Establishment of Novel Embryonic Stem Cell Lines Derived from the Common Marmoset (Callithrix jacchus)
http://www.100md.com
《干细胞学杂志》
a Division of Laboratory Animal Science, Central Institute for Experimental Animals, Kawasaki, Kanagawa, Japan;
b Department of Urology, Urayasu Hospital, Juntendo University, Urayasu, Chiba, Japan;
c Department of Molecular Genetics, Division of Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Hakata, Fukuoka, Japan;
d Tokai University School of Medicine, Isehara, Kanagawa, Japan;
e Department of Physiology, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan;
f Research Project Center,
g Department of Genetics, Central Institute for Experimental Animals, Kawasaki, Kanagawa, Japan;
h Division of Molecular Therapy, Institute of Medical Science, University of Tokyo,
i Institute of Obstetrics & Gynecology in Clinical Medicine, University of Tsukuba,
j Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center,
k Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
Key Words. Embryonic stem cells ? Common marmoset ? Embryoid body ? Nonhuman primate ? Teratoma formation
Correspondence: Kenzaburo Tani, M.D., Ph.D., Department of Molecular Genetics, Division of Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Hakata, Fukuoka 812-8582, Japan. Telephone: 81-92-642-6434; Fax: 81-92-642-6444; e-mail: taniken@bioreg.kyushu-u.ac.jp; for CMES cell distribution, contact Erika Sasaki at esasaki@ciea.or.jp
ABSTRACT
Embryonic stem cells (ESCs) are derived from preimplantation embryos. Owing to their pluripotency, ESCs have been widely used for the production of transgenic and gene knockout mice to elucidate the molecular mechanisms of various genes. In 1998, the successful establishment of human ESC (hESC) lines enhanced the role of ESCs in science and medicine. The most promising application of ESCs in the clinical setting is in the repair of defective organ function by differentiated ESCs. However, before differentiated hESCs can be used in clinical applications, the short-term and long-term safety and efficacy of ESCs must be thoroughly examined. Due to ethical considerations, these studies cannot be performed in vivo in humans. Therefore, preclinical studies using the ESCs of nonhuman primates are essential.
The common marmoset (Callithrix jacchus) is a New World primate species with reproductive characteristics that are appropriate for ESC studies. More specifically, these animals are small (weighing approximately 350–400 g), they have a short gestation period (approximately 144 days), and reach sexual maturity at 12–18 months. Unlike macaques, marmosets routinely deliver twins or triplets for each pregnancy. In addition, it is possible to synchronize the marmoset ovarian cycle with prostaglandin analogs, collect age-matched embryos from multiple females, and transfer embryos to synchronized recipients with success rates in the range of 70%–80% . Because these reproductive characteristics allow routine efficient transfer of multiple embryos, marmosets constitute an excellent primate species for the generation of transgenic and knockout animal models of human diseases.
In addition to these reproductive benefits, we have shown previously that marmosets are suitable laboratory animals for preclinical studies of stem cell therapies, owing to the similarities between the hematopoietic and immune systems of humans and marmosets . In 1996, Thomson et al. established pluripotent common marmoset cell lines, which are considered powerful tools for understanding the regulatory mechanisms of ESC differentiation both in vitro and in vivo. However, the differentiation abilities of the pluripotent common marmoset cells in terms of in vitro teratoma formation assays and certain common properties of ESC lines, such as differentiation to the cell lineages of three germ layers, have not been defined fully. The establishment of totipotent common marmoset ESC (CMESC) lines would facilitate the construction, by gene targeting, of nonhuman primate models for human disease. To achieve our goal of establishing the common marmoset as a human disease model, we have established novel CMESC lines and characterized their differentiation capacities.
MATERIALS AND METHODS
Establishment of CMESC Lines
The marmosets ovulated 10.7 ± 1.3 days (n = 70) after PGF2 administration. Our ovarian cycle control system made it possible to obtain fertilized eggs every 3 weeks from the same animals. Sixty immunosurgically isolated ICMs from 70 blastocysts (the ICM isolation rate was 85.7%) were plated on the irradiated MEF feeder layer, and 11 ICMs were cultured for more than 10 passages (18.3% derivation rate). To date, 3/11 ICM-derived cells have been cultured for more than 1 year. All ICM-derived cells showed flat, packed, and tight colony morphology and a high nucleus:cytoplasm ratio (Figs. 1A, 1B). The morphology of these derived cells was very similar to that of reported primate ESCs, including those from humans and from rhesus and cynomolgus monkeys . It has been reported that primate ESCs exhibit spontaneous differentiation during culture and that leukemia inhibitory factor does not maintain the ESCs in the undifferentiated state . The common marmoset ICM-derived cell lines also showed low frequency of differentiation. However, most ICM-derived cells maintained undifferentiated morphology of the cells. It is known that the quality of FBS is critical to the maintenance of undifferentiated primate ESCs; different lots of FBS from the same manufacturer can vary in this respect. Therefore, the chemically defined serum-free supplement KSR was used for the establishment and culture of the marmoset ESCs. The CMESC colonies appeared more packed and tight when KSR was used in the culture; they were flatter in appearance when FBS was used in the medium. To maintain stem cells, the dissociation procedure for cynomolgus ESCs was adopted for subculture of the marmoset ICM-derived cells . With this culture system, continuous cultures of CMESCs have been sustained for more than 1 year.
Figure 1. Expression of alkaline phosphatase and cell surface markers on CMESCs. Unstained CMESCs (A, B), cells stained for ALP (C), SSEA-1 (D), SSEA-3 (E), SSEA-4 (F), TRA-1-60 (G), and TRA-1-81 (H). Abbreviations: ALP, alkaline phosphatase; CMESC, common marmoset embryonic stem cell; SSEA, stage-specific embryonic antigen.
Characterization of Undifferentiated CMESC Lines
To confirm the undifferentiated status of the ICM-derived cells, all three lines (CMESC 20, 30, and 40) were examined for the expression of cell surface markers that are specific for undifferentiated ESCs. As shown in Figures 1C–1G, the ICM-derived cell lines showed alkaline phosphatase activity (Fig. 1C) and expressed SSEA-3 (Fig. 1E), SSEA-4 (Fig. 1F), TRA-1-60 (Fig. 1G), and TRA-1-81 (Fig. 1H) but not SSEA-1 (Fig. 1D). All three cell lines retained the normal 46, XX karyotype (Fig. 2) and telomerase (Fig. 3A) activity. These three ICM-derived cell lines also expressed Nanog, Oct3/4, Sox2, gp130, mCG, HEB, and Bex1/Rex3 mRNA and low levels of FoxD3 and Nestin mRNA (Fig. 4A). Conversely, LIFR mRNA was not detected by RT-PCR. All three lines showed identical expression patterns of the genes. Therefore, we believe that these cells are CMESCs. To compare the gene expression patterns of fresh ICMs and the ICM-derived cell lines, RT-PCR analysis was conducted using fresh ICMs. As a result, the expression of Nanog, Oct3/4, and Sox2 mRNA was observed, whereas FoxD3 and Nestin mRNA was not detected in fresh ICMs (Fig. 4B).
Figure 2. Karyotype analysis of CMESCs. The results for CIEA-CMESC #20 are shown. All three cell lines show the normal 46, XX karyotype after more than 6 months of culture. Abbreviations: CIEA, Central Institute for Experimental Animals; CMESC, common marmoset embryonic stem cell.
Figure 3. (A): Telomerase activities of CMESC lines. All three CMESC lines show high telomerase activity. On the other hand, MEF feeder layer does not show telomerase activity. (B): SSCP analysis of CMESCs for the MHC-DRB1 gene. The results of SSCP indicate that all three lines were derived independently. Abbreviations: CMESC, common marmoset embryonic stem cell; MEF, mouse embryonic fibroblast; SSCP, single strand conformation polymorphism.
Figure 4. (A): RT-PCR analysis of CMESCs and EBs. Undifferentiated CMESCs expressed the Nanog, Oct3/4, Sox2, FoxD3, Bex1/ Rex3, Heb and mCG, GP130, and low level of Nestin genes. Two-week cultures of EBs display expression of Nestin and CD34. Three-week cultures of EBs show expression of Nestin, CD34, and -fetoprotein. Oct3/4 and Nanog gene expression is shut down in the EBs. (B): RT-PCR analysis of fresh ICMs. ICMs expressed the Nanog, Oct3/4, and Sox2 genes. Expression of FoxD3 and Nestin was not detected. Abbreviations: CMESC, common marmoset embryonic stem cell; EB, embryoid body; ICM, inner cell mass; RT-PCR, reverse transcription–polymerase chain reaction.
All three CMESC lines were examined for MHC-DRB1 genotypes using PCR-based single strand conformation polymorphism (SSCP) methods. As shown in Figure 3B, the three CMESC lines were confirmed, based on different SSCP patterns, as having been established independently. Of these three CMESC lines, nos. 30 and 40 were derived from the same parents.
Differentiation Potency
Similar to other primate ESCs, CMESCs differentiate spontaneously during culturing on MEF feeder layer. However, the complete differentiation of CMESCs was suppressed by some growth factors or inhibitory factors from MEF feeder layer. To estimate the degree of differentiation, 50 CMESC clusters were seeded onto MEF feeder layer. As a result, 22%–74% (n = 6) of the colonies (average 42.6%) were morphologically undifferentiated ESCs (data not shown). However, the differentiation rate of each cell was unclear because CMESCs need to be cell clusters to maintain undifferentiated status.
To assess the spontaneous differentiation potency of CMESCs, the formation of EBs and teratomas was examined. The suspension cultures of all three CMESC lines formed EBs (Figs. 5A, 5B). Simple EBs formed several days after the start of the suspension culture, and cystic EBs formed within 2 weeks. These EBs expressed mRNA for the Nestin, CD34, and -fetoprotein genes, which are marker genes for the three germ layers, and mCG, Bex1/Rex3, and Heb, which are marker genes for trophectoderm (Fig. 4A). Furthermore, expression of LIFR and gp130 was observed. However, Nanog, Oct3/4, and Sox2 gene expression was shut off after 2 weeks of EB culture. In contrast, the expression level of FoxD3 in EBs was greater than in undifferentiated CMESCs. To examine the differentiation potency in more detail, cells of CMESC 20 were injected subcutaneously into five immunodeficient NOG mice . Eight weeks after injection, subcutaneous tumors were rescued from these mice and subjected to histological analysis. The tumor formation rate was 100% (5/5). The tumors were found to be teratomas that consisted of embryonic germ layers of ectodermal, mesodermal, and endodermal tissues (Figs. 6A–6M). Teratomas formed in all five NOG mice (100% teratoma formation rate). In the teratomas, the ectodermal tissue consisted of keratinized epidermis (Figs. 6B, 6G, and 7F) and neuronal cells (Fig. 6M); the mesodermal tissue was comprised of muscle (Figs. 6C, 6H) and blood vessels (Figs. 6I, 6J), and the endodermal tissue contained columnar epithelium (Figs. 6A, 6K, and 6L). Furthermore, cartilage-like tissue (Figs. 6A, 6D, and 6E) and adipose-like tissue (Fig. 6D) were also observed. These blood vessels were distinguished from murine blood vessels by immunohistochemical staining with human anti-CD31 antibody. Bronchus-like structures and gut-like structures were occasionally found in the teratomas (Figs. 6A, 6K, and 6L). Differentiation was confirmed by immunohistochemical analysis with several tissue-specific antibodies. As evidence for ESC differentiation into ectodermal cells, the GFAP-positive cells were observed as neuronal cells (Fig. 6M), and the keratinized epidermis-like structures in the teratomas expressed WSS keratin (Fig. 6G). The teratomas differentiated frequently into mesodermal tissues such as muscle, blood vessels, and cartilage. The muscle-like structure showed desmin expression, and CD31-positive cells were located in the hemangioendothelium of the blood vessel–like structures (Fig. 6J). The presence of the gut-like structures suggests endodermal differentiation. Alcian blue and periodic acid-Schiff (PAS) staining revealed mucus secretion from the columnar epithelium (Fig. 6L).
Figure 5. Spontaneous differentiation potency of CMESCs. A suspension culture of CMESCs shows the formation of EBs: (A) simple EB, (B) cystic EB. Abbreviations: CMESC, common marmoset embryonic stem cell; EB, embryoid body.
Figure 6. Differentiated CMESCs in teratomas and the expression of tissue-specific markers. (A): A bronchus-like structure that consists of columnar epithelium surrounded by cartilage-like tissue. (B–M): Keratinizing squamous epidermis (B), striated muscle (C), adipose-like tissue (high magnification) (D), cartilage-like and columnar epithelium (high magnification) (E), epidermis (F), reactivity for cyto-keratin WSS (G), muscle-expressing desmin (H), capillary blood vessels (I), CD31-positive vascular endothelial cells (J), columnar epithelium (K), Alcian blue-PAS–positive columnar epithelia (L), and GFAP-expressing neural cells (M). Abbreviations: CMESC, common marmoset embryonic stem cell; GFAP, glial fibrillary acidic protein; PAS, periodic acid-Schiff; WSS, wide specific screening.
Figure 7. Neuronal cell differentiation of CMESCs in vitro. In vitro–differentiated ESCs using the SDIA method express TH protein and class III ?-tubulin protein. Tyrosine hydroxylase: green; class III ?-tubulin: red; Hoechst: blue. Scale bar = 80 mm. Abbreviations: CMESC, common marmoset embryonic stem cell; ESC, embryonic stem cell; SDIA, stromal cell–derived inducing activity.
To investigate the in vitro differentiation potency of the CMESCs, the measurement of stromal cell–derived inducing activity (SDIA) was performed. After culture on PA6 cells for 20 days, extensive neurites appeared in the majority of the primate ESC colonies (67%, n = 30), which contained a large number of postmitotic neurons positive for class III ?-tubulin (red; Fig. 7). SDIA has been reported to induce the production of tyrosine hydroxylase (TH)–positive dopaminergic neurons in mouse and cynomolgus monkey ESCs and hESCs . Therefore, we tested the marmoset cells for similar activities. After 2 weeks of induction, 14% of the class III ?-tubulin–positive postmitotic neurons were TH-positive at the cellular level (n = 50). These cells were plated from trypsinized ESCs at passage. Most of the cells were derived from single cells, but only 15% of them expressed the TH protein. In the teratoma-like tumor induced by subcutaneous transplantation into NOG mice, some III ?-tubulin–positive cells were found in colonies. Among III ?-tublin–positive neurons, TH-positive neurons were found in 21% of them (n = 113; data not shown).
To induce hematopoietic cells, EB formation was allowed to proceed in the cytokine-free medium, and CFU assays were performed. CFU-M (CFU-monocyte/macrophage) colonies were mainly observed under these conditions (Fig. 8A), and the main population of macrophages was confirmed microscopically using May-Giemsa staining of cytospun preparations (Fig. 8B).
Figure 8. Hematopoietic CMESC differentiation in vivo. (A): CFU-M–like colonies predominate under these conditions. (B): May-Giemsa staining of colony-forming cells, confirming that the major population consists of macrophages. Abbreviations: CFU-M, colony-forming unit-monocyte/macrophage; CMESC, common marmoset embryonic stem cell.
DISCUSSION
We thank Dr. Sumiko Watanabe, Dr. Chieko Kai, and Dr. Kenichi Arai for very helpful advice and persistent support. This work was supported by grants from the Japan Society for the Promotion of Science and Research for the Future Program and partially by the Ministry of Education, Culture, Sports, Science, and Technology.
DISCLOSURES
The authors indicate no potential conflicts of interest.
REFERENCES
Lopata A, Summers PM, Hearn JP. Births following the transfer of cultured embryos obtained by in vitro and in vivo fertilization in the marmoset monkey (Callithrix jacchus). Fertil Steril 1988;50:503–509.
Summers PM, Shephard AM, Taylor CT et al. The effects of cryopreservation and transfer on embryonic development in the common marmoset monkey, Callithrix jacchus. J Reprod Fertil 1987;79:241–250.
Marshall VS, Kalishman J, Thomson JA. Nonsurgical embryo transfer in the common marmoset monkey. J Med Primatol 1997;26:241–247.
Hibino H, Tani K, Ikebuchi K et al. The common marmoset as a target preclinical primate model for cytokine and gene therapy studies. Blood 1999;93:2839–2848.
Mansfield K. Marmoset models commonly used in biomedical research. Comp Med 2003;53:383–392.
Thomson JA, Kalishman J, Golos TG et al. Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts. Biol Reprod 1996;55:254–259.
Summers PM, Wennink CJ, Hodges JK. Cloprostenol-induced luteolysis in the marmoset monkey (Callithrix jacchus). J Reprod Fertil 1985;73:133–138.
Harlow CR, Gems S, Hearn JP et al. The relationship between plasma progesterone and the timing of ovulation and early embryonic development in the marmoset monkey (Callithrix jacchus). J Zool 1983;201:273–282.
Solter D, Knowles BB. Immunosurgery of mouse blastocyst. Proc Natl Acad Sci U S A 1975;72:5099–5102.
Wu MS, Tani K, Sugiyama H et al. MHC (major histocompatibility complex)-DRB genes and polymorphisms in common marmoset. J Mol Evol 2000;51:214–222.
Ito M, Hiramatsu H, Kobayashi K et al. NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells. Blood 2002;100:3175–3182.
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.
Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.
Suemori H, Tada T, Torii R et al. Establishment of embryonic stem cell lines from cynomolgus monkey blastocysts produced by IVF or ICSI. Dev Dyn 2001;222:273–279.
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
Mitalipova M, Calhoun J, Shin S et al. Human embryonic stem cell lines derived from discarded embryos. STEM CELLS 2003;21:521–526.
Kawasaki H, Mizuseki K, Nishikawa S et al. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 2000;28:31–40.
Kawasaki H, Suemori H, Mizuseki K et al. Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity. Proc Natl Acad Sci U S A 2002;99:1580–1585.
Perrier AL, Tabar V, Barberi T et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A 2004;101:12543–12548.
Ginis I, Luo Y, Miura T et al. Differences between human and mouse embryonic stem cells. Dev Biol 2004;269:360–380.
Stojkovic M, Lako M, Stojkovic P et al. Derivation of human embryonic stem cells from day-8 blastocysts recovered after three-step in vitro culture. STEM CELLS 2004;22:790–797.
Stojkovic M, Lako M, Strachan T et al. Derivation, growth and applications of human embryonic stem cells. Reproduction 2004;128:259–267.
Toyooka Y, Tsunekawa N, Akasu R et al. Embryonic stem cells can form germ cells in vitro. Proc Natl Acad Sci U S A 2003;100:11457–11462.
Hubner K, Fuhrmann G, Christenson LK et al. Derivation of oocytes from mouse embryonic stem cells. Science 2003;300:1251–1256.
Simerly C, Navara C, Hyun SH et al. Embryogenesis and blastocyst development after somatic cell nuclear transfer in nonhuman primates: overcoming defects caused by meiotic spindle extraction. Dev Biol 2004;276:237–252.
Assady S, Maor G, Amit M et al. Insulin production by human embryonic stem cells. Diabetes 2001;50:1691–1697.
Schuldiner M, Eiges R, Eden A et al. Induced neuronal differentiation of human embryonic stem cells. Brain Res 2001;913:201–205.
Kaufman DS, Hanson ET, Lewis RL et al. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A 2001;98:10716–10721.
Kehat I, Kenyagin-Karsenti D, Snir M et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest 2001;108:407–414.
Umeda K, Heike T, Yoshimoto M et al. Development of primitive and definitive hematopoiesis from nonhuman primate embryonic stem cells in vitro. Development 2004;131:1869–1879.
Haruta M, Sasai Y, Kawasaki H et al. In vitro and in vivo characterization of pigment epithelial cells differentiated from primate embryonic stem cells. Invest Ophthalmol Vis Sci 2004;45:1020–1025.
Mizuseki K, Sakamoto T, Watanabe K et al. Generation of neural crest-derived peripheral neurons and floor plate cells from mouse and primate embryonic stem cells. Proc Natl Acad Sci U S A 2003;100:5828–5833.
Kuo HC, Pau KY, Yeoman RR et al. Differentiation of monkey embryonic stem cells into neural lineages. Biol Reprod 2003;68:1727–1735.
Calhoun JD, Lambert NA, Mitalipova MM et al. Differentiation of rhesus embryonic stem cells to neural progenitors and neurons. Biochem Biophys Res Commun 2003;306:191–197.
Lang KJ, Rathjen J, Vassilieva S et al. Differentiation of embryonic stem cells to a neural fate: a route to re-building the nervous system? J Neurosci Res 2004;76:184–192.
Li F, Lu S, Vida L et al. Bone morphogenetic protein 4 induces efficient hematopoietic differentiation of rhesus monkey embryonic stem cells in vitro. Blood 2001;98:335–342.
Nara Y, Muramatsu S, Nakano I. ES cell therapy for Parkinson’s disease. Nippon Rinsho 2004;62:1643–1647.(Erika Sasakia, Kisaburo H)
b Department of Urology, Urayasu Hospital, Juntendo University, Urayasu, Chiba, Japan;
c Department of Molecular Genetics, Division of Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Hakata, Fukuoka, Japan;
d Tokai University School of Medicine, Isehara, Kanagawa, Japan;
e Department of Physiology, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan;
f Research Project Center,
g Department of Genetics, Central Institute for Experimental Animals, Kawasaki, Kanagawa, Japan;
h Division of Molecular Therapy, Institute of Medical Science, University of Tokyo,
i Institute of Obstetrics & Gynecology in Clinical Medicine, University of Tsukuba,
j Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center,
k Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
Key Words. Embryonic stem cells ? Common marmoset ? Embryoid body ? Nonhuman primate ? Teratoma formation
Correspondence: Kenzaburo Tani, M.D., Ph.D., Department of Molecular Genetics, Division of Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Hakata, Fukuoka 812-8582, Japan. Telephone: 81-92-642-6434; Fax: 81-92-642-6444; e-mail: taniken@bioreg.kyushu-u.ac.jp; for CMES cell distribution, contact Erika Sasaki at esasaki@ciea.or.jp
ABSTRACT
Embryonic stem cells (ESCs) are derived from preimplantation embryos. Owing to their pluripotency, ESCs have been widely used for the production of transgenic and gene knockout mice to elucidate the molecular mechanisms of various genes. In 1998, the successful establishment of human ESC (hESC) lines enhanced the role of ESCs in science and medicine. The most promising application of ESCs in the clinical setting is in the repair of defective organ function by differentiated ESCs. However, before differentiated hESCs can be used in clinical applications, the short-term and long-term safety and efficacy of ESCs must be thoroughly examined. Due to ethical considerations, these studies cannot be performed in vivo in humans. Therefore, preclinical studies using the ESCs of nonhuman primates are essential.
The common marmoset (Callithrix jacchus) is a New World primate species with reproductive characteristics that are appropriate for ESC studies. More specifically, these animals are small (weighing approximately 350–400 g), they have a short gestation period (approximately 144 days), and reach sexual maturity at 12–18 months. Unlike macaques, marmosets routinely deliver twins or triplets for each pregnancy. In addition, it is possible to synchronize the marmoset ovarian cycle with prostaglandin analogs, collect age-matched embryos from multiple females, and transfer embryos to synchronized recipients with success rates in the range of 70%–80% . Because these reproductive characteristics allow routine efficient transfer of multiple embryos, marmosets constitute an excellent primate species for the generation of transgenic and knockout animal models of human diseases.
In addition to these reproductive benefits, we have shown previously that marmosets are suitable laboratory animals for preclinical studies of stem cell therapies, owing to the similarities between the hematopoietic and immune systems of humans and marmosets . In 1996, Thomson et al. established pluripotent common marmoset cell lines, which are considered powerful tools for understanding the regulatory mechanisms of ESC differentiation both in vitro and in vivo. However, the differentiation abilities of the pluripotent common marmoset cells in terms of in vitro teratoma formation assays and certain common properties of ESC lines, such as differentiation to the cell lineages of three germ layers, have not been defined fully. The establishment of totipotent common marmoset ESC (CMESC) lines would facilitate the construction, by gene targeting, of nonhuman primate models for human disease. To achieve our goal of establishing the common marmoset as a human disease model, we have established novel CMESC lines and characterized their differentiation capacities.
MATERIALS AND METHODS
Establishment of CMESC Lines
The marmosets ovulated 10.7 ± 1.3 days (n = 70) after PGF2 administration. Our ovarian cycle control system made it possible to obtain fertilized eggs every 3 weeks from the same animals. Sixty immunosurgically isolated ICMs from 70 blastocysts (the ICM isolation rate was 85.7%) were plated on the irradiated MEF feeder layer, and 11 ICMs were cultured for more than 10 passages (18.3% derivation rate). To date, 3/11 ICM-derived cells have been cultured for more than 1 year. All ICM-derived cells showed flat, packed, and tight colony morphology and a high nucleus:cytoplasm ratio (Figs. 1A, 1B). The morphology of these derived cells was very similar to that of reported primate ESCs, including those from humans and from rhesus and cynomolgus monkeys . It has been reported that primate ESCs exhibit spontaneous differentiation during culture and that leukemia inhibitory factor does not maintain the ESCs in the undifferentiated state . The common marmoset ICM-derived cell lines also showed low frequency of differentiation. However, most ICM-derived cells maintained undifferentiated morphology of the cells. It is known that the quality of FBS is critical to the maintenance of undifferentiated primate ESCs; different lots of FBS from the same manufacturer can vary in this respect. Therefore, the chemically defined serum-free supplement KSR was used for the establishment and culture of the marmoset ESCs. The CMESC colonies appeared more packed and tight when KSR was used in the culture; they were flatter in appearance when FBS was used in the medium. To maintain stem cells, the dissociation procedure for cynomolgus ESCs was adopted for subculture of the marmoset ICM-derived cells . With this culture system, continuous cultures of CMESCs have been sustained for more than 1 year.
Figure 1. Expression of alkaline phosphatase and cell surface markers on CMESCs. Unstained CMESCs (A, B), cells stained for ALP (C), SSEA-1 (D), SSEA-3 (E), SSEA-4 (F), TRA-1-60 (G), and TRA-1-81 (H). Abbreviations: ALP, alkaline phosphatase; CMESC, common marmoset embryonic stem cell; SSEA, stage-specific embryonic antigen.
Characterization of Undifferentiated CMESC Lines
To confirm the undifferentiated status of the ICM-derived cells, all three lines (CMESC 20, 30, and 40) were examined for the expression of cell surface markers that are specific for undifferentiated ESCs. As shown in Figures 1C–1G, the ICM-derived cell lines showed alkaline phosphatase activity (Fig. 1C) and expressed SSEA-3 (Fig. 1E), SSEA-4 (Fig. 1F), TRA-1-60 (Fig. 1G), and TRA-1-81 (Fig. 1H) but not SSEA-1 (Fig. 1D). All three cell lines retained the normal 46, XX karyotype (Fig. 2) and telomerase (Fig. 3A) activity. These three ICM-derived cell lines also expressed Nanog, Oct3/4, Sox2, gp130, mCG, HEB, and Bex1/Rex3 mRNA and low levels of FoxD3 and Nestin mRNA (Fig. 4A). Conversely, LIFR mRNA was not detected by RT-PCR. All three lines showed identical expression patterns of the genes. Therefore, we believe that these cells are CMESCs. To compare the gene expression patterns of fresh ICMs and the ICM-derived cell lines, RT-PCR analysis was conducted using fresh ICMs. As a result, the expression of Nanog, Oct3/4, and Sox2 mRNA was observed, whereas FoxD3 and Nestin mRNA was not detected in fresh ICMs (Fig. 4B).
Figure 2. Karyotype analysis of CMESCs. The results for CIEA-CMESC #20 are shown. All three cell lines show the normal 46, XX karyotype after more than 6 months of culture. Abbreviations: CIEA, Central Institute for Experimental Animals; CMESC, common marmoset embryonic stem cell.
Figure 3. (A): Telomerase activities of CMESC lines. All three CMESC lines show high telomerase activity. On the other hand, MEF feeder layer does not show telomerase activity. (B): SSCP analysis of CMESCs for the MHC-DRB1 gene. The results of SSCP indicate that all three lines were derived independently. Abbreviations: CMESC, common marmoset embryonic stem cell; MEF, mouse embryonic fibroblast; SSCP, single strand conformation polymorphism.
Figure 4. (A): RT-PCR analysis of CMESCs and EBs. Undifferentiated CMESCs expressed the Nanog, Oct3/4, Sox2, FoxD3, Bex1/ Rex3, Heb and mCG, GP130, and low level of Nestin genes. Two-week cultures of EBs display expression of Nestin and CD34. Three-week cultures of EBs show expression of Nestin, CD34, and -fetoprotein. Oct3/4 and Nanog gene expression is shut down in the EBs. (B): RT-PCR analysis of fresh ICMs. ICMs expressed the Nanog, Oct3/4, and Sox2 genes. Expression of FoxD3 and Nestin was not detected. Abbreviations: CMESC, common marmoset embryonic stem cell; EB, embryoid body; ICM, inner cell mass; RT-PCR, reverse transcription–polymerase chain reaction.
All three CMESC lines were examined for MHC-DRB1 genotypes using PCR-based single strand conformation polymorphism (SSCP) methods. As shown in Figure 3B, the three CMESC lines were confirmed, based on different SSCP patterns, as having been established independently. Of these three CMESC lines, nos. 30 and 40 were derived from the same parents.
Differentiation Potency
Similar to other primate ESCs, CMESCs differentiate spontaneously during culturing on MEF feeder layer. However, the complete differentiation of CMESCs was suppressed by some growth factors or inhibitory factors from MEF feeder layer. To estimate the degree of differentiation, 50 CMESC clusters were seeded onto MEF feeder layer. As a result, 22%–74% (n = 6) of the colonies (average 42.6%) were morphologically undifferentiated ESCs (data not shown). However, the differentiation rate of each cell was unclear because CMESCs need to be cell clusters to maintain undifferentiated status.
To assess the spontaneous differentiation potency of CMESCs, the formation of EBs and teratomas was examined. The suspension cultures of all three CMESC lines formed EBs (Figs. 5A, 5B). Simple EBs formed several days after the start of the suspension culture, and cystic EBs formed within 2 weeks. These EBs expressed mRNA for the Nestin, CD34, and -fetoprotein genes, which are marker genes for the three germ layers, and mCG, Bex1/Rex3, and Heb, which are marker genes for trophectoderm (Fig. 4A). Furthermore, expression of LIFR and gp130 was observed. However, Nanog, Oct3/4, and Sox2 gene expression was shut off after 2 weeks of EB culture. In contrast, the expression level of FoxD3 in EBs was greater than in undifferentiated CMESCs. To examine the differentiation potency in more detail, cells of CMESC 20 were injected subcutaneously into five immunodeficient NOG mice . Eight weeks after injection, subcutaneous tumors were rescued from these mice and subjected to histological analysis. The tumor formation rate was 100% (5/5). The tumors were found to be teratomas that consisted of embryonic germ layers of ectodermal, mesodermal, and endodermal tissues (Figs. 6A–6M). Teratomas formed in all five NOG mice (100% teratoma formation rate). In the teratomas, the ectodermal tissue consisted of keratinized epidermis (Figs. 6B, 6G, and 7F) and neuronal cells (Fig. 6M); the mesodermal tissue was comprised of muscle (Figs. 6C, 6H) and blood vessels (Figs. 6I, 6J), and the endodermal tissue contained columnar epithelium (Figs. 6A, 6K, and 6L). Furthermore, cartilage-like tissue (Figs. 6A, 6D, and 6E) and adipose-like tissue (Fig. 6D) were also observed. These blood vessels were distinguished from murine blood vessels by immunohistochemical staining with human anti-CD31 antibody. Bronchus-like structures and gut-like structures were occasionally found in the teratomas (Figs. 6A, 6K, and 6L). Differentiation was confirmed by immunohistochemical analysis with several tissue-specific antibodies. As evidence for ESC differentiation into ectodermal cells, the GFAP-positive cells were observed as neuronal cells (Fig. 6M), and the keratinized epidermis-like structures in the teratomas expressed WSS keratin (Fig. 6G). The teratomas differentiated frequently into mesodermal tissues such as muscle, blood vessels, and cartilage. The muscle-like structure showed desmin expression, and CD31-positive cells were located in the hemangioendothelium of the blood vessel–like structures (Fig. 6J). The presence of the gut-like structures suggests endodermal differentiation. Alcian blue and periodic acid-Schiff (PAS) staining revealed mucus secretion from the columnar epithelium (Fig. 6L).
Figure 5. Spontaneous differentiation potency of CMESCs. A suspension culture of CMESCs shows the formation of EBs: (A) simple EB, (B) cystic EB. Abbreviations: CMESC, common marmoset embryonic stem cell; EB, embryoid body.
Figure 6. Differentiated CMESCs in teratomas and the expression of tissue-specific markers. (A): A bronchus-like structure that consists of columnar epithelium surrounded by cartilage-like tissue. (B–M): Keratinizing squamous epidermis (B), striated muscle (C), adipose-like tissue (high magnification) (D), cartilage-like and columnar epithelium (high magnification) (E), epidermis (F), reactivity for cyto-keratin WSS (G), muscle-expressing desmin (H), capillary blood vessels (I), CD31-positive vascular endothelial cells (J), columnar epithelium (K), Alcian blue-PAS–positive columnar epithelia (L), and GFAP-expressing neural cells (M). Abbreviations: CMESC, common marmoset embryonic stem cell; GFAP, glial fibrillary acidic protein; PAS, periodic acid-Schiff; WSS, wide specific screening.
Figure 7. Neuronal cell differentiation of CMESCs in vitro. In vitro–differentiated ESCs using the SDIA method express TH protein and class III ?-tubulin protein. Tyrosine hydroxylase: green; class III ?-tubulin: red; Hoechst: blue. Scale bar = 80 mm. Abbreviations: CMESC, common marmoset embryonic stem cell; ESC, embryonic stem cell; SDIA, stromal cell–derived inducing activity.
To investigate the in vitro differentiation potency of the CMESCs, the measurement of stromal cell–derived inducing activity (SDIA) was performed. After culture on PA6 cells for 20 days, extensive neurites appeared in the majority of the primate ESC colonies (67%, n = 30), which contained a large number of postmitotic neurons positive for class III ?-tubulin (red; Fig. 7). SDIA has been reported to induce the production of tyrosine hydroxylase (TH)–positive dopaminergic neurons in mouse and cynomolgus monkey ESCs and hESCs . Therefore, we tested the marmoset cells for similar activities. After 2 weeks of induction, 14% of the class III ?-tubulin–positive postmitotic neurons were TH-positive at the cellular level (n = 50). These cells were plated from trypsinized ESCs at passage. Most of the cells were derived from single cells, but only 15% of them expressed the TH protein. In the teratoma-like tumor induced by subcutaneous transplantation into NOG mice, some III ?-tubulin–positive cells were found in colonies. Among III ?-tublin–positive neurons, TH-positive neurons were found in 21% of them (n = 113; data not shown).
To induce hematopoietic cells, EB formation was allowed to proceed in the cytokine-free medium, and CFU assays were performed. CFU-M (CFU-monocyte/macrophage) colonies were mainly observed under these conditions (Fig. 8A), and the main population of macrophages was confirmed microscopically using May-Giemsa staining of cytospun preparations (Fig. 8B).
Figure 8. Hematopoietic CMESC differentiation in vivo. (A): CFU-M–like colonies predominate under these conditions. (B): May-Giemsa staining of colony-forming cells, confirming that the major population consists of macrophages. Abbreviations: CFU-M, colony-forming unit-monocyte/macrophage; CMESC, common marmoset embryonic stem cell.
DISCUSSION
We thank Dr. Sumiko Watanabe, Dr. Chieko Kai, and Dr. Kenichi Arai for very helpful advice and persistent support. This work was supported by grants from the Japan Society for the Promotion of Science and Research for the Future Program and partially by the Ministry of Education, Culture, Sports, Science, and Technology.
DISCLOSURES
The authors indicate no potential conflicts of interest.
REFERENCES
Lopata A, Summers PM, Hearn JP. Births following the transfer of cultured embryos obtained by in vitro and in vivo fertilization in the marmoset monkey (Callithrix jacchus). Fertil Steril 1988;50:503–509.
Summers PM, Shephard AM, Taylor CT et al. The effects of cryopreservation and transfer on embryonic development in the common marmoset monkey, Callithrix jacchus. J Reprod Fertil 1987;79:241–250.
Marshall VS, Kalishman J, Thomson JA. Nonsurgical embryo transfer in the common marmoset monkey. J Med Primatol 1997;26:241–247.
Hibino H, Tani K, Ikebuchi K et al. The common marmoset as a target preclinical primate model for cytokine and gene therapy studies. Blood 1999;93:2839–2848.
Mansfield K. Marmoset models commonly used in biomedical research. Comp Med 2003;53:383–392.
Thomson JA, Kalishman J, Golos TG et al. Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts. Biol Reprod 1996;55:254–259.
Summers PM, Wennink CJ, Hodges JK. Cloprostenol-induced luteolysis in the marmoset monkey (Callithrix jacchus). J Reprod Fertil 1985;73:133–138.
Harlow CR, Gems S, Hearn JP et al. The relationship between plasma progesterone and the timing of ovulation and early embryonic development in the marmoset monkey (Callithrix jacchus). J Zool 1983;201:273–282.
Solter D, Knowles BB. Immunosurgery of mouse blastocyst. Proc Natl Acad Sci U S A 1975;72:5099–5102.
Wu MS, Tani K, Sugiyama H et al. MHC (major histocompatibility complex)-DRB genes and polymorphisms in common marmoset. J Mol Evol 2000;51:214–222.
Ito M, Hiramatsu H, Kobayashi K et al. NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells. Blood 2002;100:3175–3182.
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.
Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.
Suemori H, Tada T, Torii R et al. Establishment of embryonic stem cell lines from cynomolgus monkey blastocysts produced by IVF or ICSI. Dev Dyn 2001;222:273–279.
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
Mitalipova M, Calhoun J, Shin S et al. Human embryonic stem cell lines derived from discarded embryos. STEM CELLS 2003;21:521–526.
Kawasaki H, Mizuseki K, Nishikawa S et al. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 2000;28:31–40.
Kawasaki H, Suemori H, Mizuseki K et al. Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity. Proc Natl Acad Sci U S A 2002;99:1580–1585.
Perrier AL, Tabar V, Barberi T et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A 2004;101:12543–12548.
Ginis I, Luo Y, Miura T et al. Differences between human and mouse embryonic stem cells. Dev Biol 2004;269:360–380.
Stojkovic M, Lako M, Stojkovic P et al. Derivation of human embryonic stem cells from day-8 blastocysts recovered after three-step in vitro culture. STEM CELLS 2004;22:790–797.
Stojkovic M, Lako M, Strachan T et al. Derivation, growth and applications of human embryonic stem cells. Reproduction 2004;128:259–267.
Toyooka Y, Tsunekawa N, Akasu R et al. Embryonic stem cells can form germ cells in vitro. Proc Natl Acad Sci U S A 2003;100:11457–11462.
Hubner K, Fuhrmann G, Christenson LK et al. Derivation of oocytes from mouse embryonic stem cells. Science 2003;300:1251–1256.
Simerly C, Navara C, Hyun SH et al. Embryogenesis and blastocyst development after somatic cell nuclear transfer in nonhuman primates: overcoming defects caused by meiotic spindle extraction. Dev Biol 2004;276:237–252.
Assady S, Maor G, Amit M et al. Insulin production by human embryonic stem cells. Diabetes 2001;50:1691–1697.
Schuldiner M, Eiges R, Eden A et al. Induced neuronal differentiation of human embryonic stem cells. Brain Res 2001;913:201–205.
Kaufman DS, Hanson ET, Lewis RL et al. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A 2001;98:10716–10721.
Kehat I, Kenyagin-Karsenti D, Snir M et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest 2001;108:407–414.
Umeda K, Heike T, Yoshimoto M et al. Development of primitive and definitive hematopoiesis from nonhuman primate embryonic stem cells in vitro. Development 2004;131:1869–1879.
Haruta M, Sasai Y, Kawasaki H et al. In vitro and in vivo characterization of pigment epithelial cells differentiated from primate embryonic stem cells. Invest Ophthalmol Vis Sci 2004;45:1020–1025.
Mizuseki K, Sakamoto T, Watanabe K et al. Generation of neural crest-derived peripheral neurons and floor plate cells from mouse and primate embryonic stem cells. Proc Natl Acad Sci U S A 2003;100:5828–5833.
Kuo HC, Pau KY, Yeoman RR et al. Differentiation of monkey embryonic stem cells into neural lineages. Biol Reprod 2003;68:1727–1735.
Calhoun JD, Lambert NA, Mitalipova MM et al. Differentiation of rhesus embryonic stem cells to neural progenitors and neurons. Biochem Biophys Res Commun 2003;306:191–197.
Lang KJ, Rathjen J, Vassilieva S et al. Differentiation of embryonic stem cells to a neural fate: a route to re-building the nervous system? J Neurosci Res 2004;76:184–192.
Li F, Lu S, Vida L et al. Bone morphogenetic protein 4 induces efficient hematopoietic differentiation of rhesus monkey embryonic stem cells in vitro. Blood 2001;98:335–342.
Nara Y, Muramatsu S, Nakano I. ES cell therapy for Parkinson’s disease. Nippon Rinsho 2004;62:1643–1647.(Erika Sasakia, Kisaburo H)