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Stem Cells 2005;23:458-462 www.StemCells.com
© 2005 AlphaMed Press

RAPID COMMUNICATIONS

Identification of Developmental Pluripotency Associated 5 Expression in Human Pluripotent Stem Cells

Soo-Kyoung Kima, Mi Ra Suha, Hyun Soo Yoone, Jung Bok Leeb, Sun Kyung Ohc, Shin Yong Moonc, Sung-Hwan Moona, Ji Yeon Leea, Jung Hye Hwangd, Wha Ja Choe, Kye-Seong Kime

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{at}hanyang.ac.kr


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Pluripotent embryonic germ cells (EGCs) can be derived from the culture of primordial germ cells (PGCs). However, there are no reports of gonocytes, following the stage of PGC development, becoming stem cell lines. To analyze the gene expression differences between PGCs and gonocytes, we performed cDNA subtractive hybridization with mouse gonads containing either of the two cell populations. We confirmed that developmental pluripotency associated 5 (Dppa5), originally found in mouse embryonic stem cells (ESCs) and mouse embryonic carcinoma cells (ECCs), was strongly expressed in mouse PGCs and the expression was rapidly downregulated during germ cell development. A human sequence homologous to Dppa5 was identified by bioinformatics approaches. Interestingly, human Dppa5 was expressed only in human PGCs, human EGCs, and human ESCs and was not detected in human ECCs. Its expression was downregulated during induced differentiation of human ESCs. These findings confirmed that Dppa5 is specifically and differentially expressed in human cells that have pluripotency. The results strongly suggest that Dppa5 may have an important role in stemness in human ESCs and EGCs and also can be used as a marker of pluripotent stem cells. Human pluripotent stem cells may have their own ways to be pluripotent, as opposed to the much uniform mouse stem cells.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
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 [1]. 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 [2, 3]. In germ cell lineages, PGCs give rise to pluripotent embryonic germ cells (EGCs) that form all three germ layers [4, 5]. However, gonocytes have unipotency and become only germ cells such as spermatogonia or oogonia [6]. 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) [7, 8]. Oct4 has a critical role in the establishment and maintenance of pluripotent cells in a pluripotent state [9–11]. Leukemia inhibitory factor (LIF) can maintain self-renewal of mESCs through activation of Stat3 [12]. Oct4 and Stat3 each interact with various cofactors and regulate the expression of multiple target genes [2]. Two other transcription factors, Sox2 and FoxD3, have been shown to be essential for pluripotency in mice embryos [13, 14]. More recently, it was found that the homeoprotein Nanog is capable of maintaining mESC self-renewal independently of LIF/Stat3 [15, 16].

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
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 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Animals
Male ICR mice (5 days; 2, 4, 6, and 10 weeks old) and pregnant female ICR mice for 12.5 dpc and 15.5 dpc embryo preparation were purchased from Daehan Biolink Co., Ltd. (Chungbuk, Korea, http://www.dhbiolink.co.kr).

Preparation of Gonads and Testis
Embryonic gonads were surgically removed from 12.5 dpc and 15.5 dpc mouse embryos as described by Cooke et al. [17]. Neonatal testis (5 days old) and adult testis (2, 4, 6, and 10 weeks old) also were collected surgically.

Gonadalridges of 7–9 weeks post fertilization human embryos (obtained as a result of therapeutic termination of pregnancy by using a protocol approved by the Institutional Review Board of the CHA General Hospital) were collected following surgery.

Collecting Gonadal Germ Cells
To collect germ cells from mouse and human, gonads were washed briefly in calcium- and magnesium-free phosphate-buffered solution and incubated in EDTA solution (20 mg EDTA, 800 mg NaCl, 20 mg KCl, 115 mg Na2HPO4, 20 mg KH2PO4 in 100-ml distilled water) for 20 minutes at room temperature. The gonads were then washed in Dulbecco’s modified Eagle’s medium (DMEM) (10% fetal bovine serum [FBS]) and gently disrupted using a fine needle. The released germ cells were picked up by a finely drawn Pasteur pipette.

Isolation of Spermatogenic Cell Populations
Mixed populations of spermatogenic cells were obtained from testes of 10-week-old male mice using the collagenase dissociation method [8]. Purified populations of spermatogenic cells were isolated following collagenase treatment of testes and trypsin digestion of isolated seminiferous tubules using unit gravity sedimentation velocity in a bovine serum albumin gradient [8, 9]. Tubes containing each one of the following each—cell population (pachytene spermatocyte, >90% pure; round spermatid, >90% pure; and condensing spermatid-residual body mixture)—were selected by phase contrast microscopy.

Human EGC, ESC, and ECC Culture
Human EGCs (hEGCs) were cultured and subsequently passaged on a mouse STO fibroblast feeder layer (CRL-1503; American Type Culture Collection [ATCC], Manassas, VA, http://www.atcc.org) mitotically inactivated with 10 µg/ml mitomycin-C (Sigma Chemical Corp., St. Louis, http://www.sigma-aldrich.com) for 1.5 hours. The cells were grown in DMEM (GIBCOBRL, Rockville, MD, http://www.invitrogen.com) supplemented with 15% FBS (HyClone, Logan, UT, http://www.hyclone.com), 0.1 mM nonessential amino acids (Life Technologies, Karlsruhe, Germany, http://www.invitrogen.com), 0.1 mM 2-mercaptoethanol (Sigma), 2 mM glutamine (Life Technologies), 1 mM sodium pyruvate (Life Technologies), 100 µg/ml of streptomycin (Life Technologies), 1,000 U/ml of human recombinant leukemia inhibitory factor (hrLIF; Sigma), 1 ng/ml of human recombinant basic fibroblast growth factor (hrbFGF), and 10 µM forskolin (Sigma). These cells were grown in 5% CO2, 95% air and were routinely passaged every 7 days.

Human ESCs (SNU-hES3, Miz-hES3, Miz-hES4: Korean Stem Research Center registered cell line; Miz-hES1: National Institutes of Health registered cell line) were maintained in DMEM/F12 supplemented with 20% (v/v) serum replacements, 100 IU/ml penicillin, 100 µg/ml streptomycin, 0.1 mM nonessential amino acids (all from Life Technologies), 0.1 mM mercapto-ethanol (Sigma), and 4 ng/ml bFGF (Life Technologies). Colonies of hESCs were cultured on a feeder layer of mouse STO cells that had been pretreated with mitomycin-C (Sigma).

Human ECCs (NCCIT; ATCC CRL-2073 and NT2; ATCC CRL-1973) were cultured in DMEM medium with 10% FBS and 1% penicillin-streptomycin.

Human ESC Differentiation
To generate embryoid bodies, whole colonies of hESCs were detached by a glass pipette and cultured on tissue culture plates coated with Pluronic F-127 (Sigma) in media without bFGF for 10 days. The cells were then transferred to tissue culture plates coated with poly-L-ornithine 0.01% (v/v)/fibronectin 5 µg/ml (w/v) in N2 supplement medium containing 20 ng/ml bFGF. After 5 days, the cells were manually detached and transferred to new coated plates. The cells were then cultured in the same medium for 5 days. When the cells reached 70% confluency, the cells were trypsinized, split into two or three parts, and transferred to plates newly coated with poly-L-ornithine/fibronectin.

Human ECC Differentiation
NTERA2.cl.D1 (NT2) ECCs were maintained at high cell densities in DMEM (high-glucose formulation) (ATCC CRL-1973), supplemented with 10% fetal calf serum at 37°C under a humidified atmosphere of 5% CO2 in air, as previously described [18]. Differentiation of NT2 cells were seeding at a density of 106 cells per 75-cm2 flask in the same medium supplemented with 3 µM trans-retinoic acids (Sigma-Aldrich Company Ltd., Dorset, U.K., http://www.sigmaaldrich.com) [19]. Cells were harvested for RNA isolation 0, 1, 3, 5, and 7 days later. To reverse transcription polymerase chain reaction (RT-PCR) analysis of Dppa5 from NT2 cells, total RNA was isolated from cell pellets using TRIzol reagent (GIBCO-BRL). cDNA was synthesized using 5 µg of total RNA in a 20-µl reaction. SuperScript II (Invitrogen), a modified Maloney murine leukemia virus RT, and Oligo(dT)12–18 primers were used according to the manufacturer’s instructions.

cDNA Subtractive Hybridization
cDNA was synthesized from 1 µg of total RNA from 12.5 dpc and 15.5 dpc mouse gonads by using the Super SMART PCR cDNA synthesis kit (BD Biosciences Clontech, Palo Alto, CA, http://www.bdbiosciences.com/clontech/) which generates high-quality cDNA from small amounts of total RNA and performed cDNA subtractive hybridization by using the PCR-select cDNA subtraction kit (BD Biosciences Clontech).

RT-PCR
RT-PCR was performed to analyze the levels of Dppa5 expression in hPGCs, hEGCs, hESCs, and hECCs. One microgram of total RNA from the cells was treated with DNase I (Life Technologies). These RNA samples were reverse-transcribed with Super Script II (Invitrogen). Primers used for the amplification for mouse Dppa5 (mDppa5) were 5'-TGAAGACCTGAAAGATCCAG-3' (forward) and 5'-GACTGAAGCATCCATTTAGC-3' (reverse; product 210 bp). 5'-TGAAAGATCCAGAGGTGTTC-3' (forward) and 5'-ACTGGTTCACTTCATCCAAG-3' (reverse) were used for PCR amplification of human Dppa5 (hDppa5; product 299 bp) (GenBank accession number: mDppa5; AF490349 [GenBank] and hDppa5; XM_291161 [GenBank] ). Amplification was achieved by 40 cycles of 943C, 40 seconds; 483C, 50 seconds; 723C, 40 seconds, followed by a final incubation for 7 minutes at 723C for mDppa5. For hDppa5, it was achieved by 30 cycles of 943C, 35 seconds; 553C, 35 seconds; 723C, 40 seconds, followed by a final incubation for 7 minutes at 723C. 5'-CGAGCAATTTGCCAAGCTCCTGAA-3' (forward) and 5'-TTCGGGCACTGCAGGAACAAATTC-3'(reverse)were used for PCR amplification of Oct4. As a loading control, the same amounts of cDNA templates were amplified using GAPDH for mouse and ß-actin for human. The oligonucleotide primers used for the amplification of mGAPDH were 5'-ACTGGTGCTGCCAAGGCTGT-3' (forward) and 5'-CGGCATCGAAGGTGGAAGAG-3' (reverse; product 262 bp); for hß-actin they were 5'-TGGCACCACACCTTCTACAA-3' (forward) and 5'-GCACAGCTTCTCCTTAATGT-3' (reverse; product 396 bp).

Northern Blot Analysis
Expression of the Dppa5 mRNA was analyzed in mouse and human tissues by Northern blot analysis. A mouse multiple tissue blot was purchased from Seegene (Seoul, Korea, http://www.see-gene.com/new_seegene/site_renewal/index.html). A human multiple tissue blot was purchased from BD Biosciences Clontech. Cells were lysed in TRIzol reagent (GIBCO-BRL), and total RNA was purified according to the manufacturer’s instructions. RNAs (20 µg) were fractionated on a denaturing formaldehyde/agarose gel and transferred to a Hybond-N+ nylon membrane (Amersham Biosciences, Piscataway, NJ, http://www.amershambiosciences.com). Blots were hybridized overnight with 32P-labeled Dppa5 and Oct4 using ExpressHyb hybridization solution (BD Biosciences Clontech).


   RESULTS AND DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
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 [GenBank] ). mDppa5 has two other names: pH 34[20] and embryonal stem cell–specific gene1 [21, 22]. In previous reports [20–23], 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. 1Go). The mDppa5 gene was also expressed in mESCs and mECCs, as previously reported [21], but no expression of mDppa5 was detected in various mice somatic tissues by Northern blot analysis (data not shown).



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  		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 [GenBank] and XM_291161 [GenBank] ). 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. 2Go).



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  		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 hß-actin and mGAPDH mRNA was used as an internal control.

 
Strikingly, hDppa5 was expressed only in hPGCs, hEGCs, and four different hESCs (Fig. 2AGo) 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 [24], and hDppa5 was also downregulated in differentiated hESCs compared with the strong expression in undifferentiated hESCs (Fig. 2BGo). 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. 3AGo). 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. 3BGo). 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 [25]. All these data taken together, we assume that there are different gene regulation mechanisms between hESCs and hECCs.



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  		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 ß-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
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 
We are grateful to Dr. George L. Gerton and Man Ryoul Lee for critical reading of the manuscript and helpful discussion. This work was supported by grants (SC12011, SC12015, and SC12021) from the Stem Cell Research Center of the 21C Frontier R & D Program funded by the Ministry of Science and Technology, Republic of Korea, and a basic research grant (R01-2001-000-00144-0) funded by Korea Science and Engineering Foundation to K.S.K.

Soo-Kyoung Kim is currently at the Department of Microbiology, Hanyang University College of Medicine, Seoul, Korea. Mi Ra Suh is currently at the School of Biological Sciences, Seoul National University, Seoul, Korea.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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Received September 21, 2004; accepted for publication December 7, 2004.



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