Human Placenta Feeder Layers Support Undifferentiated Growth of Primate Embryonic Stem Cells
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《干细胞学杂志》
a Tokyo Metropolitan Institute of Technology, Systems Engineering Science, Hino, Tokyo, Japan;
b Okayama University Graduate School of Medicine and Dentistry, Department of Pathology, Okayama, Okayama, Japan;
c Hino Municipal Hospital, Hino, Tokyo, Japan;
d Kibi International University, Health Science, Takahashi, Okayama, Japan;
e Kanagawa Academy of Science and Technology, Kawasaki, Kanagawa, Japan
Key Words. Primate ES cells ? Feeder layer ? Human placenta ? Teratoma
Correspondence: Kanji Miyamoto, Ph.D., Department of Systems Engineering Science, Tokyo Metropolitan Institute of Technology, Asahigaoka 6-6, Hino, Tokyo 191-0065, Japan. Telephone: 81-42-585-8641; Fax: 81-42-585-8641; e-mail: kmiyamot@cc.tmit.ac.jp
ABSTRACT
Embryonic stem (ES) cells are derived from the inner cell mass of preimplantation embryos, and embryonic germ (EG) cells are derived from primordial germ cells (PGCs) . Both ES and EG cells are pluripotent and form immature teratomas that contain derivatives of all three embryonic germ layers. In the absence of a feeder cell layer or a leukemia inhibitory factor (LIF), ES cells assume an apparently differentiated state. Mouse embryonic fibroblast (MEF) feeder cells can support the growth of undifferentiated mouse, rat, bovine, primate, and human embryonic stem (HES) cells , and a fibroblast cell line (STO), derived from mouse embryos, has been used as a feeder layer for the growth of human primordial germ cells . However, animal pathogens can be transferred from MEF feeders to both human ES and EG cells. Therefore, it was necessary to show that human fetal muscle, fetal skin, human adult fallopian tube epithelial feeder layers, and human adult marrow stromal cells supported the prolonged undifferentiated growth of HES cells and are superior to cell-free matrices such as collagen 1, human extracellular matrix, matrigel, and laminin supplemented with human or MEF feeder–conditioned medium . However, human fetal and adult cells may still be unsuitable feeder layers because of ethical and practical limitations.
We evaluated the growth of primate ES cells (cynomolgus monkey ES cells; CMK6) on human amniotic epithelial (HAE) feeder cells and human chorionic plate (HCP) feeder cells derived from human placentas. Primate ES cells were used because they were of less ethical concern and human ES cells could not be used without the permission of the Japanese Government. The primate ES cells have many biological characteristics similar to human ES cells . As the cynomolgus and Rhesus monkeys are closely related to humans, and because they are widely used for medical research, cynomolgus monkey ES cells would be valuable for preclinical research before the clinical usage of human ES cells .
MATERIALS AND METHODS
The monkey ES cell line, CMK6, has been biologically characterized by Suemori et al. . Its behavior on HAE feeder cells, HAE feeder cells treated with DAS, HCP feeder layers, and HCP feeder layers treated with DAS was very similar to its undifferentiated growth on MEF feeder cells (Fig. 1B, D, F, H, and J). HAE feeder cells have supported the growth of undifferentiated CMK6 ES cells for at least 18 passages. CMK6 ES cell colonies grown in this manner require passage about every 8 days and remain morphologically intact. CMK6 ES cell colonies on HAE feeder cells and HAE feeder cells treated with DAS appeared to be more homologous with undifferentiated clones. CMK6 ES cells on HAE feeder cells were also cultured with F-12/D-MEM containing 10% inactivated human cord serum instead of 20% KSR. The CMK6 ES cells showed the same morphological characteristics and growth behaviors.
Figure 1. Morphology of primate embryonic stem (ES) cells (CMK6) on mouse embryonic fibroblast (MEF), human amniotic epithelial (HAE), and human chorionic plate (HCP) feeder layers. (A): MEF feeder cells. (B): Colony of CMK6 ES cells on MEF feeder cells. (C): HAE feeder layers cultured for 3 days. (D): Colonies of CMK6 ES cells after 18 passages (about 180 days) on HAE feeder cells. (E): HAE feeder layers treated with diluted ammonia solution (DAS). (F): Colonies of CMK6 ES cells on HAE treated with DAS. (G): HCP feeder layers cultured for 3 days. (H): Colonies of CMK6 ES cells on HCP feeder layers. (I): HCP feeder layers treated with DAS. (J): Colonies of CMK6 ES cells on HCP feeder layers with DAS. Magnification (A–J): x80.
CMK6 ES cell colonies grown on HAE feeder layers, HAE feeder layers treated with DAS, HCP feeder layers, and HCP feeder layers treated with DAS expressed cell surface markers that characterize undifferentiated CMK6 ES cells—a membrane alkaline phosphatase activity (ALP) (Fig. 2F), stage-specific embryonic antigen (SSEA)-1 negative (Fig. 2H), SSEA-4 positive (Fig. 2I), the transcription factor Oct-4 expression positive by RT-PCR, and they displayed the normal karyotype, 40, XY. Oct-4 expression in CMK6 ES cells was assessed using Rhesus monkey Oct-4 primers and human Oct-4 primers . The RT-PCR product bands were 697 bp and 241 bp, respectively (Fig. 3). Rhesus monkey Oct-4 primers were demonstrated to work on a cynomolgus monkey by Mitalipov et al. , since this Oct-4 mRNA sequence has more than 98% homology with the human Oct-4 sequence. The positive control used for the RT-PCRs was CMK6 ES cells on MEF feeder cells (Fig. 2E, G, and Fig. 3).
Figure 2. Morphology of biological characterization human amniotic epithelial (HAE), human chorionic plate (HCP) feeder layers, and primate embryonic stem (ES; CMK6) cells with undifferentiated markers on HAE, HCP feeder cells, and feeder-free matrices. (A): Cultured HAE feeder cells reacted with 1:50 diluted monoclonal mouse antihuman cytokeratin 18 antibodies. (B): Cultured HCP feeder cells with 1:50 diluted monoclonal mouse antihuman cytokeratin 18 antibodies. (C): Cultured HAE feeder cells negative reacted with 1:50 diluted monoclonal mouse antivimentin clone V9 antibodies. (D): Cultured HCP feeder cells reacted with 1:50 diluted monoclonal mouse antivimentin clone V9 antibodies. (E): Expression of alkaline phosphatase (ALP)–positive CMK6 ES cells on mouse embryonic fibroblast (MEF) feeder layers. (F): Expression of ALP-positive CMK6 ES cells on HCP feeder layers. (G): Stage-specific embryonic antigen (SSEA)-1–negative CMK6 ES cells on MEF feeder layers. (H): SSEA-1–negative CMK6 ES cells on HAE feeder layers. (I): SSEA-4–positive CMK6 ES cells on HAE feeder layers. (J): Differentiated CMK6 ES cells on gelatin coat dish. Magnification (A–J): x80.
Figure 3. Oct-4 expression of CMK6 embryonic stem (ES) cells on mouse embryonic fibroblast (MEF), human amniotic epithelial (HAE), and human chorionic plate (HCP) feeder layers by reverse transcription polymerase chain reaction (RT-PCR). Lane M: X174 DNA/Hinf I markers. Lane 1: CMK6 ES cells on HAE feeder layers by use of human Oct-4 primers; Lane 2, CMK6 ES cells on HAE feeder layers by use of monkey Oct-4 primers. Lane 3: CMK6 ES cells on HCP feeder layers by use of human Oct-4 primers. Lane 4: CMK6 ES cells on HCP feeder layers by use of monkey Oct-4 primers. Lane 5: CMK6 ES cells on MEF feeder layers by use of human Oct-4 primers. Lane 6: CMK6 ES cells on MEF feeder layers by use of monkey Oct-4 primers. Lane 7: ?-actin of CMK6 ES cells on HAE feeder layers. Lane 8: Negative control CMK6 ES cells on HAP feeder layers by use of human Oct-4 primers. Human Oct-4, 241-bp product; monkey Oct-4, 697-bp product; ?-actin, 400-bp product.
To investigate the biological characteristics of human placenta feeder cells, cultured HAE and HCP feeder cells were examined with monoclonal mouse antihuman cytokeratin 18 antibodies or monoclonal mouse antivimentin clone V9 antibodies. HAE and HCP feeder cells were positive for cytokeratin 18 antibodies (Fig. 2A, B) and had the characteristics of epithelial cells. In contrast, HCP feeder cells have the characteristics of mesenchymal cells (Fig. 2D, C). The doubling time for HCP feeder cells was about 4.5 days, but HAE feeder cells were slower growing, dividing only 1.5 times within 2 weeks.
The cultured CMK6 ES cells on HAE feeder cells produced typical immature teratomas in vivo after injection into SCID mice. The teratoma had abundant, unambiguous derivatives of all three embryonic germ layers, including immature neural tissues with melanin, cartilage, muscle, and glands (Fig. 4).
Figure 4. Histology of CMK6 embryonic stem (ES) cells that developed into tumors when transplanted into severe combined immun-odeficient mice. (A, B, C): CMK6 ES cells cultured on human amniotic feeder cells were histologically identified as immature teratoma containing immature neural tissues with melanin, cartilage, muscle, and glands. (D, E, F): Immunohistochemistry of the tumors revealed cytokeratin-positive glands (D), glial fibrillary acidic protein (GFAP)–positive tissues (E), and myoglobin-positive striated muscle (F). A, B, C: Hematoxylin and eosin stain 550; D: cytokeratin 550; E: GFAP550; F: myoglobin 5100.
Previously, Gospodarowicz et al. reported that corneal endothelial cells maintained in tissue culture retain their abilities to synthesize and to secrete an extracellular matrix (ECM) along their basal cell surface. They showed that treatment of confluent cells with 0.5% Triton X-100 resulted in the removal of the cell monolayer, thereby exposing the ECM, which had the permissive effect on cell proliferation as a substrate. Therefore, to investigate the activity of membrane proteins on HAE and HCP feeder cells, the cells were treated with a 2% glutaraldehyde solution, with methanol-acetic acid solution (3:1 v/v), with 0.5% Triton X-100 solution, or with DAS. As a result, CMK6 ES cells were primarily differentiated on HAE feeder layers treated with glutaralde-hyde solution, with methanol-acetic acid solution, and with Triton X-100 solution. However, CMK6 ES cells on HAE feeder cells treated with DAS, and HCP treated with DAS, were undifferentiated and formed colonies (Fig. 1F, J). But CMK6 ES cells cultured on cell-free feeder matrices such as gelatin, laminin, fibronectin, collagen type 1 coated dishes, and Matrigel were shown to differentiate in these culture media (Fig. 2J). These data suggest that human feeder layers are superior to the matrices .
We found that both HAE and HCP feeder cells in the presence of animal-free culture media could support primate ES cells in an undifferentiated state. The HAE and HCP feeder cells may secrete some proteins in culture medium, or they may maintain some membrane proteins for undifferentiated growth of ES cells. Koizumi et al. reported eight growth factors (epidermal growth factor, keratinocyte growth factor, hepatocyte growth factor, basic fibroblast growth factor, transforming growth factor alpha , TGF-?1, TGF-?2, and TGF-?3) and two growth receptors (keratinocyte growth factor receptor and hepatocyte growth factor receptor) in preserved human amniotic membrane. Recently, Sakuragawa et al. characterized neuron-like cells that expressed choline acetyltransferase and acetylcholine among HAE cells. Tsai et al. have reconstructed damaged corneas by transplantation with autologous limbal epithelial cells cultured on HAE feeder cells. Considering that HAE cells have no major histocompatibility complex (MHC) class II and fewer MHC class I molecules, no significant graft versus host reaction should occur in patients. Human placenta is quite novel and important because it would provide a relatively available source of feeders for the growth of human ES cells for therapeutic purposes that are also free of ethical complications. Considering the similarity of primate and human ES cells, the feeder cells should be effective on human ES cells. We are now planning to culture human ES cells on the HAE or HCP cells.
REFERENCES
Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditions by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 1981;78:7634–7638.
Matsui Y, Toksoz D, Nishikawa S et al. Effect of steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture. Nature 1991;353:750–752.
Resnick JL, Bixler LS, Cheng L et al. Long-term proliferation of mouse primordial germ cell in culture. Nature 1992;359:550–551.
Shamblott MJ, Axelman, J, Wang S et al. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci U S A 1998;95:13726–13731.
Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981;292:154–156.
Saito S, Strelchenko N, Niemann H. Bovine embryonic stem cell-like cell lines cultured over several passages. Dev Biol 1992;201:134–141.
Suemori H, Tada T, Torii R et al. Establishment of embryonic stem cell lines from cynomologus monkey blastocytes produced IVF or ICSI. Dev Dyn 2001;22:273–279.
Thomson JA, Kalishman J, Golos TG et al. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci U S A 1995;92:7844–7848.
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
Vassilieva S, Guan K, Pich U et al. Establishment of SSEA-1 and Oct-4 expressing rat embryonic stem-like cell lines and effects of cytokines of the IL-6 family on clonal growth. Exp Cell Res 2000;258:361–373.
Yagi T, Tokunaga T, Fukuta Y et al. Anovel ES cell line, TT2, with high germline-differentiating potency. Anal Biochem 1993;214:70–76.
Cheng L, Hammond H, Ye Z et al. Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. STEM CELLS 2003;21:131–142.
Richards M, Fong C-Y, Chan W-K et al. Human feeder support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 2002;20:933–936.
Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19:971–974.
Mitalipov SM, Kuo HC, Hennebold JD et al. Oct-4 expression in pluripotent cells of the Rhesus monkey. Biol Reprod 2003;69:1785–1792.
Gospodarowicz D, Delgado D, Vlodavsky I. Permissive effect of the extracellular matrix on cell proliferation in vitro. Proc Natl Acad Sci U S A 1980;77:4094–4098.
Koizumi NJ, Inatomi TJ, Sotozono CJ et al. Growth factor mRNA and protein in preserved human amniotic membrance. Curr Eye Res 2000;20:173–177.
Sakuragawa N, Misawa H, Ohsugi K et al. Evidence for active acetylcholine metabolism in human AE cells applicable to introcerebral allografting for neurologic disease. Neurosci Lett 1997;232:53–56.
Tsai RJ-F, Li L-M, Chen J-K. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med 2000;343:86–93.(Kanji Miyamotoa, Kazuhiko)
b Okayama University Graduate School of Medicine and Dentistry, Department of Pathology, Okayama, Okayama, Japan;
c Hino Municipal Hospital, Hino, Tokyo, Japan;
d Kibi International University, Health Science, Takahashi, Okayama, Japan;
e Kanagawa Academy of Science and Technology, Kawasaki, Kanagawa, Japan
Key Words. Primate ES cells ? Feeder layer ? Human placenta ? Teratoma
Correspondence: Kanji Miyamoto, Ph.D., Department of Systems Engineering Science, Tokyo Metropolitan Institute of Technology, Asahigaoka 6-6, Hino, Tokyo 191-0065, Japan. Telephone: 81-42-585-8641; Fax: 81-42-585-8641; e-mail: kmiyamot@cc.tmit.ac.jp
ABSTRACT
Embryonic stem (ES) cells are derived from the inner cell mass of preimplantation embryos, and embryonic germ (EG) cells are derived from primordial germ cells (PGCs) . Both ES and EG cells are pluripotent and form immature teratomas that contain derivatives of all three embryonic germ layers. In the absence of a feeder cell layer or a leukemia inhibitory factor (LIF), ES cells assume an apparently differentiated state. Mouse embryonic fibroblast (MEF) feeder cells can support the growth of undifferentiated mouse, rat, bovine, primate, and human embryonic stem (HES) cells , and a fibroblast cell line (STO), derived from mouse embryos, has been used as a feeder layer for the growth of human primordial germ cells . However, animal pathogens can be transferred from MEF feeders to both human ES and EG cells. Therefore, it was necessary to show that human fetal muscle, fetal skin, human adult fallopian tube epithelial feeder layers, and human adult marrow stromal cells supported the prolonged undifferentiated growth of HES cells and are superior to cell-free matrices such as collagen 1, human extracellular matrix, matrigel, and laminin supplemented with human or MEF feeder–conditioned medium . However, human fetal and adult cells may still be unsuitable feeder layers because of ethical and practical limitations.
We evaluated the growth of primate ES cells (cynomolgus monkey ES cells; CMK6) on human amniotic epithelial (HAE) feeder cells and human chorionic plate (HCP) feeder cells derived from human placentas. Primate ES cells were used because they were of less ethical concern and human ES cells could not be used without the permission of the Japanese Government. The primate ES cells have many biological characteristics similar to human ES cells . As the cynomolgus and Rhesus monkeys are closely related to humans, and because they are widely used for medical research, cynomolgus monkey ES cells would be valuable for preclinical research before the clinical usage of human ES cells .
MATERIALS AND METHODS
The monkey ES cell line, CMK6, has been biologically characterized by Suemori et al. . Its behavior on HAE feeder cells, HAE feeder cells treated with DAS, HCP feeder layers, and HCP feeder layers treated with DAS was very similar to its undifferentiated growth on MEF feeder cells (Fig. 1B, D, F, H, and J). HAE feeder cells have supported the growth of undifferentiated CMK6 ES cells for at least 18 passages. CMK6 ES cell colonies grown in this manner require passage about every 8 days and remain morphologically intact. CMK6 ES cell colonies on HAE feeder cells and HAE feeder cells treated with DAS appeared to be more homologous with undifferentiated clones. CMK6 ES cells on HAE feeder cells were also cultured with F-12/D-MEM containing 10% inactivated human cord serum instead of 20% KSR. The CMK6 ES cells showed the same morphological characteristics and growth behaviors.
Figure 1. Morphology of primate embryonic stem (ES) cells (CMK6) on mouse embryonic fibroblast (MEF), human amniotic epithelial (HAE), and human chorionic plate (HCP) feeder layers. (A): MEF feeder cells. (B): Colony of CMK6 ES cells on MEF feeder cells. (C): HAE feeder layers cultured for 3 days. (D): Colonies of CMK6 ES cells after 18 passages (about 180 days) on HAE feeder cells. (E): HAE feeder layers treated with diluted ammonia solution (DAS). (F): Colonies of CMK6 ES cells on HAE treated with DAS. (G): HCP feeder layers cultured for 3 days. (H): Colonies of CMK6 ES cells on HCP feeder layers. (I): HCP feeder layers treated with DAS. (J): Colonies of CMK6 ES cells on HCP feeder layers with DAS. Magnification (A–J): x80.
CMK6 ES cell colonies grown on HAE feeder layers, HAE feeder layers treated with DAS, HCP feeder layers, and HCP feeder layers treated with DAS expressed cell surface markers that characterize undifferentiated CMK6 ES cells—a membrane alkaline phosphatase activity (ALP) (Fig. 2F), stage-specific embryonic antigen (SSEA)-1 negative (Fig. 2H), SSEA-4 positive (Fig. 2I), the transcription factor Oct-4 expression positive by RT-PCR, and they displayed the normal karyotype, 40, XY. Oct-4 expression in CMK6 ES cells was assessed using Rhesus monkey Oct-4 primers and human Oct-4 primers . The RT-PCR product bands were 697 bp and 241 bp, respectively (Fig. 3). Rhesus monkey Oct-4 primers were demonstrated to work on a cynomolgus monkey by Mitalipov et al. , since this Oct-4 mRNA sequence has more than 98% homology with the human Oct-4 sequence. The positive control used for the RT-PCRs was CMK6 ES cells on MEF feeder cells (Fig. 2E, G, and Fig. 3).
Figure 2. Morphology of biological characterization human amniotic epithelial (HAE), human chorionic plate (HCP) feeder layers, and primate embryonic stem (ES; CMK6) cells with undifferentiated markers on HAE, HCP feeder cells, and feeder-free matrices. (A): Cultured HAE feeder cells reacted with 1:50 diluted monoclonal mouse antihuman cytokeratin 18 antibodies. (B): Cultured HCP feeder cells with 1:50 diluted monoclonal mouse antihuman cytokeratin 18 antibodies. (C): Cultured HAE feeder cells negative reacted with 1:50 diluted monoclonal mouse antivimentin clone V9 antibodies. (D): Cultured HCP feeder cells reacted with 1:50 diluted monoclonal mouse antivimentin clone V9 antibodies. (E): Expression of alkaline phosphatase (ALP)–positive CMK6 ES cells on mouse embryonic fibroblast (MEF) feeder layers. (F): Expression of ALP-positive CMK6 ES cells on HCP feeder layers. (G): Stage-specific embryonic antigen (SSEA)-1–negative CMK6 ES cells on MEF feeder layers. (H): SSEA-1–negative CMK6 ES cells on HAE feeder layers. (I): SSEA-4–positive CMK6 ES cells on HAE feeder layers. (J): Differentiated CMK6 ES cells on gelatin coat dish. Magnification (A–J): x80.
Figure 3. Oct-4 expression of CMK6 embryonic stem (ES) cells on mouse embryonic fibroblast (MEF), human amniotic epithelial (HAE), and human chorionic plate (HCP) feeder layers by reverse transcription polymerase chain reaction (RT-PCR). Lane M: X174 DNA/Hinf I markers. Lane 1: CMK6 ES cells on HAE feeder layers by use of human Oct-4 primers; Lane 2, CMK6 ES cells on HAE feeder layers by use of monkey Oct-4 primers. Lane 3: CMK6 ES cells on HCP feeder layers by use of human Oct-4 primers. Lane 4: CMK6 ES cells on HCP feeder layers by use of monkey Oct-4 primers. Lane 5: CMK6 ES cells on MEF feeder layers by use of human Oct-4 primers. Lane 6: CMK6 ES cells on MEF feeder layers by use of monkey Oct-4 primers. Lane 7: ?-actin of CMK6 ES cells on HAE feeder layers. Lane 8: Negative control CMK6 ES cells on HAP feeder layers by use of human Oct-4 primers. Human Oct-4, 241-bp product; monkey Oct-4, 697-bp product; ?-actin, 400-bp product.
To investigate the biological characteristics of human placenta feeder cells, cultured HAE and HCP feeder cells were examined with monoclonal mouse antihuman cytokeratin 18 antibodies or monoclonal mouse antivimentin clone V9 antibodies. HAE and HCP feeder cells were positive for cytokeratin 18 antibodies (Fig. 2A, B) and had the characteristics of epithelial cells. In contrast, HCP feeder cells have the characteristics of mesenchymal cells (Fig. 2D, C). The doubling time for HCP feeder cells was about 4.5 days, but HAE feeder cells were slower growing, dividing only 1.5 times within 2 weeks.
The cultured CMK6 ES cells on HAE feeder cells produced typical immature teratomas in vivo after injection into SCID mice. The teratoma had abundant, unambiguous derivatives of all three embryonic germ layers, including immature neural tissues with melanin, cartilage, muscle, and glands (Fig. 4).
Figure 4. Histology of CMK6 embryonic stem (ES) cells that developed into tumors when transplanted into severe combined immun-odeficient mice. (A, B, C): CMK6 ES cells cultured on human amniotic feeder cells were histologically identified as immature teratoma containing immature neural tissues with melanin, cartilage, muscle, and glands. (D, E, F): Immunohistochemistry of the tumors revealed cytokeratin-positive glands (D), glial fibrillary acidic protein (GFAP)–positive tissues (E), and myoglobin-positive striated muscle (F). A, B, C: Hematoxylin and eosin stain 550; D: cytokeratin 550; E: GFAP550; F: myoglobin 5100.
Previously, Gospodarowicz et al. reported that corneal endothelial cells maintained in tissue culture retain their abilities to synthesize and to secrete an extracellular matrix (ECM) along their basal cell surface. They showed that treatment of confluent cells with 0.5% Triton X-100 resulted in the removal of the cell monolayer, thereby exposing the ECM, which had the permissive effect on cell proliferation as a substrate. Therefore, to investigate the activity of membrane proteins on HAE and HCP feeder cells, the cells were treated with a 2% glutaraldehyde solution, with methanol-acetic acid solution (3:1 v/v), with 0.5% Triton X-100 solution, or with DAS. As a result, CMK6 ES cells were primarily differentiated on HAE feeder layers treated with glutaralde-hyde solution, with methanol-acetic acid solution, and with Triton X-100 solution. However, CMK6 ES cells on HAE feeder cells treated with DAS, and HCP treated with DAS, were undifferentiated and formed colonies (Fig. 1F, J). But CMK6 ES cells cultured on cell-free feeder matrices such as gelatin, laminin, fibronectin, collagen type 1 coated dishes, and Matrigel were shown to differentiate in these culture media (Fig. 2J). These data suggest that human feeder layers are superior to the matrices .
We found that both HAE and HCP feeder cells in the presence of animal-free culture media could support primate ES cells in an undifferentiated state. The HAE and HCP feeder cells may secrete some proteins in culture medium, or they may maintain some membrane proteins for undifferentiated growth of ES cells. Koizumi et al. reported eight growth factors (epidermal growth factor, keratinocyte growth factor, hepatocyte growth factor, basic fibroblast growth factor, transforming growth factor alpha , TGF-?1, TGF-?2, and TGF-?3) and two growth receptors (keratinocyte growth factor receptor and hepatocyte growth factor receptor) in preserved human amniotic membrane. Recently, Sakuragawa et al. characterized neuron-like cells that expressed choline acetyltransferase and acetylcholine among HAE cells. Tsai et al. have reconstructed damaged corneas by transplantation with autologous limbal epithelial cells cultured on HAE feeder cells. Considering that HAE cells have no major histocompatibility complex (MHC) class II and fewer MHC class I molecules, no significant graft versus host reaction should occur in patients. Human placenta is quite novel and important because it would provide a relatively available source of feeders for the growth of human ES cells for therapeutic purposes that are also free of ethical complications. Considering the similarity of primate and human ES cells, the feeder cells should be effective on human ES cells. We are now planning to culture human ES cells on the HAE or HCP cells.
REFERENCES
Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditions by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 1981;78:7634–7638.
Matsui Y, Toksoz D, Nishikawa S et al. Effect of steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture. Nature 1991;353:750–752.
Resnick JL, Bixler LS, Cheng L et al. Long-term proliferation of mouse primordial germ cell in culture. Nature 1992;359:550–551.
Shamblott MJ, Axelman, J, Wang S et al. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci U S A 1998;95:13726–13731.
Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981;292:154–156.
Saito S, Strelchenko N, Niemann H. Bovine embryonic stem cell-like cell lines cultured over several passages. Dev Biol 1992;201:134–141.
Suemori H, Tada T, Torii R et al. Establishment of embryonic stem cell lines from cynomologus monkey blastocytes produced IVF or ICSI. Dev Dyn 2001;22:273–279.
Thomson JA, Kalishman J, Golos TG et al. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci U S A 1995;92:7844–7848.
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
Vassilieva S, Guan K, Pich U et al. Establishment of SSEA-1 and Oct-4 expressing rat embryonic stem-like cell lines and effects of cytokines of the IL-6 family on clonal growth. Exp Cell Res 2000;258:361–373.
Yagi T, Tokunaga T, Fukuta Y et al. Anovel ES cell line, TT2, with high germline-differentiating potency. Anal Biochem 1993;214:70–76.
Cheng L, Hammond H, Ye Z et al. Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. STEM CELLS 2003;21:131–142.
Richards M, Fong C-Y, Chan W-K et al. Human feeder support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 2002;20:933–936.
Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19:971–974.
Mitalipov SM, Kuo HC, Hennebold JD et al. Oct-4 expression in pluripotent cells of the Rhesus monkey. Biol Reprod 2003;69:1785–1792.
Gospodarowicz D, Delgado D, Vlodavsky I. Permissive effect of the extracellular matrix on cell proliferation in vitro. Proc Natl Acad Sci U S A 1980;77:4094–4098.
Koizumi NJ, Inatomi TJ, Sotozono CJ et al. Growth factor mRNA and protein in preserved human amniotic membrance. Curr Eye Res 2000;20:173–177.
Sakuragawa N, Misawa H, Ohsugi K et al. Evidence for active acetylcholine metabolism in human AE cells applicable to introcerebral allografting for neurologic disease. Neurosci Lett 1997;232:53–56.
Tsai RJ-F, Li L-M, Chen J-K. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med 2000;343:86–93.(Kanji Miyamotoa, Kazuhiko)