Mol. Hum. Reprod. Advance Access originally published online on August 12, 2008
Molecular Human Reproduction 2008 14(9):521-529; doi:10.1093/molehr/gan044
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Multipotent adult germline stem cells and embryonic stem cells have similar microRNA profiles
1Institute of Human Genetics, University of Goettingen, Heinrich-Dueker- Weg 12, Goettingen D-37073, Germany 2Institute of Human Genetics, University of Mainz, Mainz 55131, Germany 3Department of Cardiology and Pneumology, University of Goettingen, Goettingen 37075, Germany 4Institute of Human Genetics, International Centre for Life, University of Newcastle, Newcastle upon Tyne NE1 3BZ, UK
5 Correspondence address. Tel: +49-551-397589; Fax: +49-551-399303; E-mail: azovoil{at}gwdg.de
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Abstract |
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Spermatogonial stem cells (SSCs) isolated from the adult mouse testis and cultured have been shown to respond to culture conditions and become pluripotent, so called multipotent adult germline stem cells (maGSCs). microRNAs (miRNAs) belonging to the 290 and 302 miRNA clusters have been previously classified as embryonic stem cell (ESC) specific. Here, we show that these miRNAs generally characterize pluripotent cells. They are expressed not only in ESCs but also in maGSCs as well as in the F9 embryonic carcinoma cell (ECC) line. In addition, we tested the time-dependent influence of different factors that promote loss of pluripotency on levels of these miRNAs in all three pluripotent cell types. Despite the differences regarding time and extent of differentiation observed between ESCs and maGSCs, expression profiles of both miRNA families showed similarities between these two cell types, suggesting similar underlying mechanisms in maintenance of pluripotency and differentiation. Our results indicate that the 290-miRNA family is connected with Oct-4 and maintenance of the pluripotent state. In contrast, members of the 302-miRNA family are induced during first stages of in vitro differentiation in all cell types tested. Therefore, detection of miRNAs of miR-302 family in pluripotent cells can be attributed to the proportion of spontaneously differentiating cells in cultures of pluripotent cells. These results are consistent with ESC-like nature of maGSCs and their potential as an alternative source of pluripotent cells from non-embryonic tissues.
Key words: multipotent adult germline stem cells/embryonic stem cells/microRNAs/Oct4/pluripotency markers
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Introduction |
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Embryonic stem cells (ESCs) are known to be pluripotent cells having the capacity to self-renew as well as the ability to generate all types of differentiated cells. However, ESCs face immune reaction after transplantation and there are ethical issues regarding the usage of embryos. Several studies have revealed that the germline lineage retains the potential to generate pluripotent cells. In 2004, ESC-like cells were found in germ stem cell cultures established from neonatal mouse testis, designated as multipotent germline stem cells (Kanatsu- Shinohara et al., 2004). In 2006, we have isolated and cultured for the first time spermatogonial stem cells (SSCs) from the adult mouse testis which respond to culture conditions and acquire ESC properties (Guan et al., 2006). We proved that the pluripotency and plasticity of these cells, which were named multipotent adult germline stem cells (maGSCs), were similar to ESCs. They are able to spontaneously differentiate into derivatives of the three embryonic germ layers in vitro, to generate teratomas in immunodeficient mice and to contribute to the development of various organs when injected into an early blastocyst. Isolation of these cells is not restricted to the transgenic Stra8-EGFP/ROSA26 mouse. We have successfully obtained ESC-like cell lines derived from testes of three different strains of mice (FVB, C57BL/6 and 129/Sv) by morphological criteria only. Our results were confirmed by other groups (Seandel et al., 2007; Izadyar et al., 2008). Interestingly, another group showed recently that SSCs are not pluripotent but that a single SSC can dedifferentiate from a highly lineage-specified state to a pluripotent state (Kanatsu- Shinohara et al., 2008). Since pluripotent cells have not been reported for human testes until now, the mouse is a necessary model system for the study of these cells.
In this study, we were interested to substantiate the ESC-like nature of maGSCs with respect to microRNA (miRNA) expression. miRNAs represent a recently identified class of cellular RNAs that regulate protein expression at the translational level. The mature miRNAs are 17–24 bp single-stranded RNA molecules which are expressed in eucaryotic cells and affect the translation or stability of target mRNAs (Bartel, 2004; Bartel and Chen, 2004). Each miRNA seems to be able to regulate multiple genes. It was shown recently that the expression of certain genes is more dependent on the level of regulatory miRNAs than on the level of mRNAs that encode the proteins (Johnson et al., 2005).
Recently, a set of miRNAs was described to be ESC-specific in mouse, with their expression being repressed during ESC differentiation and undetectable in adult mouse organs. This set of miRNAs consists of miR-290, miR-291a-3p, miR-292-3p, miR-293, miR-294 and miR-295 (miR-290 family), and miR-302a, miR-302b, miR-302c and miR-302d (miR-302 family). In a previous work, miRNAs of the miR-290 family were repressed in embryoid bodies (EBs) prepared by culturing ESC for 14 days in either the presence or absence of retinoic acid (RA), and it was suggested that their expression is specific for pluripotent ES cells and is either silenced or down-regulated upon differentiation (Houbaviy et al., 2003, 2005). Another group confirmed the expression of these miRNAs as well as of those of miR-302 family only in mESCs and mEBs, and not in somatic tissues. In addition, they reported a negative correlation in EBs between miRNAs of miR-302 family and differentiation time (Strauss et al., 2006; Chen et al., 2007). These miRNAs are expressed in clusters (members of each miR-family are transcribed as parts of the same pri-miRNA) and they have close homologues in human ESCs with the same expression profile during differentiation (Suh et al., 2004). However, their role in pluripotency is still not well defined. In this study, we show that these miRNAs generally characterize pluripotent cells, since maGSCs share with ESCs the unique characteristic of expressing these miRNAs. Furthermore, we show that members of miR-302 family are induced during first stages of in vitro differentiation.
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Materials and Methods |
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Culture of mouse maGSC and ESC lines
The culture of maGSC lines from mouse lines 129/Sv (maGSC 129SV), C57BL/6 (maGSC C57BL), FVB (maGSC FVB) and from the transgenic line Stra8-EGFP/ROSA26 (maGSC Stra8) was described previously (Guan et al., 2006). The ESC R1 line was derived from the 129/Sv mouse line (Wurst and Joyner, 1993). The ESC line ESC Stra8 was generated from the transgenic Stra8-EGFP/ROSA26 mouse as described previously (Cheng et al., 2004). To maintain maGSCs and ESCs in an undifferentiated state, the cells were cultured under standard ESC culture conditions: DMEM (PAN, Aidenbach, Germany) supplemented with 20% fetal calf serum (PAN), 2 mM L-glutamine (Pan), 50 µ M β-mercaptoethanol (Gibco/Invitrogen, Eggenstein, Germany), 1x non-essential amino acids (Gibco/Invitrogen), sodium pyruvate (Gibco/Invitrogen), penicillin/streptomycin (PAN). maGSCs and ESCs were cultured on a feeder layer of mitomycin C-inactivated mouse embryonic fibroblasts (MEFs) in the presence of 1000 U/ml recombinant mouse leukaemia inhibitory factor (LIF) (Chemicon, Temecula, USA). For the differentiation studies, the following culture conditions were used: (A) ESC medium with fibroblasts (FL) and LIF (FL+LIF); (B) ESC medium with FL, LIF and RA (10–6 M) (Sigma-Aldrich, Steinheim, Germany) (FL+LIF+RA); (C) cells were cultured in 0.1% gelatine-coated culture flasks with ESC medium, without LIF (Gel); (D) cells cultured in 0.1% gelatine-coated culture flasks with ESC medium, without LIF but with RA (Gel+RA). In order to eliminate the impact of FL on the accuracy of the results from cells cultured under conditions A and B, cells were cultured for 5 days (two passages) on 0.1% gelatine instead of FL prior to miRNA and protein extraction. F9 cells were obtained from ATCC (Manassas, USA) and cultured as described previously (Nayernia et al., 2004).
miRNA and mRNA analysis
Total RNA including miRNAs was isolated from cultured cells and from testes of wild-type 129/Sv mouse using the miRNeasy mini Kit (Qiagen, Hilden, Germany). Conversion of miRNA and mRNA into cDNA and real-time PCR detection of miRNAs was carried out according to the manufacturer's protocols using the miScript Reverse Transcription Kit and miScript SYBR Green PCR Kit (Qiagen) on an ABI Prism 7900HT Sequence Detection System. Optimized miRNA-specific primers for each miRNA as well as for the endogenous control RNU6B are also commercially available (miScript Primer Assays, Qiagen). All experiments were performed in duplicate and PCR specificity was checked by melting curves, gel electrophoresis and sequencing of the PCR products after gel extraction and cloning into a pGEM-T Easy vector (Promega, Madison, USA). On the basis of preliminary results, we decided not to include miR-302c in our study, since the high amount of unspecific products observed for this miRNA could not guarantee reliability of the results. The ESC R1 line was used to prepare the standard curve for both the target miRNA and RNU6B, to which all quantities were further normalized, and as calibrator. The ESC R1 (Fig. 1b) was of a higher passage number of the ESC R1 used in all other experiments. Moreover, RNA from MEFs was used to exclude the possibility of contamination due to FL. For real-time quantitative RT–PCR of Nestin, Vimentin, Hnf4, Nkx2.5 and Sdha, to which all quantities were further normalized, the QuantiTect SYBR-Green PCR MasterMix (Qiagen) was used with gene-specific primers provided in Supplementary Table S1.
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Protein isolation, western blotting and immunofluorescence
For isolation of proteins from cultured cells, cell pellets were resuspended in lysis buffer (10 mM Tris/HCl, pH 8, 1 mM EDTA, 2.5% SDS) containing 1 mM phenylmethanesulphonylfluoride and proteinase inhibitors and were sonificated. For protein isolation from mouse testis, 30 mg of tissue was homogenized in the lysis buffer. Protein extracts (20 µg) were denaturated at 70°C in NuPage SDS sample buffer (Invitrogen, Karlsruhe, Germany) containing 0.1 mM dithiothreitol (DTT), separated on NuPage 10% Bis-Tris Gel (Invitrogen) and transferred on a Hybond-C extra membrane (GE Healthcare Europe, Freiburg, Germany). Blots were blocked for unspecific binding and were incubated overnight at 4°C with primary and for 1 h at 4°C with secondary HRP-conjugated antibody. Protein bands were visualized using enhanced chemiluminescence as described by the manufacturer (Santa Cruz Biotechnology, USA). When the expected band size allowed it, membranes were reused for one more time and were incubated with another primary antibody after blocking. The following antibodies were used:
-Tubulin dilution 1:5000 (Sigma-Aldrich, T5168), anti-rabbit and anti-mouse IgG-peroxidase antibodies (Sigma-Aldrich), Oct-4 dil 1:500 (Abcam, Cambridge, UK, ab19857), Sox-2 dil 1:1000 (Abcam, ab15830), Zfp-206 (gift from Dr L. Stanton, Singapore) and Sall-4 dil 1:500 (Abcam, ab29112). For immunofluorescence staining of SSEA-1, the ES Cell Characterization Kit (Chemicon) was used as described by the manufacturer. An anti-rabbit IgG Cy3-conjugated antibody (Sigma-Aldrich) was used as secondary antibody and slides were stained with DAPI (Vectashield, Vector Laboratories, Burlingame, USA). Slides were viewed in a BX60 fluorescence microscope (Olympus, Hamburg, Germany). Levels of Oct-4 and Sox-2 from western blots of two independent experiments were quantified densitometrically with QuantityOne software (Bio-Rad, Muenchen, Germany) and normalized to -Tubulin.
Statistical analysis
Data are expressed as the mean ± SD. A one-way analysis of variance (ANOVA) followed by Fisher LSD's multiple comparison tests was used for statistical analysis with P < 0.05 considered statistically significant.
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maGSCs express standard pluripotency markers as well as Sall-4 and Zfp-206
In order to evaluate the pluripotency of the cells used in the experiments, the expression of pluripotency markers Oct-4, Sox-2, Zfp-206 and Sall-4 was determined at the protein level (Schöler et al., 1989; Rodda et al., 2005; Buitrago and Roop, 2007; Masui et al., 2007; Pan and Thomson, 2007). Zfp-206 and Sall-4 have been shown recently to be expressed in ESCs and become down-regulated during ESC differentiation (Zhang et al., 2006; Wang et al., 2007). As it can be seen in Supplementary Fig. S1a, b and S2a, the pluripotency markers are highly expressed in all maGSC lines derived from different mouse strains (maGSC 129SV, maGSC Stra8, maGSC FVB and maGSC C57BL). The expression of Oct-4, Sox-2 in ESC R1 and ESC Stra8 and of Sall-4 and Zfp-206 in ESC R1 was used as control. The pluripotency marker proteins could not be detected by western analysis in testis nor in inactivated MEFs.
As can be seen in Supplementary Fig. S2a, the expression of Oct-4 and Sox-2 in ESC and maGSC lines remains unchanged during passages 15–25. When cultured under differentiation conditions for 35 days (cells in 0.1% gelatine-coated flasks with 10–6 M RA), Oct-4, Sox-2 and SSEA-1 are down-regulated in ESC lines as well as in maGSC lines (Supplementary Fig. S2a and b).
ESC-specific miRNAs are expressed in maGSCs
A specific set of miRNAs is known to be present in pluripotent ESCs. These miRNAs can be demonstrated in ESC and maGSC lines of different mouse strains, whereas no expression was detected in MEFs, NIH 3T3 cells and testis (Fig. 1a). Interestingly, miRNA expression pattern of maGSC Stra8 differed from maGSCs derived from other mouse strains by demonstrating lower and higher levels of miR-290 and miR-302 family, respectively. In addition, differences were observed between maGSC 129SV and ESCs from the same mouse strain (ESC R1). We examined whether ESC and maGSC lines retain the expression of the specific miRNAs after culture for many passages. Cells of passage 15 from the mouse strain 129/Sv were cultivated for 35 days (10 passages; P25) under standard ESC culture conditions. miRNA expression levels were found to remain relatively stable despite slight differences between both cell types (Fig. 1b). Under differentiation conditions for 35 days (cells on 0.1% gelatine in the presence of 10–6 M RA), however, ESCs as well as maGSCs lost their specific miRNA signature (Fig. 1c).
Members of miR-290 family are connected with maintenance of pluripotency
We examined the effects of different factors that are commonly used for in vitro differentiation of pluripotent cells on the expression of members of ESC-specific miRNA families 290 and 302 in maGSCs in comparison with ESCs. Figure 2a summarizes the strategy we followed. ESCs and maGSCs of passage P16 from the mouse strain 129/Sv (ESC R1 and maGSC 129SV, respectively) were cultivated for 5, 10 and 21 days under different culture conditions: feeder layer (FL), LIF and RA (FL+LIF+RA); 0.1% gelatine-coated flasks (Gel); 0.1% gelatine-coated flasks and RA (Gel+RA). We also studied cells that were cultivated for 5 days in 0.1% gelatine-coated flasks and then induced by RA for 5 days (Gel+RA from Day 5). Cells were collected at Day 5, 10 and 21 and expression of miRNAs was determined.
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To assess the degree of differentiation, we determined the levels of Oct-4 and Sox-2 proteins by western analysis, and the expression of differentiation markers like Nestin, Vimentin, Hnf4 and Nkx2.5 was analysed by qRT–PCR. Figure 2b shows that, after 5 days under FL+LIF+RA condition, the expression of Oct-4 and Sox-2 is strongly down-regulated in ESCs and maGSCs. After culture of the cells for 5 days under Gel+RA condition, Oct-4 expression is hardly detectable in maGSCs and absent in ESCs. Furthermore, no Sox-2 expression is detected. However, culture under Gel condition for 5 days was found to result in down-regulation of both pluripotency marker proteins only in ESCs, but not in maGSCs. In maGSCs, Oct-4 protein levels are similar to those of untreated cells. Only after cultivation of maGSCs under Gel condition for a longer period (21 days), Oct-4 expression is down-regulated (Fig. 2c).
Expression pattern of the differentiation markers tested differed between ESCs and maGSCs. In ESCs (Fig. 3a), Vimentin and Nestin are significantly increased under Gel+RA condition at Day 5 (Vimentin also under FL+LIF+RA condition), and in all three differentiation conditions at Day 10. At Day 21, they are down-regulated under all conditions, and only Nkx2.5 is increased under Gel and Gel+RA conditions at that day. Expression of Hnf4 is significantly up-regulated only at Day 5 under Gel+RA condition. In contrast in maGSCs (Fig. 3b), no significant change in the expression of these markers takes place at Day 5 and 10, with the exceptions of Nestin, Vimentin and Hnf4 under FL+LIF+RA condition at Day 10 and a slight increase of Nkx2.5 under Gel condition at Day 10. Only at Day 21, an increase of Vimentin and Nestin expression under Gel+RA condition and of Nestin under Gel condition is observed. At Day 21, expression of Nkx2.5 and Hnf4 were restricted to under Gel and Gel+RA conditions, respectively.
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ESCs and maGSCs differed also concerning expression levels between Gel+RA and Gel+RA from Day 5 condition. Compared with Gel+RA condition, levels of Nestin and Vimentin in ESCs were lower when RA was added from Day 5, whereas in maGSCs Nestin, Vimentin and Nkx2.5 were increased under this condition and only Hnf4 levels were lower (Supplementary Fig. S3c).
We then studied the effects of the different culture conditions on the expression of members of miRNA families 290 and 302 in ESCs and maGSCs. Figure 4 shows the expression profile of miRNAs of the 290-family in ESCs and maGSCs during culture (5–21 days) under all differentiation conditions used as well as in untreated cells. In both ESCs and maGSCs, all members of the 290-family are constantly expressed or even increased in untreated cells, although in the case of maGSCs at lower levels (50% of ESC expression in some cases) comparing with ESCs. In ESCs, these miRNAs are down-regulated at Day 5 of culture under all differentiation conditions with the strongest effect observed in Gel+RA condition (Fig. 4a). At Day 10 and 21, miRNA levels can hardly be detected under all differentiation conditions (Fig. 4b and c). In maGSCs at Day 5, levels of miRNAs do not decrease in Gel and Gel+RA conditions (with the exception of miR-290 in Gel+RA). Their expression is the same or even higher than in untreated cells (Fig. 4a). At Day 10, miRNA levels have further increased in Gel condition (Fig. 4b). In Gel+RA condition at Day 10, miRNA levels do not increase further but they are still high, whereas a strong down-regulation at Day 10 is observed only under FL+LIF+RA condition (Fig. 4b). At Day 21, miRNA levels of cells in Gel condition are lower than those of untreated cells (with the exception of miR-290), but remain still high in comparison with the other two conditions (Gel+RA and FL+LIF+RA), where miRNAs are hardly detectable (Fig. 4c).
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Finally, in maGSCs if RA is added from Day 5 onwards (Gel+RA from Day 5), miRNA levels at Day 10 are lower compared with Gel+RA condition, where RA was added from the beginning, contrasting with ESC R1 (Supplementary Fig. S3a).
Members of miR-302 family are induced during first stages of in vitro differentiation
The expression profiles of members of the 302-family were found to differ significantly from those of miR-290 family members (Fig. 5). In ESCs, the Gel condition has an extreme effect on the expression of miRNAs 302 at Day 5 (Fig. 5a). They become strongly up-regulated (20–100-fold increase). Their levels decrease rapidly after Day 5, but even at Day 10 and 21 of culture, the miRNA expression is still higher than that in untreated cells (Fig. 5b and c). In Gel+RA, miRNA levels increase temporally to levels higher than that in untreated cells around Day 10 (Fig. 5b). Then they decrease leading to expression levels lower than that in untreated cells at Day 21. In contrast, when RA is added from Day 5 onwards, such an increase at Day 10 does not occur (Supplementary Fig. S3b). miRNAs 302 also become up-regulated in maGSCs under Gel condition. During the culture period of 21 days, expression levels increase 10–30-fold (Fig. 5c). However, in the case of maGSCs, levels increase gradually at least until Day 21, and not only at Day 5 like in ESCs. In the other two conditions (Gel+RA and FL+LIF+RA), miRNA levels at Day 5 are higher than those in untreated cells (Fig. 5a) and become similar to them thereafter (Fig. 5b and c). The increase from Day 5 to Day 10 observed in Gel is weaker (especially for mir-302b and d) when RA is added from Day 5 onwards (Gel+RA from Day 5), but miRNA levels in this condition are still higher compared with Gel+RA at the same day (Supplementary Fig. S3b).
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ESC-specific miRNAs are expressed in teratocarcinoma cell line F9
In addition, we studied the expression of ESC-specific miRNAs in the teratocarcinoma cell line F9 [embryonic carcinoma cell (ECCs)] that was found to share many similarities with pluripotent cells (Andrews, 2002). As can be seen from Supplementary Fig. S1a and Fig. 1a, ECCs express the pluripotency markers Oct-4 and Sox-2 as well as the ESC-specific set of miRNA families 290 and 302. In the past, RA has been used to induce differentiation of these cells (Alonso et al., 1991). When ECCs are treated with 10–6 M RA for 25 days, miR-290 and miR-291 levels decrease slightly, miR-292, miR-293 and miR-294 levels remain relatively stable and only miR-295 increases. In contrast, the levels of all miR-302 family members increase significantly (3–5-fold increase) (Fig. 6a). Both treated and untreated cells express the pluripotency markers Oct-4 and Sox-2 (Fig. 6b), but in treated cells an increase in the levels of differentiation markers Nestin and Hnf4 is observed (Fig. 6c).
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Discussion |
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Previously, several authors have described a unique miRNA expression signature in mouse ESCs. Members of 290 and 302 miRNA families were previously classified as ESC-specific, since they are expressed only in undifferentiated ESCs. Expression of these miRNAs in ESC EBs is strongly down-regulated when ESCs are induced to differentiate and undetectable in adult organs (this, however, does not apply to miR-302 family during early in vitro differentiation as we show in the present study). Our results show that maGSCs share this unique miRNA expression signature with ESC lines. These miRNAs are also constantly expressed in maGSCs and down-regulated after long exposure to differentiation conditions. However, expression levels differed between maGSCs from different mouse strains, as well as between ESCs and maGSCs from the same mouse strain. A possible explanation for this, apart from the different genetic background, could be the different passage number of the cell lines tested. As shown in Figs 1b, 4 and 5, even under standard ESC culture conditions miRNA expression levels vary between different passage numbers (which, for example, in the case of miR-293 demonstrates an increase in expression levels of more than 50% in untreated ESCs at Day 21, Fig. 4c, compared with untreated ESCs at Day 5, Fig. 4a). maGSCs Stra8 was the maGSC line of the highest passage number used in this study, which could explain the different miRNA expression pattern compared with the other maGSC lines that are of lower passage number. For this reason in all other experiments, cell lines of the same passage number and the same mouse strain (129/Sv) were used to eliminate this effect. In this case, when cell lines of the same passage number are used, maGSCs 129SV seem to express these miRNAs in lower levels than ESCs from the same background (ESC R1). We also detected these miRNAs in high levels in F9 teratocarcinoma cells, which have also been shown to be pluripotent (Andrews, 2002). Thus, it is shown that these miRNAs generally characterize pluripotent cells. However, in contrast to F9 cells, proliferation and expression of these markers in maGSCs are restricted to standard ESC culture conditions. This is an important similarity between ESCs and maGSCs that distinguishes them from ECCs.
Several authors have studied expression profiles of members of miRNA families 290 and 302 during ESC differentiation. They found a negative correlation between their expression levels and differentiation over time (Houbaviy et al., 2003; Strauss et al., 2006; Chen et al., 2007). Because ESCs and maGSCs share great similarities in pluripotency (Kanatsu-Shinohara et al., 2004, 2008; Guan et al., 2006; Seandel et al., 2007) (Supplementary Figs S1 and S2), we decided to study the profiles of both miRNA families during differentiation of both cell types. Owing to the high number of differentiation strategies so far described, we concentrated on the most important factors that prevent or induce differentiation in ESC culture, namely LIF and RA, respectively (Rohwedel et al., 1999; Rao, 2004; Kurosawa, 2007; Tighe and Gudas, 2004; Liu et al., 2007). Loss of pluripotent state of the cells tested was evaluated by determining expression levels of well-known pluripotency markers as well as differentiation markers like Nestin (neural stem cell marker) (Lin et al., 1995; Lendahl, 1997; Wiese et al., 2004), Vimentin (early neuro-ectoderm formation and cells of mesodermal origin) (Franke et al., 1982; Boisseau and Simonneau, 1989; Colucci-Guyon et al., 1999), Hnf4 (endoderm) (Taraviras et al., 1994; Duncan et al., 1997) and Nkx2.5 (early embryo heart formation) (Liberatore et al., 2002).
The observation of other authors that the members of miRNA family 290 are down-regulated in ESCs during differentiation is supported by our results and a down-regulation was found to be realized during maGSCs differentiation. However, in maGSCs under Gel and Gel+RA condition, miRNA levels remain high for a longer period than in ESCs or even increase transiently (Gel condition). These differences seem to correlate with the differences in the differentiation status of these cells. Under Gel condition, Nestin and Vimentin are up-regulated earlier in ESCs, whereas Oct-4 expression decreases later in maGSCs. In addition, FL+LIF+RA, which is the only condition in maGSCs at Day 10 with a significant increase in most differentiation markers, is characterized by a strong down-regulation of miRNA levels. Since the expression profile of Oct-4 corresponds to that of members of miRNA family 290 in both ESCs and maGSCs, our results indicate that expression of these miRNAs is more connected with maintenance of pluripotency than with differentiation.
Chen et al. have studied expression of members of the miRNA 302 family in ESCs at Days 3, 6 and 9 during EB formation in the absence of LIF. They found that these miRNAs are negatively correlated to differentiation time (Chen et al., 2007). This expression profile in ESCs is different from that we obtained in our study, since during the first 5 days of differentiation under Gel condition, all members of miRNA family 302 are strongly up-regulated. Transient up-regulation of these miRNAs in ESCs is also observed in the presence of RA, although not so strongly as in Gel condition. Expression profiles of these miRNAs in maGSCs demonstrate similarities and differences compared with ESCs. Under Gel condition, strong up-regulation of miRNA levels is also observed, and addition of RA was found to result also in an up-regulation of these miRNAs. However, in maGSCs up-regulation under Gel condition takes place slowly. At Day 21, miRNA levels in maGSCs depict 10–30-fold increase, whereas in ESCs 20–100-fold increase is reached already at Day 5. This gradual increase in maGSCs correlates to the differences in differentiation status between ESCs and maGSCs mentioned above. In addition, the increase under Gel+RA condition in maGSCs occurs at Day 5 and not at Day 10 as in ESCs.
Since the expression profile of Oct-4 does not correspond to that of 302 miR-family members, our results suggest that these miRNAs are more connected with response of pluripotent cells to differentiation than with the undifferentiated state itself. This is in contradiction to the observation that, even in undifferentiated cells, miRNAs 302 are present but can be explained by the observation that cultures of pluripotent cells contain spontaneously differentiated cells (Houbaviy et al., 2003).
The connection of members of miRNA family 290 with pluripotency and that of members of miRNA family 302 with the process of differentiation is further supported by our miRNA analysis in ECCs and by comparing Gel+RA condition with Gel+RA from Day 5 condition. In ECCs (Fig. 6), where addition of RA is followed by an increase in Nestin and Hnf4 expression, miR-302 family is up-regulated. At the same time, treated cells retain expression of miR-290 family as they do for Oct-4. When Gel+RA and Gel+RA from Day 5 conditions are compared, high levels of miR-290 family are connected with low levels of miR-302 family and most of differentiation markers (with the exception of Hnf4) and vice versa. This connection is also supported by the findings of Tang et al. They have shown that the miRNA 290 family belongs to the most significant miRNAs strongly up-regulated in early mouse embryogenesis from 2-cell stage onwards (Tang et al., 2007). This is exactly the stage when Oct-4 expression increases (Schöler et al., 1989). In contrast, miRNA family 302 does not show significant expression changes. However, the exact correlation of miR-302 expression with a specific lineage commitment requires differentiation strategies that are beyond the scope of this study and the simple differentiation model used here. In addition, it was recently shown that miRNAs of the miR-290 family control de novo DNA methylation through regulation of transcriptional repressors in mouse ESCs (Sinkkonen et al., 2008) which implies that differences observed during differentiation between ESCs and maGSCs may be connected with the differences observed in the miRNA level.
miRNAs are believed to play a crucial role in development by regulating expression of hundreds of genes simultaneously. Members of miRNA families 290 and 302, which were previously classified as ESC specific, are candidates for such a role in pluripotent stem cells and not only in mouse, since they have close homologues in human ESCs with similar expression profile during differentiation. Our results support further the connection of miR-290 family with maintenance of pluripotency and provide indirect evidence for a possible role of members of miR-302 family during first stages of in vitro differentiation of pluripotent cells. Moreover, detection of these miRNAs in maGSCs is consistent with the ESC-like nature of maGSCs and their potential as an alternative source of pluripotent cells.
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Author's contribution |
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A.Z.: Conception and design, provision of study material, collection and assembly of data, data analysis and interpretation, manuscript writing.
J.N.: Conception and design, provision of study material, manuscript writing.
N.D.: Data analysis and interpretation.
U.Z.: Manuscript writing, final approval of manuscript.
H.H.: Collection and assembly of data.
K.G.: Provision of study material, final approval of manuscript.
G.H.: Provision of study material, final approval of manuscript.
K.N.: Provision of study material, final approval of manuscript.
W.E.: Conception and design, financial support, administrative support, manuscript writing, final approval of manuscript.
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Funding |
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This work was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft: SPP 1356; EN 84/22-1, ZE 442/4-1).
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Acknowledgements |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAuthor's contributionFundingAcknowledgementsReferences |
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We would like to thank Dr Stanton (Singapore) for providing the Zfp-206 antibody. We also thank Dr A. Zibat for technical assistance with real-time PCR and Britta Kaltwasser for cell culture work.
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References |
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Alonso A, Breuer B, Steuer B, Fischer J. The F9-EC cell line as a model for the analysis of differentiation. Int J Dev Biol (1991) 35:389–397.[CrossRef][Medline]
Andrews PW. From teratocarcinomas to embryonic stem cells. Philos Trans R Soc Lond B Biol Sci (2002) 357:405–417.
Bartel DP. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell (2004) 116:281–297.[CrossRef][Web of Science][Medline]
Bartel DP, Chen CH. Micromanagers of gene expression: the potentially widespread influence of metazoan microRNAs. Nat Rev Genet (2004) 5:396–400.[Web of Science][Medline]
Boisseau S, Simonneau M. Mammalian neuronal differentiation: early expression of a neuronal phenotype from mouse neural crest cells in a chemically defined culture medium. Development (1989) 106:665–674.
Buitrago W, Roop DR. Oct-4: The almighty POUripotent regulator? J Invest Dermatol (2007) 127:260–262.[CrossRef][Web of Science][Medline]
Chen C, Ridzon D, Lee CT, Blake J, Sun Y, Strauss WM. Defining embryonic stem cell identity using differentiation-related microRNAs and their potential targets. Mamm Genome (2007) 18:316–327.[CrossRef][Web of Science][Medline]
Cheng J, Dutra A, Takesono A, Garrett-Beal L, Schwartzberg PL. Improved generation of C57BL/6J mouse embryonic stem cells in a defined serum-free media. Genesis (2004) 39:100–104.[CrossRef][Web of Science][Medline]
Colucci-Guyon E, Gimenez Y, Ribotta M, Maurice T, Babinet C, Privat A. Cerebellar defect and impaired motor coordination in mice lacking vimentin. Glia (1999) 25:33–43.[CrossRef][Web of Science][Medline]
Duncan SA, Nagy A, Chan W. Murine gastrulation requires HNF-4 regulated gene expression in the visceral endoderm: tetraploid rescue of Hnf-4(-/-) embryos. Development (1997) 124:279–287.[Abstract]
Franke WW, Grund C, Kuhn C, Jackson BW, Illmensee K. Formation of cytoskeletal elements during mouse embryogenesis. III. Primary mesenchymal cells and the first appearance of vimentin filaments. Differentiation (1982) 23:43–59.[CrossRef][Web of Science][Medline]
Guan K, Nayernia K, Maier LS, Wagner S, Dressel R, Lee JH, Nolte J, Wolf F, Li M, Engel W, et al. Pluripotency of spermatogonial stem cells from adult mouse testis. Nature (2006) 440:1199–1203.[CrossRef][Web of Science][Medline]
Houbaviy H, Murray M, Sharp P. Embryonic stem cell-specific microRNAs. Dev Cell (2003) 5:351–358.[CrossRef][Web of Science][Medline]
Houbaviy H, Dennis L, Jaenisch R, Sharp PA. Characterization of a highly variable eutherian microRNA gene. RNA (2005) 11:1245–1257.
Izadyar F, Pau F, Marh J, Slepko N, Wang T, Gonzalez R, Ramos T, Howerton K, Sayre C, Silva F. Generation of multipotent cell lines from a distinct population of male germ line stem cells. Reproduction (2008) 135:771–784.
Johnson SM, Grosshans H, Shingara J. RAS is regulated by the let-7 microRNA family. Cell (2005) 120:635–647.[CrossRef][Web of Science][Medline]
Kanatsu-Shinohara M, Inoue K, Lee J, Yoshimoto M, Ogonuki N, Miki H, Baba S, Kato T, Kazuki Y, Toyokuni S, et al. Generation of pluripotent stem cells from neonatal mouse testis. Cell (2004) 119:1001–1012.[CrossRef][Web of Science][Medline]
Kanatsu-Shinohara M, Lee J, Inoue K, Ogonuki N, Miki H, Toyokuni S, ikawa M, Tomoyuki N, Ogura A, Shinohara T. Pluripotency of a single spermatogonial stem cell in mice. Biol Reprod (2008) 78:681–687.
Kurosawa H. Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. J Biosci Bioeng (2007) 103:389–398.[CrossRef][Web of Science][Medline]
Lendahl U. Transgenic analysis of central nervous system development and regeneration. Acta Anaesthesiol Scand Suppl (1997) 110:116–118.[Medline]
Liberatore CM, Searcy-Schrick RD, Vincent EB, Yutzey KE. Nkx-2.5 gene induction in mice is mediated by a Smad consensus regulatory region. Dev Biol (2002) 244:243–256.[CrossRef][Web of Science][Medline]
Lin RC, Matesie DF, Marvin M, McKay RD, Bruestle O. Re-expression of the intermediate filament nestin in reactive astrocytes. Neurobiol Dis (1995) 2:79–85.[CrossRef][Web of Science][Medline]
Liu N, Lu M, Tian X, Han Z. Molecular mechanisms involved in self-renewal and pluripotency of embryonic stem cells. J Cell Physiol (2007) 211:279–286.[CrossRef][Web of Science][Medline]
Masui S, Nakatake Y, Toyooka Y, Shimosato D, Yagi R, Takahashi K, Okochi H, Okuda A, Matoba R, Sharov AA, et al. Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nat Cell Biol (2007) 9:625–635.[CrossRef][Web of Science][Medline]
Nayernia K, Li M, Jaroszynski L, Khusainov R, Wulf G, Schwandt I, Korabiowska M, Michelmann HW, Meinhardt A, Engel W. Stem cell based therapeutical approach of male infertility by teratocarcinoma derived germ cells. Hum Mol Genet (2004) 13:1451–1460.
Pan G, Thomson JA. Nanog and transcriptional networks in embryonic stem cell pluripotency. Cell Res (2007) 17:42–49.[CrossRef][Web of Science][Medline]
Rao M. Conserved and divergent paths that regulate self-renewal in mouse and human embryonic stem cells. Dev Biol (2004) 275:269–286.[CrossRef][Web of Science][Medline]
Rodda DJ, Chew JL, Lim LH, Loh YH, Wang B, Ng HH, Robson P. Transcriptional regulation of Nanog by Oct4 and Sox2. J Biol Chem (2005) 280:24731–24737.
Rohwedel J, Guan K, Wobus A. Induction of cellular differentiation by retinoic acid in vitro. Cells tissues Organs (1999) 165:190–202.[CrossRef][Web of Science][Medline]
Schöler HR, Hatzopoulos AK, Balling R, Suzuki N, Gruss P. A family of octamer-specific proteins present during mouse embryogenesis: evidence for germline-specific expression of an Oct factor. EMBO J (1989) 8:2543–2550.[Web of Science][Medline]
Seandel M, James D, Shmelkov SV, Falciatori I, Kim J, Chavala S, Scherr DS, Zhang F, Torres R, Gale NW, et al. Generation of functional multipotent adult stem cells from GPR125+ germline progenitors. Nature (2007) 449:346–350.[CrossRef][Web of Science][Medline]
Sinkkonen L, Hugenschmidt T, Berninger P, Gaidatzis D, Mohn F, Artus-Revel CG, Zavolan M, Svoboda P, Filipowicz W. MicroRNAs control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells. Nat Struct Mol Biol (2008) 15:259–267.[CrossRef][Web of Science][Medline]
Strauss W, Chen C, Lee CT, Ridzon D. Nonrestrictive developmental regulation of microRNA gene expression. Mamm Genome (2006) 17:833–840.[CrossRef][Web of Science][Medline]
Suh MR, Lee Y, Kim JY, Kim SK, Moon SH, Lee JY, Cha KY, Chung HM, Yoon HS, Moon SY, et al. Human embryonic stem cells express a unique set of microRNAs. Dev Biol (2004) 270:488–498.[CrossRef][Web of Science][Medline]
Tang F, Kaneda M, O'Carroll D, Hajkova P, Barton SC, Sun YA, Lee C, Tarakhovsky A, Lao K, Surani MA. Maternal microRNAs are essential for mouse zygotic development. Genes Dev (2007) 21:644–648.
Taraviras S, Monaghan AP, Schütz G, Kelsey G. Characterization of the mouse HNF-4 gene and its expression during mouse embryogenesis. Mech Dev (1994) 48:67–79.[CrossRef][Web of Science][Medline]
Tighe A, Gudas LJ. Retinoic acid inhibits leukemia inhibitory factor signaling pathways in mouse embryonic stem cells. J Cell Physiol (2004) 198:223–229.[CrossRef][Web of Science][Medline]
Wang ZX, Kueh JL, Teh CH, Rossbach M, Lim L, Li P, Wong KY, Lufkin T, Robson P, Stanton LW. Zfp206 is a transcription factor that controls pluripotency of embryonic stem cells. Stem Cells (2007) a 25:2173–2182.
Wang ZX, Teh Ch, Kueh JL, Lufkin T, Robson P, Stanton LW. Oct4 and Sox2 directly regulate expression of another pluripotency transcription factor, Zfp206, in embryonic stem cells. J Biol Chem (2007) b 282:12822–12830.
Wiese C, Rolletschek A, Kania G, Blyszczuk P, Tarasov KV, Tarasova Y, Wersto RP, Boheler KR, Wobus AM. Nestin expression—a property of multi-lineage progenitor cells? In: Cell Mol Life Sci (2004) 61:2510–2522.[CrossRef][Web of Science][Medline]
Wurst W, Joyner AL. Production of targeted embryonic stem cell clones. In: Gene Targeting: A Practical Approach.—Joyner AL, ed. (1993) Oxford, UK: IRL Press. 33–61.
Zhang J, Tam WL, Tong GQ, Wu Q, Chan HY, Soh BS, Lou Y, Yang J, Ma Y, Chai L, et al. Sall4 modulates embryonic stem cell pluripotency and early embryonic development by the transcriptional regulation of Pou5f1. Nat Cell Biol (2006) 8:1114–1123.[CrossRef][Web of Science][Medline]
Submitted on March 29, 2008; resubmitted on July 24, 2008; accepted on August 6, 2008.
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