Human STELLAR, NANOG, and GDF3 Genes Are Expressed in Pluripotent Cells and Map - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:38 |只看该作者 |倒序浏览 |打印

a Department of Obstetrics, Gynecology, and Reproductive Sciences, Center for Reproductive Sciences, San Francisco, California;
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b Departments of Physiology and
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c Urology,8 |! {/ a( W4 p

d Programs in Human Genetics, Cancer Genetics and Development, and Stem Cell Biology, University of California at San Francisco, San Francisco, California, USA

Key Words. Human embryonic stem cells ? Pluripotent ? Germ cell ? OCT-4 ? STELLAR ? NANOG ? GDF3 ? Ovary ? Testis ? Embryonal carcinoma ? Teratoma

Renee A. Reijo Pera, Ph.D., Center for Reproductive Sciences, HSE1656, Box 0556 UCSF, San Francisco, California 94143-0556, USA. Telephone: 415-476-3178; Fax: 415-476-3121; e-mail: reijo@itsa.ucsf.edu; Q- R$ W  _! E
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ABSTRACT
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Little is known of the determinants of pluripotency in human cells. However, one of the best characterized genes is the POU-domain class-5 transcription factor, POU5f1 (OCT-4). OCT-4 is a classic marker of pluripotency in human embryonic stem (hES) cells; expression has been shown to coordinately decrease with differentiation in vitro into embryoid bodies (EBs) and in vivo with the formation of teratocarcinomas . Additional pluripotency factors have not yet been identified in humans, though several candidate factors that function in murine ES (mES) cells, such as Fgf4, Foxd3, and the recently identified Nanog genes, have been reported . Although we might assume that a program similar to that in mES cells is required for maintenance of hES cell pluripotency and self-renewal, there are some obvious differences between the two mammalian systems. For example, it is well known that the critical leukemia inhibitory factor-signaling pathways are required for undifferentiated self-renewal of mES cells under normal physiological conditions , yet they are not sufficient for the same function in hES cells . As a result, novel approaches will be necessary in order to identify the candidate genes associated with the maintenance of hES cell pluripotency.
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A common biological link among hES cells, the epiblast, and premeiotic germ cells is pluripotency, or the ability of these cell types to contribute to multiple embryonic lineages . Interestingly, Oct-4 is enriched in each of these three cell populations in mice; with respect to germ cells, expression is reduced as meiotic differentiation is initiated . Thus, we hypothesized that genes with restricted expression patterns similar to OCT-4 also may be critical regulators of pluripotency in humans.
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Examination of Unigene EST databases for tissues that express human OCT-4 revealed that it is largely expressed in human germ cell tumors. The elevated expression may reflect an increase in the germ cell (OCT-4 ) to somatic cell (OCT-4-) ratio in these tumors. Based on these observations, we sought to identify novel genes that are expressed exclusively in pluripotent cell types by taking advantage of an observation that a consistent structural chromosomal abnormality associated with the formation and/or overproliferation of germ cell tumors in men is the formation of 12p isochromosomes. These isochromosomes may overexpress 12p genes that are associated with excessive growth of undifferentiated germ lineage cells . As a result, we chose to identify novel genes that may be associated with the molecular regulation of pluripotency by focusing on human chromosome 12p. The region of the mouse genome syntenic to human 12p is the distal end of mouse chromosome 6.

A search for loci present on the mouse chromosome 6 identified stella . stella encodes a putative DNA-binding protein that is expressed differentially between nascent germ cells and their somatic neighbors at E7.25 in mice. As development progresses, stella expression is restricted to germ line stem cells. Given that mouse stella passed our criteria of having Oct-4-like expression and mapped to a chromosomal location syntenic to human chromosome 12p, we explored this genomic locus in more detail.
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MATERIALS AND METHODS( c8 Q2 M: D5 h' H8 C

Characterization of Syntenic Human STELLAR and Surrounding Genomic Sequences

We localized the mouse genomic locus of stella to BAC RP23-180N22 (house mouse) and RP23-117I23 (C57BL/6J) by BLAST analysis of the NCBI HTGS database. All predicted and known sequences homologous with BAC RP23-117I23 and RP23-180N22 were then identified using ENSEMBL. From this analysis, a RIKEN deposited sequence (2410075G02Rik ) corresponding to the gene PGC7  was also identified. Using synteny maps in the Mouse Genome Informatics and Mouse Genome Resource databases, together with the location of the neighboring MIT marker D6MIT151 as an anchor, we identified the region of the short arm of human chromosome 12p, which was syntenic to the mouse stella locus (mouse chromosome 6 at 59.3cM). Analysis of the human map at this site (BAC RP11-277J24) revealed that the homologous region contained a predicted gene that had no previous experimental evidence for transcription. Analysis of this gene revealed that, like mouse stella (Fig. 1A), the predicted syntenic human gene (which we called STELLAR) contains four exons of length similar to the mouse gene (Fig. 1A). Further sequence analysis of BAC RP11-277J24 revealed a novel locus 72.5 kb distal to STELLAR called FLJ12581 fis (AK022643  ; Unigene Hs.329296) (Fig. 1B).5 S: C, a+ M$ k5 x0 i" Y0 k

Figure 1. Genomic organization and exon/intron structure of the mouse stella and human STELLAR locus on mouse chromosome 6 and human chromosome 12, respectively (A). Both mouse stella and human STELLAR are composed of four exons and the exon/intron boundaries are shown. B) The mouse physical map in ENSEMBL reveals that the stella locus is approximately 71.4 kb distal to the mouse Gdf3 locus and 53.7 kb proximal to nanog. Mouse BAC RP23-117I23 contains two sequence gaps represented by arrow heads. In humans, the STELLAR locus is 15.6 kb distal to GDF3 and 72.5 kb proximal to the predicted gene, FLJ12581fis (NANOG), which is syntenic to the RIKEN sequence 241000E02Rik (nanog). The two complementing human BACs (RP11-44J21 and RP11-277J24), which were used to generate the human genomic contig, are fully sequenced. C) Genomic organization and exon/intron structure of the mouse nanog and human NANOG locis. Both syntenic loci are composed of four exons. Nucleotide sequences of exon/intron junctions are shown. Uppercase letters refer to exon sequences. Intron sequences are represented by lowercase letters. All introns begin with the dinucleotide "gt" and end with the dinucleotide "ag" at both the mouse and human locis. (______) represents intronic sequence between each boundary.
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We originally named this novel human locus Neighbor Of STELLAR (NOSTEL); however, recent work has identified this novel locus as the human homolog of the mouse nanog gene . The proximal end of BAC RP11-277J24 hybridizes with a second BAC called RP11-44J21, which contains the growth and differentiation factor 3 (Gdf3) locus (Fig. 1B). By forming a contig between the two BACs, it can be determined that human STELLAR is 15.6 kb distal to GDF3 (Fig. 1B). In the mouse, Gdf3, stella, and nanog are represented in BAC RP23-117I23. The current ENSEMBL order is shown in Figure 1B; however, as more sequence information becomes available, the distances between STELLAR, NANOG, and GDF3 in the mouse genome will potentially change. Currently, stella is placed 71.4 kb distal to Gdf3 and 53.7 kb proximal to nanog. In the human genome, NANOG is annotated as a four-exon gene comparable to the mouse chromosome 6 homolog annotated in the ENSEMBL database (Fig. 1C).

To obtain the full-length cDNA sequence of human STELLAR and NANOG, we performed PCR amplification of human testis and hES cell (HSF-6) cDNA and compared the sequences of the cloned fragments with their respective annotated genomic loci. The accession numbers of cloned STELLAR and NANOG obtained in the current analysis are AY230136  and AY230262  , respectively.

STELLAR and NANOG Expression in Adult and Embryonic Tissues4 G+ }1 P" k5 c8 a3 G

Northern blot analysis was used to examine expression of NANOG and STELLAR in adult human tissues (Fig. 2A and Fig. 2B). Expression of GDF3 in adult human tissues was previously reported . Expression of both STELLAR and NANOG in all human adult tissues examined was extremely low (Fig. 2A and 2B). In both cases, a minimum of 10 days of exposure was required in order to observe the weak signals. STELLAR probe hybridized to a 1.1-kb band in ovary, testis, and thymus consistent with the length of the cDNA transcript identified from human testis (Fig. 2A). In addition, a larger 4.4-kb STELLAR band of unknown origin was also detected in testis and ovary; 3' RACE failed to identify this STELLAR transcript, although it may represent an alternatively spliced variant or novel 5' structure not identified in the current analysis. NANOG was detected as a faint 2.2-kb band exclusively in the testis (Fig. 2B). Given this extremely low expression level, we used a more sensitive RT-PCR approach to examine expression of these three loci, and OCT-4, in human fetal tissues (Fig. 2C). We found that OCT-4, STELLAR, and NANOG were expressed only in fetal ovary. All other fetal tissues had basal levels of transcription comparable to OCT-4 and lacked detectable expression. By comparison, GDF3 was enriched in fetal ovaries; however, expression was also detected in fetal kidney, lung, skeletal muscle, and thymus.
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Figure 2. Northern blot analysis of normal human adult tissues and RT-PCR of human embryonic tissues. STELLAR is expressed in testis, thymus, and ovary (A). NANOG is expressed exclusively in the adult human testis, albeit at very low levels (B). C) RT-PCR on mRNA isolated from human fetal tissues. OCT-4, STELLAR, and NANOG are expressed exclusively in the fetal ovaries at 20–29 weeks of gestation, whereas GDF3 is expressed in fetal ovary, kidney, skeletal muscle, and thymus.0 C. t; L8 z; @# |) d

Given that GDF3 was previously cloned from human teratocarcinoma libraries  and human chromosome 12p is an apparent hotspot for chromosomal abnormalities associated with teratocarcinoma, we used real-time PCR analysis to compare expression of STELLAR and NANOG in normal human testis with that in two independently isolated testicular germ cell tumors (seminomas) (Fig. 3). We determined that transcription of all three genes was greater in germ cell tumors than in normal adult testis. In particular, STELLAR was greater by approximately seven- and fourfold, GDF3 was greater by 12- and fourfold, and NANOG was greater by four- and threefold, respectively, in the two seminomas (Fig. 3). Furthermore, OCT-4, which in silico can be identified largely in germ cell tumor libraries, was greater by approximately ninefold compared with normal testis.

Figure 3. Real Time RT-PCR of STELLAR (A), GDF3 (B), NANOG (C), and OCT-4 (D) from two different seminoma specimens. In all cases, the seminomas had greater expression than normal testis. 50 ng of first-strand cDNA were used in each reaction.
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Amino Acid Identity of Human STELLAR

Translation of human STELLAR revealed a highly basic protein (pI = 8.3) of 159 amino acids, with a predicted mass of 17.9 kD (Fig. 4), similar to mouse stella (150 amino acids; 18 kD) . Analysis of the human STELLAR peptide sequence using the pSORT prediction program suggests that human STELLAR, like mouse STELLA, has no known functional motifs. Furthermore, amino acid analysis predicted that, like mouse STELLA, the human syntenic STELLAR locus also encodes a nuclear protein. However, given these similarities, the degree of amino acid identity between the mouse and human proteins is remarkably low, with just 32.1% identity over the protein sequences. Comparisons of NANOG and GDF3 proteins with their respective mouse proteins have been described  and also demonstrate low identity between these homologs (Table 1).

Figure 4. Amino acid sequence of mouse stella compared with human STELLAR cloned from a testis cDNA library with a predicted protein identity (mouse versus human) of 32.1% and similarity of 21.7%. The symbols between each alignment indicate residue conservation, (letter for amino acid identity; colon for strongly conserved; and period for weakly conserved).

Table 1. Germ cell and ES cell restricted loci at human chromosome 12p. K1 G+ \8 N1 q% {9 o% }* p0 d

Human STELLAR and NANOG Are Enriched in Pluripotent Cell Populations9 P% j$ K! {1 r7 {
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Given the expression of the STELLAR, NANOG, and GDF3 genes, albeit at low levels in normal adult human testis and ovary, we sought to further characterize their expression in somatic cells and/or germ cells of the gonads. We compared expression of human STELLAR, NANOG, and GDF3 in adult ovary, testis, and fetal ovary at 20–29 weeks of gestation. We also further defined expression in tissues with and without germ cells (Fig. 5A–5C). Our results show that all three genes and the positive control, DAZL, are expressed in the adult ovary; however, expression of NANOG in adult ovary was much lower than that of STELLAR or GDF3 (Fig. 5A). Similarly, GDF3 expression in the adult testis was lower than that of STELLAR and NANOG. RT-PCR on mRNA extracted from tissues of men who lack all germ cells (and were diagnosed with Sertoli cell only syndrome ) was used to determine if STELLAR, GDF3, and NANOG are germ cell specific in the human testis (Fig. 5B).
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Figure 5. RT-PCR analysis showing expression of NANOG, GDF3, and STELLAR in human testis, ovary, and fetal ovary at 20–29 weeks of gestation (A). Two samples from patients diagnosed with SCOS #1 and #2 were used to show that, in the absence of germ cells (therefore, absence of DAZL), STELLAR, GDF3, and NANOG are also absent (B). C) Expression in adult human ovary and isolated postovulatory oocytes. DAZL, STELLAR, GDF3, NANOG, and NCAM1 were all identified in the adult ovary, whereas only STELLAR and DAZL were expressed in mature oocytes (C). NCAM1 was used as a positive control for the presence of somatic tissue and a negative control for germ cells. GAPDH was used as a positive control for first strand cDNA production.! `, ?/ C! s# E7 O1 A$ G
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Our results indicate that, in the absence of DAZL (in biopsies that lack germ cells), STELLAR, GDF3, and NANOG are also absent (Fig. 5B). Finally, we performed RT-PCR on ovulated, unfertilized, mature oocytes from women (Fig. 5C). We found that STELLAR was expressed in isolated oocytes together with the positive control, DAZL , whereas, the somatic marker, neural cell adhesion molecule-1 (NCAM-1), was not expressed. Notably, unlike STELLAR, expression of both GDF3 and NANOG were not detected in ovulated oocytes.
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Human STELLAR, NANOG, and GDF3 Expression Decreases in hES Cells with Differentiation

Given the similarities of expression of the STELLAR, GDF3, and NANOG genes to those of OCT-4 in germ cells and germ cell tumors, we compared the relative levels of STELLAR, GDF3, and NANOG in three undifferentiated hES cell lines (HSF-6, HSF-1, and H9; Fig. 6). We found that all three genes were expressed in undifferentiated ES cells. NANOG was expressed at significantly higher levels than STELLAR and GDF3 in HSF-1 (p = 0.023), H9 (p = 0.004), and HSF-6 (p = 0.002); there was no statistically significant difference in expression between STELLAR and GDF3 in any of the three cell lines. In particular, NANOG was expressed at approximately 10-fold higher levels than GDF3 and threefold higher levels than STELLAR in the hES cell line HSF-1 (Fig. 6B). Similar expression was observed in the H9 and HSF-6 cell lines.
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Figure 6. Real-time PCR was used to measure the relative levels of human STELLAR, GDF3, and NANOG in undifferentiated HSF-1, HSF-6, and H9 hES cells. Mean normalized expression was calculated relative to GAPDH. Real-time PCR was performed in triplicate on three independently isolated samples. 50 ng of first-strand cDNA was used in each PCR reaction. The difference between A and B and A and C are significant.$ A" q+ @8 k7 |, `& R
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In order to ensure that the hES cells in the current study were not expressing differentiated germ cell markers, we examined expression of the SYnaptonemal Complex 1 Protein (SYCP1), which is expressed only during meiosis I; it was not expressed at detectable levels (Fig. 6A). Finally, we examined expression of STELLAR, GDF3, and NANOG in differentiating hES cells in suspension culture at days 0, 3, 7, and 14 of EB formation (Fig. 7). Expression of OCT-4, NANOG, GDF3, and STELLAR were all decreased as differentiation progressed. In particular, OCT-4 was significantly decreased by day 7 (p = 0.003), NANOG was significantly decreased by day 14 (p = 0.02), STELLAR was significantly decreased by day 3 (p = 0.005), and GDF3 was significantly decreased by day 14 (p = 0.009). Although there was an initial apparent rise in GDF3 expression on day 3 compared with day 0, this was not statistically significant. In contrast, the somatic lineage markers NCAM-1 and  fetoprotein (AFP) were significantly (p = 0.009 and p
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Figure 7. Real-time PCR was performed on hES cells undergoing differentiation into EBs in suspension culture. Over the 14-day culture period, OCT-4 mRNA expression decreased (A) together with a coordinate decrease in STELLAR (B), NANOG, (C) and GDF3 (D), whereas the markers of somatic lineage differentiation (NCAM-1 and AFP) increased (E). Real-time PCR was performed in triplicate with 50 ng of first-strand cDNA used in each PCR reaction. Significant difference in expression from day 0 was indicated by *(A-D). In (E), a significant difference in expression from day 0 is indicated by * (NCAM-1) ** (AFP)., h. W& j7 V, X- v* Y

DISCUSSION
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We thank the UCSF Fertility Clinic, in particular, Dr. Shehua Shen, for obtaining the human oocyte samples. We also thank Professor Robert N. Taylor, M.D., Ph.D., and Eugene Y. Xu for helpful comments on the manuscript. This work was supported by grants from the Lance Armstrong Foundation (P.J.T.), the Sandler Foundation, and the National Institute of Child Health and Human Development (Grant R01-HD37095) (R.A.R.P.).5 J' v- T0 I+ U* L' U' J3 A$ n8 d

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