Identification of Developmental Pluripotency Associated 5 Expression in Human Pluripotent Stem Cells
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《干细胞学杂志》
a Cell and Gene Therapy Research Institute, Pochon CHA University College of Medicine, Seoul, Korea;
b Medical Research Center, MizMedi Hospital, Seoul, Korea;
c Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, Seoul, Korea;
d Department of Obstetrics and Gynecology, Hospital of Hanyang University, Guri, Korea;
e Department of Anatomy and Cell Biology, Hanyang University College of Medicine, Seoul, Korea
Key Words. Developmental pluripotency associated 5 ? Primordial germ cells ? Embryonic germ cells ? Embryonic stem cells ? Pluripotency
Correspondence: Kye-Seong Kim, D.V.M., Ph.D., Hanyang University College of Medicine, 17 Haengdang-dong, Sungdong-gu, Seoul 133-791, Korea. Telephone: 82-2-2290-0607; Fax: 82-2-2281-7841; e-mail: ks66kim@hanyang.ac.kr
ABSTRACT
In mammals, primordial germ cells (PGCs) are first specified in the extraembryonic mesoderm. Subsequently, PGCs migrate to gonads and proliferate to form gonocytes. Gonocyte identity is expressed by transition into a nonmitotic state, growth in cell size, and the onset of specific gene expression . Cellular pluripotency can be defined as the ability of a cell to differentiate into diverse cell types. During mammalian development, only particular subsets of cells in embryos transiently possess pluripotency . In germ cell lineages, PGCs give rise to pluripotent embryonic germ cells (EGCs) that form all three germ layers . However, gonocytes have unipotency and become only germ cells such as spermatogonia or oogonia . To analyze the gene expression differences between PGCs and gonocytes, we performed cDNA subtractive hybridization with mouse gonads that contained either of the two cell populations and found candidate gene developmental pluripotency associated 5 (Dppa5). Currently a few molecular regulators are known to participate in the self-renewal and pluripotency of mouse embryonic stem cells (mESCs). A POU family transcription factor Oct4, the classical marker of all pluripotent cells, is specifically expressed in pre-implantation embryos, epiblast, germ cells, and pluripotent stem cell lines, including ESCs, EGCs, and embryonic carcinoma cells (ECCs) . Oct4 has a critical role in the establishment and maintenance of pluripotent cells in a pluripotent state . Leukemia inhibitory factor (LIF) can maintain self-renewal of mESCs through activation of Stat3 . Oct4 and Stat3 each interact with various cofactors and regulate the expression of multiple target genes . Two other transcription factors, Sox2 and FoxD3, have been shown to be essential for pluripotency in mice embryos . More recently, it was found that the homeoprotein Nanog is capable of maintaining mESC self-renewal independently of LIF/Stat3 .
In this study, we clearly showed Dppa5 expressed in mouse and human pluripotent cells. Based on the expression patterns, we assumed that the Dppa5 is closely related in cell pluripotency and is able to serve as an informative marker for human pluripotent cell types.
MATERIALS AND METHODS
To begin characterizing the distinction between cells that have or do not have the potential to be a pluripotent cell line, we performed cDNA subtractive hybridization and identified several candidate genes that were expressed differentially between mouse PGCs and gonocytes. We accessed the GenBank database and searched using the BLAST algorithm at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST). One of the genes is Dppa5. This gene has exactly the same sequence as mDppa5 (GenBank; AF490349 ). mDppa5 has two other names: pH 34 and embryonal stem cell–specific gene1 . In previous reports , mDppa5 is expressed in mESCs, mouse embryonic carcinoma cells (mECCs), preimplantation embryos, and developing germ cells. However, in our study, the mDppa5 gene was strongly expressed only in PGCs at the transcriptional level, and the expression was rapidly downregulated in gonocytes and developing germ cells, including spermatogonial stem cells (Fig. 1). The mDppa5 gene was also expressed in mESCs and mECCs, as previously reported , but no expression of mDppa5 was detected in various mice somatic tissues by Northern blot analysis (data not shown).
Figure 1. Northern blot analysis of mDppa5 expression in cells and tissues of mouse. Total RNA was extracted from mouse feeder cell (STO), primordial germ cells (PGCs), gonocytes, spermatogonial stem cells (SGs), testis (2, 4, 6, and 10 weeks), pachytene spermatocyte (PS), round spermatid (RS), condensing spermatid (CS), and mouse embryonic stem cells (mESCs). The expression of Dppa5 is specifically shown in PGCs and mESCs, respectively. 18S ribosomal RNA is shown as a loading control.
To identify the human homologue of mDppa5, bioinformatics approaches found two candidates (the GenBank accession numbers are BX092581 and XM_291161 ). Recently, one report showed hDppa5 expression in hESCs, but there is no report of hDppa5 expression patterns in various cells and tissues in comparison with currently reported mDppa5. The sequences of mDppa5 and hDppa5 were highly conserved (77% identical; data not shown). Using RT-PCR, we analyzed the expression of hDppa5 in hPGCs, hEGCs, hESCs, hECCs (NCCIT and NT2), and HeLa cells, as well as mESCs and STOs (Fig. 2).
Figure 2. Reverse transcription polymerase chain reaction analysis of hDppa5 expression. (A): From human primordial germ cells (hPGCs), human embryonic germ cells (Miz-hEG1), human embryonic stem cells (Miz-hES4, Miz-hES3, Miz-hES1, and SNU-hES3), HeLa, and human embryonic carcinoma cells (NCCIT and NT2), compared with mouse embryonic stem cells (mESCs) and mouse STO. hDppa5 expression was detected in hPGCs, hEGCs, and hESCs but not in hECCs. (B): Expression of hDppa5 and hOct4 in undifferentiated (SNU-hES3) and differentiated hESCs. hDppa5 is downregulated in differentiated hESCs, as well as hOct4. The h?-actin and mGAPDH mRNA was used as an internal control.
Strikingly, hDppa5 was expressed only in hPGCs, hEGCs, and four different hESCs (Fig. 2A) but not in hECCs (NCCIT and NT2) and HeLa cells. The primers for hDppa5 did not amplify a product from RNA of mouse cells (mESC and STO). We checked the expression of hDppa5 and Oct4 in differentiated hESCs induced by in vitro fertilization. The Oct4 was downregulated in differentiated cells , and hDppa5 was also downregulated in differentiated hESCs compared with the strong expression in undifferentiated hESCs (Fig. 2B). We examined the hDppa5 expression in primary cultured human embryonic fibroblast cells, HeLa cells, hECCs (NCCIT and NT2), and hESCs by Northern blot analysis. As mentioned earlier, the hDppa5 was detected only in hESCs but not in hECCs (Fig. 3A). This finding is in contrast with the pattern of mDppa5 which is expressed in both mESCs and mECCs. However, the primary marker of pluripotent stem cells, Oct4, is expressed in hESCs and hECCs (NCCIT and NT2; Fig. 3B). To investigate more specifically whether hDppa5 is expressed in hECCs, we prepared retinoic acid (RA)–treated hECC samples and checked the expression patterns of Oct4 and hDppa5. Oct4 was expressed in undifferentiated hECCs and downregulated in response to RA-induced differentiation. However, hDppa5 was not expressed in undifferentiating and differentiating hECCs. Recently, we reported that there was a set of miRNAs specifically expressed in hESCs but not in hECCs . All these data taken together, we assume that there are different gene regulation mechanisms between hESCs and hECCs.
Figure 3. Northern blot analysis of hDppa5 and hOct4 expression. (A): Expression of hDppa5 and hOct4 was analyzed in mouse embryonic fibroblast cells, STO cells, mouse embryonic stem cells (mESCs), human embryonic fibroblast cells (hEFCs), HeLa cells, human embryonic carcinoma cells (hECCs), and human ESCs (hESCs). Total RNAs were prepared from mEF, STO, mES, hEF, HeLa, NCCIT, NT2, and SNU-hES3 cells. (B): Although hDppa5 was specifically expressed only in hESCs not in hECCs, Oct4 was expressed in hESCs and hECCs, respectively. 18S ribosomal RNA is shown as a loading control. (C): Validation of gene transcription during retinoic acid–induced differentiation of hECCs (NT2). Reverse transcription polymerase chain reaction analysis with pluripotent stem cell marker (Oct4) and identify hDppa5 expression from NT2 cells (undifferentiating-NT2 and differentiating-NT2). Expression of ?-actin was used as a loading control.
We investigated the expression pattern of hDppa5 in various types of human cells in this study. Human somatic tissues (MTC Panels, BD Biosciences Clontech; colon, leukocyte, ovary, prostate, small intestine, spleen, testis, and thymus) do not express hDppa5 as detected by RT-PCR and Northern blot analysis (data not shown).
Based on expression pattern results in human PGCs, EGCs, and ESCs, we assumed that the Dppa5 is closely related in cell pluripotency and is able to serve as an informative marker for human pluripotent cell types. Although the biological mechanisms of this gene have yet to be elucidated, Dppa5 can be accurately used in the analysis of the pluripotency of human PGCs, EGCs, and ESCs.
ACKNOWLEDGMENTS
Kiger AA, Fuller MT. Male germ-line stem cell. In: Marshak DR, Gardner RL, Gottlieb D, eds. Stem Cell Biology. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2001:149–187.
Niwa H. Molecular mechanism to maintain stem cell renewal of ES cells. Cell Struct Funct 2001;26:137–148.
Donovan PJ, Gearhart J. The end of the beginning for pluripotent stem cells. Nature 2001;414:92–97.
Matsui Y, Zsebo K, Hogan BL. Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 1992;70:841–847.
McLaren A. Primordial germ cells in the mouse. Dev Biol 2003;262:1–15.
Buehr M. The primordial germ cells of mammals: some current perspectives. Exp Cell Res 1997;232:194–207.
Palmieri SL, Peter W, Hess H et al. Oct-4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. Dev Biol 1994;166:259–267.
Yeom YI, Fuhrmann G, Ovitt CE et al. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 1996;122:881–894.
Nichols J, Zevnik B, Anastassiadis K et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 1998;95:379–391.
Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000;24:372–376.
Pesce M, Gross MK, Scholer HR. In line with our ancestors Oct-4 and the mammalian germ. Bioessays 1998;20:722–732.
Niwa H, Burdon T, Chambers I etal. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev 1998;12:2048–2060.
Avilion AA, Nicolis SK, Pevny LH et al. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev 2003;17:126–140.
Hanna LA, Foreman RK, Tarasenko IA et al. Requirement for Foxd3 in maintaining pluripotent cells of the early mouse embryo. Genes Dev 2002;16:2650–2661.
Chambers I, Colby D, Robertson M et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003;113:643–655.
Mitsui K, Tokuzawa Y, Itoh H et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 2003;113:631–642.
Cooke JE, Godin I, Ffrench-Constant C et al. Culture and manipulation of primordial germ cells. Methods Enzymol 1993;225:37–58.
Andrews PW, Damjanov I, Simon D et al. Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2: differentiation in vivo and in vitro. Lab Invest 1984;50:147–162.
Andrews PW, Damjanov I, Simon D et al. A pluripotent human stem-cell clone isolated from the TERA-2 teratocarcinoma line lacks antigens SSEA-3 and SSEA-4 in vitro, but expresses these antigens when grown as a xenograft tumor. Differentiation 1985;29:127–135.
Astigiano S, Barkai U, Abarzua P et al. Changes in gene expression following exposure of nulli-SCCl murine embryonal carcinoma cells to inducers of differentiation: characterization of a down-regulated mRNA. Differentiation 1991;46:61–67.
Bierbaum P, MacLean-Hunter S, Ehlert F et al. Cloning of embryonal stem cell-specific genes: characterization of the transcriptionally controlled gene esg-1. Cell Growth Differ 1994;5:37–46.
Tanaka TS, Kunath T, Kimber WL et al. Gene expression profiling of embryo-derived stem cells reveals candidate genes associated with pluripotency and lineage specificity. Genome Res 2002;12:1921–1928.
Bortvin A, Eggan K, Skaletsky H et al. Incomplete reactivation of Oct4-related genes in mouse embryos cloned from somatic nuclei. Development 2003;130:1673–1680.
Pesce M, Scholer HR. Oct-4: gatekeeper in the beginnings of mammalian development. STEM CELLS 2001;19:271–278.
Suh MR, Lee Y, Kim JY et al. Human embryonic stem cells express a unique set of microRNAs. Dev Biol 2004;270:488–498.(Soo-Kyoung Kima, Mi Ra Su)
b Medical Research Center, MizMedi Hospital, Seoul, Korea;
c Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, Seoul, Korea;
d Department of Obstetrics and Gynecology, Hospital of Hanyang University, Guri, Korea;
e Department of Anatomy and Cell Biology, Hanyang University College of Medicine, Seoul, Korea
Key Words. Developmental pluripotency associated 5 ? Primordial germ cells ? Embryonic germ cells ? Embryonic stem cells ? Pluripotency
Correspondence: Kye-Seong Kim, D.V.M., Ph.D., Hanyang University College of Medicine, 17 Haengdang-dong, Sungdong-gu, Seoul 133-791, Korea. Telephone: 82-2-2290-0607; Fax: 82-2-2281-7841; e-mail: ks66kim@hanyang.ac.kr
ABSTRACT
In mammals, primordial germ cells (PGCs) are first specified in the extraembryonic mesoderm. Subsequently, PGCs migrate to gonads and proliferate to form gonocytes. Gonocyte identity is expressed by transition into a nonmitotic state, growth in cell size, and the onset of specific gene expression . Cellular pluripotency can be defined as the ability of a cell to differentiate into diverse cell types. During mammalian development, only particular subsets of cells in embryos transiently possess pluripotency . In germ cell lineages, PGCs give rise to pluripotent embryonic germ cells (EGCs) that form all three germ layers . However, gonocytes have unipotency and become only germ cells such as spermatogonia or oogonia . To analyze the gene expression differences between PGCs and gonocytes, we performed cDNA subtractive hybridization with mouse gonads that contained either of the two cell populations and found candidate gene developmental pluripotency associated 5 (Dppa5). Currently a few molecular regulators are known to participate in the self-renewal and pluripotency of mouse embryonic stem cells (mESCs). A POU family transcription factor Oct4, the classical marker of all pluripotent cells, is specifically expressed in pre-implantation embryos, epiblast, germ cells, and pluripotent stem cell lines, including ESCs, EGCs, and embryonic carcinoma cells (ECCs) . Oct4 has a critical role in the establishment and maintenance of pluripotent cells in a pluripotent state . Leukemia inhibitory factor (LIF) can maintain self-renewal of mESCs through activation of Stat3 . Oct4 and Stat3 each interact with various cofactors and regulate the expression of multiple target genes . Two other transcription factors, Sox2 and FoxD3, have been shown to be essential for pluripotency in mice embryos . More recently, it was found that the homeoprotein Nanog is capable of maintaining mESC self-renewal independently of LIF/Stat3 .
In this study, we clearly showed Dppa5 expressed in mouse and human pluripotent cells. Based on the expression patterns, we assumed that the Dppa5 is closely related in cell pluripotency and is able to serve as an informative marker for human pluripotent cell types.
MATERIALS AND METHODS
To begin characterizing the distinction between cells that have or do not have the potential to be a pluripotent cell line, we performed cDNA subtractive hybridization and identified several candidate genes that were expressed differentially between mouse PGCs and gonocytes. We accessed the GenBank database and searched using the BLAST algorithm at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST). One of the genes is Dppa5. This gene has exactly the same sequence as mDppa5 (GenBank; AF490349 ). mDppa5 has two other names: pH 34 and embryonal stem cell–specific gene1 . In previous reports , mDppa5 is expressed in mESCs, mouse embryonic carcinoma cells (mECCs), preimplantation embryos, and developing germ cells. However, in our study, the mDppa5 gene was strongly expressed only in PGCs at the transcriptional level, and the expression was rapidly downregulated in gonocytes and developing germ cells, including spermatogonial stem cells (Fig. 1). The mDppa5 gene was also expressed in mESCs and mECCs, as previously reported , but no expression of mDppa5 was detected in various mice somatic tissues by Northern blot analysis (data not shown).
Figure 1. Northern blot analysis of mDppa5 expression in cells and tissues of mouse. Total RNA was extracted from mouse feeder cell (STO), primordial germ cells (PGCs), gonocytes, spermatogonial stem cells (SGs), testis (2, 4, 6, and 10 weeks), pachytene spermatocyte (PS), round spermatid (RS), condensing spermatid (CS), and mouse embryonic stem cells (mESCs). The expression of Dppa5 is specifically shown in PGCs and mESCs, respectively. 18S ribosomal RNA is shown as a loading control.
To identify the human homologue of mDppa5, bioinformatics approaches found two candidates (the GenBank accession numbers are BX092581 and XM_291161 ). Recently, one report showed hDppa5 expression in hESCs, but there is no report of hDppa5 expression patterns in various cells and tissues in comparison with currently reported mDppa5. The sequences of mDppa5 and hDppa5 were highly conserved (77% identical; data not shown). Using RT-PCR, we analyzed the expression of hDppa5 in hPGCs, hEGCs, hESCs, hECCs (NCCIT and NT2), and HeLa cells, as well as mESCs and STOs (Fig. 2).
Figure 2. Reverse transcription polymerase chain reaction analysis of hDppa5 expression. (A): From human primordial germ cells (hPGCs), human embryonic germ cells (Miz-hEG1), human embryonic stem cells (Miz-hES4, Miz-hES3, Miz-hES1, and SNU-hES3), HeLa, and human embryonic carcinoma cells (NCCIT and NT2), compared with mouse embryonic stem cells (mESCs) and mouse STO. hDppa5 expression was detected in hPGCs, hEGCs, and hESCs but not in hECCs. (B): Expression of hDppa5 and hOct4 in undifferentiated (SNU-hES3) and differentiated hESCs. hDppa5 is downregulated in differentiated hESCs, as well as hOct4. The h?-actin and mGAPDH mRNA was used as an internal control.
Strikingly, hDppa5 was expressed only in hPGCs, hEGCs, and four different hESCs (Fig. 2A) but not in hECCs (NCCIT and NT2) and HeLa cells. The primers for hDppa5 did not amplify a product from RNA of mouse cells (mESC and STO). We checked the expression of hDppa5 and Oct4 in differentiated hESCs induced by in vitro fertilization. The Oct4 was downregulated in differentiated cells , and hDppa5 was also downregulated in differentiated hESCs compared with the strong expression in undifferentiated hESCs (Fig. 2B). We examined the hDppa5 expression in primary cultured human embryonic fibroblast cells, HeLa cells, hECCs (NCCIT and NT2), and hESCs by Northern blot analysis. As mentioned earlier, the hDppa5 was detected only in hESCs but not in hECCs (Fig. 3A). This finding is in contrast with the pattern of mDppa5 which is expressed in both mESCs and mECCs. However, the primary marker of pluripotent stem cells, Oct4, is expressed in hESCs and hECCs (NCCIT and NT2; Fig. 3B). To investigate more specifically whether hDppa5 is expressed in hECCs, we prepared retinoic acid (RA)–treated hECC samples and checked the expression patterns of Oct4 and hDppa5. Oct4 was expressed in undifferentiated hECCs and downregulated in response to RA-induced differentiation. However, hDppa5 was not expressed in undifferentiating and differentiating hECCs. Recently, we reported that there was a set of miRNAs specifically expressed in hESCs but not in hECCs . All these data taken together, we assume that there are different gene regulation mechanisms between hESCs and hECCs.
Figure 3. Northern blot analysis of hDppa5 and hOct4 expression. (A): Expression of hDppa5 and hOct4 was analyzed in mouse embryonic fibroblast cells, STO cells, mouse embryonic stem cells (mESCs), human embryonic fibroblast cells (hEFCs), HeLa cells, human embryonic carcinoma cells (hECCs), and human ESCs (hESCs). Total RNAs were prepared from mEF, STO, mES, hEF, HeLa, NCCIT, NT2, and SNU-hES3 cells. (B): Although hDppa5 was specifically expressed only in hESCs not in hECCs, Oct4 was expressed in hESCs and hECCs, respectively. 18S ribosomal RNA is shown as a loading control. (C): Validation of gene transcription during retinoic acid–induced differentiation of hECCs (NT2). Reverse transcription polymerase chain reaction analysis with pluripotent stem cell marker (Oct4) and identify hDppa5 expression from NT2 cells (undifferentiating-NT2 and differentiating-NT2). Expression of ?-actin was used as a loading control.
We investigated the expression pattern of hDppa5 in various types of human cells in this study. Human somatic tissues (MTC Panels, BD Biosciences Clontech; colon, leukocyte, ovary, prostate, small intestine, spleen, testis, and thymus) do not express hDppa5 as detected by RT-PCR and Northern blot analysis (data not shown).
Based on expression pattern results in human PGCs, EGCs, and ESCs, we assumed that the Dppa5 is closely related in cell pluripotency and is able to serve as an informative marker for human pluripotent cell types. Although the biological mechanisms of this gene have yet to be elucidated, Dppa5 can be accurately used in the analysis of the pluripotency of human PGCs, EGCs, and ESCs.
ACKNOWLEDGMENTS
Kiger AA, Fuller MT. Male germ-line stem cell. In: Marshak DR, Gardner RL, Gottlieb D, eds. Stem Cell Biology. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2001:149–187.
Niwa H. Molecular mechanism to maintain stem cell renewal of ES cells. Cell Struct Funct 2001;26:137–148.
Donovan PJ, Gearhart J. The end of the beginning for pluripotent stem cells. Nature 2001;414:92–97.
Matsui Y, Zsebo K, Hogan BL. Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 1992;70:841–847.
McLaren A. Primordial germ cells in the mouse. Dev Biol 2003;262:1–15.
Buehr M. The primordial germ cells of mammals: some current perspectives. Exp Cell Res 1997;232:194–207.
Palmieri SL, Peter W, Hess H et al. Oct-4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. Dev Biol 1994;166:259–267.
Yeom YI, Fuhrmann G, Ovitt CE et al. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 1996;122:881–894.
Nichols J, Zevnik B, Anastassiadis K et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 1998;95:379–391.
Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000;24:372–376.
Pesce M, Gross MK, Scholer HR. In line with our ancestors Oct-4 and the mammalian germ. Bioessays 1998;20:722–732.
Niwa H, Burdon T, Chambers I etal. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev 1998;12:2048–2060.
Avilion AA, Nicolis SK, Pevny LH et al. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev 2003;17:126–140.
Hanna LA, Foreman RK, Tarasenko IA et al. Requirement for Foxd3 in maintaining pluripotent cells of the early mouse embryo. Genes Dev 2002;16:2650–2661.
Chambers I, Colby D, Robertson M et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003;113:643–655.
Mitsui K, Tokuzawa Y, Itoh H et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 2003;113:631–642.
Cooke JE, Godin I, Ffrench-Constant C et al. Culture and manipulation of primordial germ cells. Methods Enzymol 1993;225:37–58.
Andrews PW, Damjanov I, Simon D et al. Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2: differentiation in vivo and in vitro. Lab Invest 1984;50:147–162.
Andrews PW, Damjanov I, Simon D et al. A pluripotent human stem-cell clone isolated from the TERA-2 teratocarcinoma line lacks antigens SSEA-3 and SSEA-4 in vitro, but expresses these antigens when grown as a xenograft tumor. Differentiation 1985;29:127–135.
Astigiano S, Barkai U, Abarzua P et al. Changes in gene expression following exposure of nulli-SCCl murine embryonal carcinoma cells to inducers of differentiation: characterization of a down-regulated mRNA. Differentiation 1991;46:61–67.
Bierbaum P, MacLean-Hunter S, Ehlert F et al. Cloning of embryonal stem cell-specific genes: characterization of the transcriptionally controlled gene esg-1. Cell Growth Differ 1994;5:37–46.
Tanaka TS, Kunath T, Kimber WL et al. Gene expression profiling of embryo-derived stem cells reveals candidate genes associated with pluripotency and lineage specificity. Genome Res 2002;12:1921–1928.
Bortvin A, Eggan K, Skaletsky H et al. Incomplete reactivation of Oct4-related genes in mouse embryos cloned from somatic nuclei. Development 2003;130:1673–1680.
Pesce M, Scholer HR. Oct-4: gatekeeper in the beginnings of mammalian development. STEM CELLS 2001;19:271–278.
Suh MR, Lee Y, Kim JY et al. Human embryonic stem cells express a unique set of microRNAs. Dev Biol 2004;270:488–498.(Soo-Kyoung Kima, Mi Ra Su)