et 152 In yet another aspect theiinventipn provides a for culturing primate stem It has a fibroblast grov supplied other than the feeder culture system is essentially animal aspect of derived using is used in directly or idirectly derived WO 25 feeder This pennitsthe culture of cells that have never been exposed either to animal cells or to media cells have been cultivation conditions no feeder cells and conditioned are here as feeder Prior culture conditions have based the use of medium feeder which are described as of conditioned does not dependence on use of feeder must be used to condition the The techniques described here permit the and feeder ndependeht the stem Techniques for the initial and characterization of the human were Thomson particular ES cells aids and distinct the at moderate levels permits the culture of undifferentiated human ES cells in a medium devoid of At this the rate of diffeentiation of compared to lower levels of but the cells eventually the addition of at higher levels the culture conditions of medium feeder pluripotsncy of human Surprisingl it has been found that for the second attribute of FGF a human ES cell the selection of the variant of FGF has some For this purpose it has been found that when the concentration of bFGF is about ng this condition is sufficient to avoid the for both serum and feeder making the culture feeder For this has been found that FGF family members FGF2 FGFl 7 and FGFl 8 are each sufficient at 100 of culture to make the human ES cell culture feeder By it has been found that FGF family members FGFl FGFl FGF and FGF 20 are not sufficient at to support feeder but do not have present that the results using these forms of FGF is not a result of concentration and higher concentrations of the particular FGF also would not succeed in supporting feeder For our data suggests that at this level ng FGF9 supports human ES cell culture but the data has been slightly more The exact minimal amount of the effective variants of FGF that to support human ES cells as feeder independent in culture is not known with precision at this but can be by empirical It is known for that 4 added to the medium alone is for the indefinite maintenance of euploid human ES cells in while 100 ng ml of FGF2 alone in the is ES cells grown in unconditioned medium containing as little as 4 will remain undifferentiated for some and perhaps a passage or the cells will begin to In bur the ability of a medium to culture ES cells to remain indefinitely undifferentiated and euploid is demonstrated when the cells are cultured for at least six passages while remaining euploid and while maintaining the characteristic morphology of As used a maintenance concentration of an FGF is the of that FGF necessary to support the maintenance of human ES cells in an euploid and proliferating state for at least For the minimal maintenance concentration is between 4 and and the exact minimal maintenance concentration can be determined by using the protocols below to interpolate those For each other effective FGFl the corresponding minimal maintenance concentration for each FGF can be determined by similar Human ES cell cultures in the defined human ES cell media described below in the examples can be cultivated in the complete absence of fibroblast feeder cells and without conditioned media while remaining The ES cells are thus truly feeder The human ES cells retain all of the characteristics of human ES cells including 3 characteristic morphology and compact with indistinct cell proliferation and ability to into if the human ES cells will also retain characteristic that they can form all primordial cell layers injected into In ths ES cells retain ability to differentiate into mesoderm and cells stilfex of ES cell such as expression of miclear factor is associated with the process and at its ES cells retain normal WG EXAMPLES In the first experiments described human ES cells were plated on irradiated gray gamma mouse embryonic Culture medium for present consisted of 1 acids supplemented either fetal serum or KnoclcOut a replacement originally op for mouse ES The components KnoclcOut SR described for serumreplacementsin WO alternative experiments medium was s upplemented with either fibroblast 4 ng the culture w as To under varying culture cultures minutes and plated m cells per well a To confirm single cells for the derivation ES individual bymicropipette to individual w fibroblasts feeders medium were expanded routine coliagenaseitype I Six H9 cells by spreads 1 inverted chromosome one with one with multiple at cells described culture cpnditionsithat included absence of We also observed that in the absence of animal serum the cloning efficiency and It has now been established that the adciiti WO FGF facilitated the cultivation of human ES cells in general and is of particular help in facilitating the cloning of human ES data expressed below is the total number of colonies resulting from individualized ES cells standard error of the mean colony cloning With fetal serum and no bFGF there was a of 240 With serum and bFGF 4 the result was about the In the absence of the serum of serum the result with no bFGF was 633 43 and the result with bFGF was S26 serum adversely affected cloning and the presence of the bFGF in the absence of serum had an added synergistic benefit insofar as cloning The lon g term culture of human ES cells in the presence of serum does not require the addition of exogenously supplied and noted the addition of bFGF to medium does not significantly increase human ES cell cloning in bFGF increased the initial cloning efficiency of human ES it has been discovered that supplying exogenous bFGF is very important for continued undifferentiated proliferation of primate embryonic stem cells in the absence of animal medium lacking exogenous human ES cells uniformly differentiated by two weeks of Addition of other factors as LIF inhibitory in the absence of did not prevent the The results are particularly applicable to clonal In this clones for expansion were selected by placing cells individually into wells of a 96 well plate under direct microscopic Of 192 cells into wells of 96 well two clones were successfully expanded and Both of these clones were subsequently cultured continuously in media supplemented with serum replacer and and cells both maintained a normal XX karyotype even after more S months of continuous culture after The and clones maintained the potential to form derivatives of all three embryonic germ layers even long culture in After 6 months of and clones were confirmed to have normal karyotypes and were then injected into Both and cells formed teratomas that contained derivatives of all three embryonic germ layers gut epithelium embryonic striated smooth cartilage and neural tissue The range of 9 cells was comparable to that observed in teratomas formed by low passage parental It should be appreciated from description above mat while animal serum is supportive of growth is complex mixture that compounds both beneficjal and serum batches vary w dely their ability to support vigorous undifferentiated proliferation of human ES serum a defined component reduces the variability of results w this serum differentiation the lower cloning efficiency in medium containing suggests the presence of when dispersed to single Avoidhig he use of compounds is therefore hi Culture Additional later were concentrations of FGF but in the absence of both serum and feeder have been used this and thosemedium formulations are referred to here S nomenclature to unconditioned medium which has been added 100 of medium does contain Gibco Serum Replacer product but does 10 WO medium was as BSA 196 ΐ Insulin Transferrin 100 ng 1 mM mM and nonessential amino acid stock were combined and osmolality was adjusted rnOsm with The medium was then filtered a nylon filler medium was prepared as Insulin ml nonessential amino acid and rng L to mg L selenium combined in the was adjusted to 340 mOsm with Itis vitamin supplements X and same M defined lipids were added to complete HI or embryoruc cells previously on MEF embryonic feeder cells with and plated onto until cell density was determimed to he adequate for Cells were passaged with dispase as described and maintained on ates To growth human ES cells plated at a density of about 5 x in tri ti additional wells treated density of about 2 x 105 The day 7 which had been trypsin were analyzed for ES cell surface markers and by FACS Growth rates were collected for 3 consecutive Growth rate experiments show that human ES cells grow as robustly as human ES Attachment Dynamics determine the attachment rate of human ES cells in the various media cells were plated at a density of 2 x in a tissue culture dish At time points ranging from minutes to 48 hours unattached cells were washed away and attached cells were removed with These experiments were performed to examine if the growth rate data was due to a combination of better cell attachment and slower growth as opposed to equivalent growth rates for UM100 and We found that attachment percentages were equivalent for both media at all time points they grow at the same FACS Analysis of human ES cells ES cells were removed from a tissue culture plate with trypsin EDTA chick serum at The cells were in an equal volume of FACS Buffer FBS Sodium and filtered through an 80 μΜ cell strainer Pellets were collected for 5 1000 RPM and resuspended in 1 ml Human ES cells were fixed for 10 at and the pellets were collected as The ES cells were resuspended in 2 FACS Buffer and total cell number was counted with a Cells were pelleted as described and permeablized for 30 on ice in Human ES cells were pelleted as described and 1 x 105 cells were diluted into 1 ml of FACS Buffer Triton in a FACS tube Di hESC were pelleted as described and resuspended in 50 of primary antibody dilated in FACS Buffer Triton Samples of appropriate control antibodies applied in hESC were incubated overnight at Supematants were poured off and cells were incubated in the dark for 30 at room temperature in 50 μΐ of secondary antibody FACS analysis was performed in a Facscalibur cell sorter with CellQuest Software This method for perforating FACS analysis allows one to detect cell surface to thus show that you have ES The result observed was that human ES cells cultured in were positive for as a This is comparable to ES cells and confirms that the cells are an ES cell For the analysis of SSEA4 and the process was performed as for except that the cells were not treated in paraformaldehyde or After cell the cells were in FACS buffer and analyzed as described with n appropriate antibodies in FACS again The undifferentiated ES cell cultures averaged about forithese two cell surface as This was demonstrated by FACS analysis discussed Results of human ES ie have now been medium over 33 passages 164 population while retaining the morphology and characteristics of human ES cells were cultivated in for over 6 H9 eells cultivated human ES ere cultivated stateTor passages passages Subsequent testing of normal line H were cultured under standard conditions conditioned medium for toee passages before For the test cells were cultured on conditioned medium for h urs then switched to day Thereafter the cells were in the respective test ES also n cond passages before being switched to the test media The cells were passaged usin the cultures were grown to took approximately 7 6 and was ml the appropriate growth Using a 5 ml the cells were mechanically removed from the tissue cultirre plate and then dispersed by The cells were centrifuge for 5 minutes at The folio wing FGF were each added to the ng FGFl FGFl FGFl FGFl All FGFs were purchased commercially or produced in recombinant 13 WO The competence of the particular FGF form to support ES cell cultures was judged after each The conditions which judged to cell culture cultures that proliferated appropriately in state in independent of feeder could be passaged and continued the human ES cell markers and The conditions which were judged not to support human ES cells in culture gave rise to cultures in which significant differentiation of the cells was apparent by cells wcre to proliferate upon colony The FGF variants which supported human ES culture were and FGEl The FG variants which not support maintenance of cells in an The results m FGF9 added were initially the fe supported undifferentiated human ES cell cultures of cells for 8 Similar replicates with and FGF18 on human ES cell lines H9 and for 3 of this concept are also intended to within the scope of the For while was used the naturally isolated Sbroblastigrowm factor also be primate cell the claims should be looked to in order to of Industrial Applicability culturm embryonic and for 14 insufficientOCRQuality

Claims (24)

Claims

1. A method of culturing human embryonic stem cells, comprising: culturing the stem cells in a culture essentially free of mammalian fetal serum and in a stem cell culture medium including amino acids, vitamins, salts, minerals, transferrin or a transferrin substitute, insulin or an insulin substitute, albumin, and a fibroblast growth factor that is supplied from a source other than just a feeder layer and is present in a concentration that is at least as high as the maintenance concentration, the medium capable of supporting the culture and proliferation of human undifferentiated proliferating euploid embryonic stem cells for at least six passages, without the need for feeder cells or for exposure of the medium to feeder cells, and wherein at least 90% of the stem cells in the culture express Oct-4, SSEA4 or Tral-60.

2. The method of claim 1 , wherein the culture is essentially free of any animal serum.

3. The method of claim 1 wherein the FGF is selected from the group consisting of FGF2, FGF4, FGF9, FGF17 and FGF18.

4. The method of claim 1 wherein the FGF is FGF2 which is present in the medium at 100 ng/ml.

5. A method of culturing human embryonic stem cells in defined media 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 insulin substitute, the culture medium essentially free of mammalian fetal serum and containing at least about 100 ng/ml of a fibroblast growth factor capable of activating a fibroblast growth factor signaling receptor, wherein the growth factor is supplied from a source other than just a feeder layer, the medium supporting the proliferation of stem cells in an undifferentiated state without feeder cells or conditioned medium, and wherein at least 90% of the stem cells in the culture express Oct-4, SSEA4 or Tral-60.

6. The method of claim 5, wherein said culturing step includes the embryonic stem cells proliferating in culture for over one month while maintaining the potential of the stem cells to differentiate into derivatives of endoderm, mesoderm, and ectoderm tissues, and while maintaining the karyotype of the stem cells. 15

7. The method of claim 5 wherein the FGF is selected from the group consisting of FGF2, FGF4, FGF9, FGF17 and FGF18.

8. A culture of human embryonic stem cells comprising: human embryonic stem cells; and a stem cell medium containing albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, at least one insulin or insulin substitute, the culture medium essentially free of mammalian fetal serum and containing at least a maintenance concentration of a fibroblast growth factor capable of activating a fibroblast growth factor signaling receptor, the medium capable of culturing stem cells indefinitely in the absence of serum and in the absence of feeder cells and also in the absence of medium exposed to feeder cells, wherein the culture is capable of maintaining the stem cells in an undifferentiated state indefinitely, while maintaining the karyotype of the stem cells, and wherein at least 90% of the stem cells in the culture express Oct-4, SSEA4 or Tral-60.

9. The culture of claim 8 wherein the fibroblast growth factor is FGF2 which is present in the medium in a concentration of at least about 100 ng/ml.

10. A culture of feeder independent human embryonic stem cells comprising human embryonic stem cells in a stem cell culture medium, the stem cell 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 essentially free of mammalian fetal serum and containing at least a maintenance concentration of a fibroblast growth factor selected from the group consisting of FGF2, FGF4, FGF9, FGF17, and FGF18, the culture being independent of feeder cells while the cells remain euploid, where at least 90% are in an undifferentiated state, and express Oct-4, SSEA4 and Tral-60.-

11. A culture as claimed in claim 10 wherein the fibroblast growth factor is present at a concentration of at least about 100 ng/ml.

12. 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 essentially free of mammalian fetal serum and containing at least a 16 maintenance concentration of a fibroblast growth factor capable of activating a fibroblast growth factor signaling receptor, the medium capable of culturing stem cells indefinitely in the absence of serum and in the absence of feeder cells and also in the absence of medium exposed to feeder cells, wherein the medium is capable of maintaining the stem cells in an undifferentiated state indefinitely, and wherein at least 90% of the stem cells in the culture medium express Oct-4, SSEA4 or Tral-60.

13. The medium of claim 12 wherein the stem cells are primate pluripotent stem cells.

14. 4. The medium of claim 12 wherein the stem cells are human pluripotent stem cells.

15. The medium of claim 12 wherein the fibroblast growth factor is selected from the group consisting of FGF2, FGF4, FGF9, FGF17 and FGF18.

16. The medium of claim 2 wherein the fibroblast growth factor is present at a concentration of at least about 100 ng/ml.

17. A cell culture medium comprising amino acids, vitamins, salts, minerals, transferrin or a transferrin substitute, insulin or an insulin substitute, albumin, and a fibroblast growth factor that is supplied from a source other than just a feeder layer and is present in a concentration that is at least as high as the maintenance concentration, the medium essentially free of mammalian fetal serum, and capable of supporting the culture and proliferation of human undifferentiated proliferating euploid stem cells for at least six passages, without the need for feeder cells or for exposure of the medium to feeder cells and wherein at least 90% of the stem cells in the culture medium express Oct-4, SSEA4 or Tral-60.

18. The medium of claim 17 wherein the stem cells are primate pluripotent stem cells.

19. The medium of claim 17 wherein the stem cells are human pluripotent stem cells.

20. The medium of claim 7 wherein the fibroblast growth factor is selected from the group consisting of FGF2, FGF4, FGF9, FGF17 and FGF18.

21. 2 . The medium of claim 17 wherein the fibroblast growth factor is present at a concentration of at least about 100 ng/ml. 17

22. A culture medium without serum and without feeder cells comprising: albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, at least one insulin or insulin substitute, the culture medium essentially free of mammalian fetal serum and containing at least about 100 ng/ml of a fibroblast growth factor capable of activating a fibroblast growth factor signaling receptor, wherein the growth factor is supplied from a source other than just a feeder layer, the medium supporting the proliferation of stem cells in an undifferentiated state without feeder cells or conditioned medium, and wherein at least 90% of the stem cells in the culture medium express Oct-4, SSEA4 or Tral-60.

23. (The medium of claim 22 wherein the stem cells are primate pluripotent stem cells.

24. A cell 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 essentially free of mammalian fetal serum and containing at least a maintenance concentration of a fibroblast growth factor selected from the group consisting of FGF2, FGF4, FGF9, FGF17, and FGF18. For the Applicant, C:61563 18