Trophoblast cell preparations
Stable pluripotent trophoblast stem (TS) cell lines and uses of the cell lines are described. The cell lines comprise cells that (i) are capable of indefinite proliferation in vitro in an undifferentiated state; and (ii) are capable of differentiation into cells of the trophoblast lineage in vivo.
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The invention relates to trophoblast cell preparations and uses of the cell preparations.
BACKGROUND OF THE INVENTIONIn mammals, the earliest developmental decision specifies the trophoblast cell lineage. In mice, this lineage appears at the blastocyst stage as the trophectoderm, a sphere of epithelial cells surrounding the inner cell mass (ICM) and the blastocoel. After implantation, the ICM gives rise to the embryo proper and some extraembryonic membranes. However, the trophectoderm is exclusively restricted to form the fetal portion of the placenta and the trophoblast giant cells. The polar trophectoderm (the subset of trophectoderm in direct contact with the ICM) maintains a proliferative capacity and gives rise to the extraembryonic ectoderm (ExE), the ectoplacental cone (EPC), and secondary giant cells of the early conceptus (1). The rest of the trophectoderm ceases to proliferate and becomes primary giant cells. Studies in primary culture and chimeric mice have suggested that stem cells exist in the extraembryonic ectoderm which contribute descendants to the EPC and the polyploid giant cells (2). Further evidence indicated that maintenance of these stem cell-like characteristics was dependent on signals from the ICM and later from the epiblast (3), since diploid trophoblast cells transformed into giant cells when removed from the embryonic environment (4). However, the nature of the embryo-derived signal was not known and all attempts at routine long-term culture of mouse trophoblast stem cells have been unsuccessful.
Expression and functional analyses indicated that Fgf4 and Fgfr2 may be involved in trophoblast proliferation (5, 6, 7). The reciprocal expression domains of Fgfr2 and Fgf4 suggested that the trophoblast could be a target tissue for an embryonic FGF signal. Fgfr2-null and Fgf4-null mice show similar peri-implantation lethal phenotypes (6, 7). This may result from defects in the ICM and its endoderm derivatives. However, it is also consistent with the possibility that FGF4 acts on the trophoblast through FGFR2 to maintain a proliferating population of trophoblast cells. Support for this latter possibility is provided by recent studies showing that inhibiting FGF signaling blocked cell division in both the ICM and trophectoderm (8).
SUMMARY OF THE INVENTIONThe present inventors have found that FGF4 can promote sustained proliferation of primary cultures of diploid trophoblast cells and it permits isolation of stable FGF4-dependent mouse trophoblast stem (TS) cell lines from both the ExE of 6.5 dpc embryos and the trophectoderm of 3.5 dpc blastocysts. TS cell lines expressed many diploid trophoblast markers and retained the capacity to differentiate into other trophoblast subtypes in vitro upon removal of FGF4. Most importantly, when these stem cells were introduced into chimeras they exclusively contributed to all trophoblast subtypes in vivo. Availability of trophoblast stem cell lines opens up new possibilities for understanding the genetic regulation of placental development and placental insufficiencies and modulating the same. The cell lines also enable the treatment of placental insufficiencies by pharmacological intervention or gene-based therapy.
Broadly stated, the present invention relates to a stable pluripotent trophoblast stem (TS) cell line. In particular, the invention relates to a purified preparation of trophoblast stem cells which (i) are capable of indefinite proliferation in vitro in an undifferentiated state; and (ii) are capable of differentiation into cells of the trophoblast lineage in vivo. The preparation of trophoblast stem cells is also characterized by expression of genetic markers of diploid trophoblast stem cells.
A trophoblast stem cell preparation of the invention may be induced to differentiate into cells of the trophoblast lineage in vitro or in vivo. The invention therefore also relates to a purified trophoblast stem cell preparation of the invention (preferably cultured in vitro) induced to differentiate into cells of the trophoblast lineage. This differentiated cell preparation is characterized by expression of genetic markers of trophoblast cell lineages (e.g. diploid trophoblast cells of the ectolacental cone (EPC), and the secondary giant cells of the early conceptus). In an embodiment of the invention a purified trophoblast cell preparation comprises cells of the tropoblast lineage including diploid trophoblast cells.
A cell preparation of the invention may be derived from or comprised of cells that have been genetically modified either in nature or by genetic engineering techniques in vivo or in vitro.
Cell preparations or cell lines of the invention can be modified by introducing mutations into genes in the cells or by introducing transgenes into the cells. Insertion or deletion mutations may be introduced in a cell using standard techniques. A transgene may be introduced into cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, or microinjection. Suitable methods for transforming and transfecting cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks. By way of example, a transgene may be introduced into cells using an appropriate expression vector including but not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses). Transfection is easily and efficiently obtained using standard methods including culturing the cells on a monolayer of virus-producing cells (Van der Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).
A gene encoding a selectable marker may be integrated into cells of a cell preparation of the invention. For example, a gene which encodes a protein such as β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or a fluorescent protein marker may be integrated into the cells. Examples of fluorescent protein markers are the Green Fluorescent Protein (GFP) from the jellyfish A. victoria, or a variant thereof that retains its fluorescent properties when expressed in vertebrate cells. (Examples of GFP variants include a variant of GFP having a Ser65Thr mutation of GFP (S65T) that has longer wavelengths of excitation and emission, 490 nm and 510 nm, respectively, compared to wild-type GFP (400 nm and 475 nm); a blue fluorescent variant of GFP (e.g. Y66H-GFP) (Heim et al. Proc. Natl. Acad. Sci. 91:12501, 1994). MmGFP (M. Zernicka-Goetz et al, Development 124:1133-1137, 1997), enhanced GFP (“EGFP”) (Okabe, M. et al. FEBS Letters 407:313-319, 1997; Clontech Palo Alto, Calif.), EGFP which has a Phe to Leu mutation at position 64 resulting in the increased stability of the protein at 37° C. and a Ser to Thr mutation at position 65 resulting in an increased fluorescence; and, EGFP commercially available from Clontech incorporating a humanised codon usage rendering it “less foreign” to mammalian transcriptional machinery and ensuring maximal gene expression.)
The invention also relates to a method for producing a purified trophoblast stem (TS) cell preparation i.e. a cell line, comprising the steps of culturing early postimplantation trophoblast cells or cells of a blastocyst, preferably from the trophectoderm on a feeder layer (e.g. a fibroblast layer or a medium conditioned by fibroblasts) in the presence of FGF4 and a co-factor. The method may additionally comprise inducing differentiation of the trophoblast stem cells by removing the FGF4, the co-factor, or the feeder layer. In an embodiment of the invention, the method comprises isolating a blastocyst, culturing the blastocyst on a fibroblast layer in the presence of FGF4 and a co-factor, removing a blastocyst outgrowth and dissociating the outgrowth, selecting flat colonies i.e. epithelial-like cells, and culturing the colonies. The invention also contemplates trophoblast cell preparations or lines derived at all stages of development under the same culture conditions.
The term “blastocyst” used herein refers to the structure during early embryonic development comprising an inner cluster of cells, the inner cell mass (ICM), which gives rise to the embryo, and an outer layer, the trophectoderm, which gives rise to extra-embryonic tissues. Preferably, cells from the trophectoderm of a 3.5 dpc blasotocyst are used in the method of the invention. The term “postimplantation trophoblasts” used herein refers to cells derived from extraembryonic extoderm (ExE) cells preferably isolated from 6.5 days post coitum conceptuses. The term “epithelial-like cells” refers to the flat colonies obtained after dissociation of a blastocyst outgrowth and which are like the cells which sometimes appear during the isolation of embryonic stem cells from blastocysts as described in B. Hogan et al (10).
The blastocysts or early postimplantation trophoblasts may be derived or isolated from any mammalian or marsupial species including but not limited to rodents (e.g. mouse, rat, hamster, etc.). rabbits, sheep, goats, pigs, cattle, primates, and humans are preferred. Mutant or transgenic blastocysts and postimplantation trophoblasts may be used to prepare a cell preparation or cell line of the invention. For example, a cell preparation or cell line of the invention may be derived from a Fgf4 or Errβ mutant blastocyst. Cells used to prepare a cell preparation or cell line of the invention can be engineered to contain a selectable marker or they may be genetically altered using techniques well known in the art.
The cells derived from a blastocyst or postimplantation trophoblast cells are cultured on a feeder layer. The feeder layer may be a confluent fibroblast layer, preferably primary mouse embryonic fibroblast (EMFI) cells. Embryonic fibroblasts may be obtained from 12 day old fetuses from outbred mice, but other strains may be used as an alternative. STO cells (i.e. a permanent line of irradiated mouse fibroblasts) can also be used as a feeder layer. The feeder layer may also comprise medium conditioned by primary embryonic fibroblast cells.
Cells from a blastocyst or early postimplantation trophoblast cells are preferably cultured in medium comprising RPMI 1640 with 20% fetal bovine serum, sodium pyruvate, β-mercaptoethanol, L-glutamine, and penicillin/streptomycin. The FGF4 used in the method of the invention may be recombinant FGF4 (preferably recombinant human FGF4) which may be produced using standard recombinant techniques or it may be obtained from commercial sources (e.g. Sigma). The co-factor used in the method of the invention is preferably heparin. Once established the cell lines may be grown on a feeder layer such as a fibroblast layer (e.g. EMFI cells) or in a conditioned medium prepared from a fibroblast layer (See for example the medium described in note 13, page 15).
Cells from the cell preparations may be introduced into a blastocyst or aggregated with an early stage embryo to produce chimeric conceptuses. A chimeric conceptus may be allowed to grow to term, or sacrificed during gestation to observe the contribution of the stem cell line. In an embodiment, the invention provides a chimeric placenta wherein the trophoblast lineage is repopulated by cells from a cell preparation of the invention. The conceptuses and placenta can be engineered to carry selectable markers or genetic alterations. Cell lines can be derived from the chimeric conceptuses and placenta. Therefore, the invention further provides a chimeric conceptus, differentiated trophoblast cells, mutant trophoblast stem cells, or a chimeric placenta derived from a purified preparation of the invention.
The cell preparations, chimeric conceptuses, and chimeric placentas may be used to screen for potential therapeutics that modulate trophoblast development or activity e.g. invasion or proliferation. In particular, the cell preparations, chimeric embryos, or chimeric placenta may be subjected to a test substance, and the effect of the test substance may be compared to a control (e.g. in the absence of the substance) to determine if the test substance modulates trophoblast development or activity. Cell preparations of the invention derived from mouse mutants can be used to identify genes and substances that are important for the trophoblast cell lineage, and in vitro differentiation of mutant cell preparations can identify genes and substances important for selected trophoblast subtypes. Selected substances may be useful in regulating trophoblasts in vivo and they may be used to treat various conditions requiring regulation of trophoblast development or activity such as the conditions described below.
The cell preparations of the invention may be transplanted into animals to treat specific conditions requiring modulation of trophoblast development or activity. For example, the cell preparations may be used to prolong fetal survival in conditions of placental insufficiency, or to reduce uncontrolled trophoblast invasion and abnormal trophoblast growth associated with conditions such as hydatiform mole and choriocarcinoma. The cell preparations may be used for therapeutic treatment of placental defects in humans by transplantation of the cell preparations at any stage of pregnancy to generate chimeric placenta.
The cell preparations may be used to prepare model systems of disease for conditions such as precclampsia, hydatiform mole, or choriocarcinoma.
The cell preparations or cell lines of the invention can be used to produce growth factors, hormones, etc. relevant to human placenta. The cell preparations or cell lines of the invention can also be used to produce therapeutics such as human Chorionic Gonadotropin (hCG).
The cell preparations or cell lines of the invention can be used to screen for genes expressed in or essential for trophoblast differentiation. Screening methods that can be used include Representational Difference Analysis (RDA) or gene trapping with for example SA-lacZ (D. P. Hill and W. Wurst, Methods in Enzymology, 225: 664, 1993). Gene trapping can be used to induce dominant mutations (e.g. by deleting particular domains of the gene product) that affect differentiation or activity of trophoblast cells and allow the identification of genes expressed in or essential for trophoblast differentiation.
DESCRIPTION OF THE DRAWINGSThe invention will now be described in relation to the drawings in which:
TS cell lines were first derived from early postimplantation embryos. ExE cells were isolated from 6.5 dpc conceptuses as previously described (4), disaggregated by trypsin, and cultured on a feeder layer of primary mouse embryonic fibroblast (EMFI) cells in the presence of various combinations of growth factors (data not shown). The combination of FGF4 (25 ng/ml) and heparin (1 μg/ml) in TS cell medium (9) proved successful in allowing the passage of colonies with a tight epithelial morphology (
Under the identical culture conditions used for isolating TS cell lines from ExE, cell lines were derived from 3.5 dpc blastocysts which exhibited a morphology and behaviour indistinguishable from that of ExE-derived TS cell lines (12).The blastocyst-derived and ExE-derived lines are referred to as TS3.5 and TS6.5 cell lines, respectively, to distinguish their tissues of origin. Generation of TS3.5 and TS6.5 cell lines was efficient and reproducible; 58 clonal TS3.5 cell lines were obtained from 91 blastocysts (64%) and 17 TS6.5 cell lines from 39 ExEs of 6.5dpc embryos (44%); they were derived from different strain backgrounds (129/sv and ICR) and of both sexes Some of these TS cell lines were stably maintained for more than 50 passages over a period of more than six months with no apparent change in their morphology or viability.
To address the possibility that FGF4 stimulated the proliferation of TS cells indirectly by inducing the secretion of mitotic factors from the feeder cells, conditioned medium from EMFI cells (EMFI-CM) was prepared in the absence of FGF4. TS cells were maintained in an undifferentiated state on gelatin-coated plates in medium supplemented with 70% EMFI-CM and FGF4/heparin; lower concentrations of EMFI-CM were not effective (13). Leukemia inhibitory factor (LIF), the critical factor produced by EMFI cells that maintains ES cells undifferentiated, could not substitute for EMFI-CM even at five-times the concentration used in ES cell medium. These results suggest that a) EMFI cells secrete an unidentified factor(s) (EMFI-factor) that acts along with FGF4 to maintain the TS cells in a proliferative and undifferentiated state, b) secretion of this factor(s) is not a result of the addition of FGF4 to the media, and c) FGF4 acts directly on the TS cells.
Chromosome spreads from two TS cell lines passaged over 20 times revealed an apparently normal euploid karyotype. The ploidy of the stem cells and differentiated giant cells were determined by FACS analysis of cells stained with propidium iodide (14). The profile for cells maintained in EMFI-CM supplemented with FGF4/heparin (13) revealed prominent peaks at 2N and 4N indicative of the G1 and G2/M DNA content of a diploid cell line (
Several genetic markers were analyzed during stem cell and differentiative culture conditions to confirm the trophoblast identity of the TS3.5 and TS6.5 cell lines and characterize their differentiation in the absence of FGF4 (15). Markers of the diploid ExE were highly expressed in TS cells. Errβ, an orphan nuclear receptor, is specifically expressed in the ExE nearest to the extraembryonic-embryonic boundary at early postimplantation stages and later in the chorionic ectoderm (16). This gene was highly expressed in TS cells grown in the presence of FGF4 and 70% EMFI-CM, but was down-regulated when differentiation was induced by removing FGF4 and EMFI-CM (
The most definitive test for the trophoblast identity and stem cell capacity of TS cells is to investigate their potential to incorporate into trophoblast lineages in vivo. Rossant et al. (2) have shown that the cells isolated from the ExE of 6.5 dpc embryos can contribute to the EPC and giant cells when directly injected into blastocysts, despite temporal asynchrony between donor and host cells. To investigate the potency of TS cells to contribute to trophoblast lineages in vivo, chimeric embryos were made by the aggregation method (26) and blastocyst injection. A TS3.5 and a TS6.5 cell line were derived from B5/EGFP transgenic mice (27) that ubiquitously express enhanced-green fluorescent protein (EGFP, Clontech) in all embryonic and extraembryonic tissues. These lines were passaged more than 20 times (two months) before they were used for the chimera experiments. Chimeras were obtained from each cell line using both methods (Table 1). EGFP-positive cells were only observed in tissues of the trophoblast lineage in the 61 chimeric embryos analyzed (
It has been proposed that the ExE is the first tissue to be formed from the polar trophectoderm and that it may act as a stem cell population that subsequently gives rise to the EPC which generates new secondary giant cells (2, 3). Successful derivation of TS cell lines expressing trophoblast markers from the ExE of 6.5 dpc embryos and 3.5 dpc blastocysts is consistent with this model. FGF4 produced by the ICM and later by the epiblast is one of the critical signals required for the maintenance of the proliferative undifferentiated state of ExE (
The above model makes a number of testable predictions about the involvement of FGF-signaling in trophoblast development. For example, the model predicts that TS cell lines could be derived from Fgf4, but not Fgfr2 mutant blastocysts. Establishing TS cell lines from other mouse mutants will reveal the genes essential for this stem cell lineage, while in vitro differentiation of mutant lines will identify genes important for other trophoblast subtypes. In summary, the establishment of FGF4-dependent TS cell lines from blastocysts and the ExE of 6.5 dpc embryos has revealed that a stem cell population exists within the trophoblast lineage for at least a 3-day window during early development and that the essential embryo-derived signals for trophoblast proliferation include FGF4. These cell lines are an invaluable tool to further dissect the function of genes and signaling pathways important to the development of the mammalian trophoblast lineage and its interactions with the embryo. The ability of wild type TS cells to make high contributions in chimeras indicates that these cells have the potential to rescue mutant embryos with placental defects. Such “TS cell rescue” analysis could be an alternative to the “tetraploid rescue” technique (27) currently used. Finally, obtaining similar trophoblast stem cell lines from human embryos opens up new avenues to future cell-based therapies for placental insufficiencies.
While the present invention has been described with reference to what is presently considered to be a preferred embodiment, it is to be understood that the invention is not limited to the disclosed embodiment. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
REFERENCES AND NOTES
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- 9. TS cell medium is RPMI 1640 supplemented with 20% fetal bovine serum (HyClone), sodium pyruvate (1 mM, GibcoBRL), β-mercaptoethanol (100 μM, Sigma), L-glutamine (2 mM, GibcoBRL), and penicillin/streptomycin (50 μg/ml each). Human recombinant FGF4 (25 ng/ml, Sigma) and heparin (1 μg/ml) were added to aliquots of TS cell medium and used immediately.
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- 12. TS3.5 cell lines were obtained using similar techniques for ES cell line derivation (10). Briefly, 3.5 dpc blastocysts were individually plated into 4-well plates on EMFI cells and cultured in TS media with FGF4 and heparin (9). The medium was changed after two days and the blastocyst outgrowth was trypsinized on the third day. On day 5 or 6, flat colonies, referred to as “epithelial-like cells” in (10), were picked and passaged. Once established, the cell lines were grown without EMFI cells, but in the presence of EMFI conditioned medium (13). Under the current culture conditions ES cell colonies were not observed.
- 13. Conditioned medium from EMFI cells (EMFI-CM) was prepared by incubating TS medium (9) without FGF4 or heparin on confluent plates of mitomycin-treated EMFI cells for 72 hours. The conditioned medium was filtered (0.45 μm) and stored at −20° C. Established TS cell lines were routinely cultured in 70% EMFI-CM, 30% TS medium, 25 ng/ml hrFGF4, and 1 μg/ml heparin on gelatin-coated plates. The medium was changed every two days and the cells were passaged (1 in 25) every four days or at 80%-90% confluency.
- 14. TS cells were grown in the absence of EMFI cells (13) and collected by cell scraping at 0, 2, 4, and 6 days after the removal of FGF4, heparin, and EMFI-CM. The cells were fixed and stained with propidium iodide (Molecular Probes) as described [Z. Darzynkiewicz and G. Juan, in Current Protocols in Cytometry (John Wiley & Sons, Inc., New York, 1997), pp. 7.5.2-7.5.3]. Cell fluorescence was measured by a flow cytometry with an argon ion laser (488 nm). The data was analyzed with Coulter EXPO Cytometer Software version 2.0 by Applied Cytometry Systems, 1998.
- 15. Total RNA was prepared from cells and embryos with TRIzol (GibcoBRL) according to the manufacturer's instructions. Northern blotting was performed by a standard protocol. Antisense RNA probes for Errβ (16), eomesodermin (18), Cdx2 [E. Suh, L. Chen, J. Taylor, P. G. Traber, Mol. Cell. Biol. 14, 7340 (1994)], Fgfr2, Mash2 (20), 4311 (19), Hand1 (22), Pl-1 [P. Colosi, F. Talamantes, D. I. H. Linzer, Mol. Endocrinol. 1, 767 (1987)], Oct-3/4 (23), Brachyury (24), and GAPDH [P. Fort et al., Nucleic Acids Res. 13, 1431 (1985)] were labeled with either [α-32P]UTP or DIG-11-UTP (Boehringer Mannheim) by using Strip-EZ RNA kit (Ambion). Blots were hybridized overnight at 65° C. in NorthernMax Prehybridizatiorn/hybridization Buffer (Ambion) and were finally washed in 0.1×SSC/0.1% SDS at 65° C. DIG-labeled probes were detected with the DIG Luminescent Detection Kit (Boehringer Mannheim). Removal of hybridized RNA probes was performed with the Strip-EZ RNA kit (Ambion) according to manufacturer's recommendations. To assess the expression of Hnf4 in the TS cell lines, first strand cDNA synthesized from 0.5 μg total RNA of TS cells and 7.5 dpc embryos with random hexamers was subjected to 35 cycles of PCR (62° C. annealing temperature) by using 0.2 μM each of Hnf4-specific primers (5′-CACGTCCCCATCTGAAGGTG-3′ and 5′-CTTCCTTCTTCATGCCAGCCC-3′) and 0.1 μM each of β-actin-specific primers (5′-GACAACGGCTCCGGCATGTGCAAAG-3′ and 5′-TTCACGGTTGGCCTTAGGGTTCAG-3). The primer sequences were adapted from D. Ioannis et al., Development 125, 1529 (1998).
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Claims
1. A stable pluripotent trophoblast stem (TS) cell line.
2. A purified preparation of trophoblast stem cells which (i) are capable of indefinite proliferation in vitro in an undifferentiated state; and (ii) are capable of differentiation into cells of the trophoblast lineage in vivo.
3. A purified preparation as claimed in claim 2 which is further characterized by expression of genetic markers of diploid trophoblast cells.
4. A purified preparation as claimed in claim 2 wherein the cells are differentiated into cells of the trophoblast lineage.
5. A purified cell preparation as claimed in claim 4 characterized by expression of genetic markers of diploid trophoblast cells of the ectoplacental cone (EPC), and the secondary giant cells of the early conceptus.
6. (canceled)
7. A purified cell preparation as claimed in claim 6 modified by introducing mutations into genes in the cells or by introducing transgenes into the cells.
8. A method for producing a trophoblast cell line comprising culturing early postimplantation trophoblast cells or cells of a blastocyst on a feeder layer in the presence of FGF4, and a co-factor.
9. A method as claimed in claim 8 additionally comprising inducing differentiation of the cells of the cell line to cells of the trophoblast lineage by removing the FGF4, the co-factor, or the feeder layer.
10. A method as claimed in claim 8 wherein the early postimplantation trophoblast cells or cells of a blastocyst are isolated from a mammalian or marsupial species.
11. A method as claimed in claim 8 wherein the early postimplantation trophoblast cells or cells of a blastocyst are isolated from a rodent, rabbit, sheep, goat, pig, cattle, primate, or human.
12. A method as claimed in claim 8 wherein the early postimplantation trophoblast cells or cells of a blastocyst are transgenic.
13. A method as claimed in claim 8 wherein the feeder layer is a confluent fibroblast layer or a medium conditioned by primary embryonic fibroblast cells.
14. A method as claimed in claim 8 wherein the feeder layer comprises primary mouse embryonic fibroblast (EMFI) cells or STO cells.
15. A method as claimed in claim 8 wherein the FDF4 is recombinant FGF4 and the cofactor is heparin.
16. A method as claimed in claim 8 which further comprises introducing cells from the cell line into a blastocyst or aggregating the cells with an early stage embryo to produce chimeric conceptuses or placenta.
17. A method as claimed in claim 16 wherein the chimeric conceptuses or placenta are engineered to carry selectable markers or genetic alterations.
18. A method as claimed in claim 16 wherein cell lines are derived from the chimeric conceptuses or chimeric placenta.
19. A chimeric conceptus derived from a purified preparation as claimed in claim 2.
20. A chimeric placenta derived from a purified preparation as claimed in claim 2.
21-22. (canceled)
23. A method as claimed in claim 22 wherein the mammal is a human.
Type: Application
Filed: Aug 6, 2003
Publication Date: Sep 1, 2005
Applicant: Mount Sinai Hospital (Toronto)
Inventors: Janet Rossant (Toronto), Satoshi Tanaka (Tokyo), Tilo Kunath (Toronto)
Application Number: 10/635,923