Mol. Hum. Reprod. Advance Access originally published online on November 17, 2006
Molecular Human Reproduction 2007 13(1):11-20; doi:10.1093/molehr/gal096
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Derivation of oocyte-like cells from a clonal pancreatic stem cell line
Fraunhofer-Institute of Biomedical Engineering, Group of Cell Differentiation and Cell Technology at the University of Luebeck, Luebeck, Germany
1 To whom correspondence should be addressed at: Fraunhofer-Institute of Biomedical Engineering, Group of Cell Differentiation and Cell Technology at the University of Luebeck, MFC Innovationscampus 1, Maria-Goeppert-Str. 1, D-23538 Luebeck, Germany. E-mail: charli.kruse{at}ibmt.fhg.de
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Abstract |
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Adult pancreatic stem cells (PSCs) are able to differentiate spontaneously in vitro into various somatic cell types. Stem cells isolated from rat pancreas show extensive self-renewal ability and grow in highly viable long-term cultures. Additionally, these cells express typical stem cell markers such as Oct-4, nestin and SSEA-1. Although differentiation potential is slightly decreasing in long-term cultures, it is possible to keep cell lines up to passage 140. Clonal cell lines could be established from different passages and showed similar characteristics. Remarkably, one clonal cell line, generated from passage 75, showed deviant properties during further culture. Clonal cells formed aggregates, which built tissue-like structures in suspension culture. These generated 3D aggregates produced permanently new cells at the outside margin. Released cells had remarkable size, and closer examination by light microscopy analysis revealed oocyte-like morphology. A comparison of the gene expression patterns between primary cultures of passages 8 and 75, the clonal cell line and the produced oocyte-like cells (OLCs) from tissue-like structures demonstrated some differences. Expression of various germ cell markers, such as Vasa, growth differentiation marker 9 and SSEA-1, increased in the clonal cell line, and OLCs showed additionally expression of meiosis-specific markers SCP3 and DMC1. We here present a first pilot study investigating the putative germ line potential of adult PSCs.
Key words: rat pancreas/adult stem cells/clonal analysis/oocyte-like cells
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Introduction |
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Adult stem cells reside in various organs in a specific cellular environment called the niche, in which they are kept in an undifferentiated state (Watt and Hogan, 2000
; Tumbar et al., 2004; Kindler, 2005; Theise, 2006). These stem cells possess an extensive self-renewal capability bearing an indefinite proliferative potential. Until recently, adult stem cells are believed to be lineage-restricted with limited differentiation potency, compared with embryonic stem cells. Pluripotent embryonic stem cells are blastocyst-derived cells and proliferate unlimited in an undifferentiated state, being capable of giving rise to cells found in all three germ layers (Thomson et al., 1998; Amit et al., 2000; Bodnar et al., 2004). However, this stem cell plasticity was recently shown also for adult stem cells by various groups (reviewed Forbes et al., 2002). Different examples of the versatility of adult stem cells have been demonstrated from bone marrow (Jiang et al., 2002; Ortiz-Gonzalez et al., 2004), umbilical cord blood (Kogler et al., 2004), testes (Guan et al., 2006) and pancreas (Choi et al., 2004; Kruse et al., 2004; Seaberg et al., 2004; Seeberger et al., 2006). They differentiated into various cell types cutting across lineage boundaries.
We have previously reported a simple but effective method for isolation of stem cells from the exocrine pancreas. These cells were formerly termed pancreatic stellate-like cells (PSLCs) because of their morphologic and immunohistochemical similarities to pancreatic stellate cells, which are located within the interlobular septa and interacinar areas of the pancreas. According to recent published studies demonstrating isolation of adult stem cells from the pancreas (Ramiya et al., 2000; Seaberg et al., 2004), we further term these PSLCs here ‘pancreatic stem cells’ (PSCs).
Primary cultures of these adult stem cells have the capacity of extended self-renewal and are able to differentiate spontaneously into cell types of all three germ layers. Stable long-term in vitro cultures were established from rat PSCs, and propagation was possible up to passage 140. Owing to the spontaneous differentiation of primary cultures, we generated cell lines from single-cell clones for characterization of stem cell properties. One rat clonal cell line established from passage 75 displayed deviant features. These clonal cells are able to form large tissue-like structures termed ‘tissue bodies’ (TBs), which generate permanently single round-shaped cells. Here, we present that some of these cells show typical oocyte-like characteristics, demonstrated by morphological, immunohistochemical and RT–PCR analyses.
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Materials and methods |
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Cell isolation and adherent cultivation
Pancreatic acini were obtained from male Sprague-Dawley rats (200–300 g) anaesthetized and bled to death from the dorsal aorta. Before the cell isolation, the pancreas was freed of adhering fatty tissue, lymph nodes and blood vessels. The pancreatic tissue was digested, and exocrine acini were purified as described previously (Kruse et al., 2004
). The acini were resuspended in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Eggenstein, Germany) supplemented with 20% fetal calf serum (FCS, PAA Laboratories, Pasching, Germany) and cultured at 37°C in a 5% CO2-humidified atmosphere. After 1–2 days of culture, spindle-shaped cells were observed surrounding the outer borders of pancreatic acini. Differentiated acinary cells were removed during each medium change. After reaching confluence, PSCs were subcultured by trypsinization and reseeded with a density of 2–4 x 105 cells/cm2. This procedure was repeated until sufficient PSCs were available. Further cultivation was carried out with DMEM (Gibco) supplemented with 10% FCS, 1 U/ml penicillin and 10 mg/ml streptomycin (both PAA Laboratories).
Generation of cell clones
In proliferating primary stem cell cultures, a subpopulation of cells spontaneously differentiates into various cell types, thus representing a heterogeneous cell mixture. Therefore, it was required to generate cell cultures from single-cell clones. Clonal cell lines were established through diluting and plating the PSCs from primary cultures, whereby single-cell deposition was possible. Hence, the primary stem cell cultures from passage 75 were plated at a density of 0.01 cells/µl in 96-well plates. Microscopical analysis followed 10 h later, whereas every single well was checked for the presence of only one single cell. After 2 weeks, 
5% of the analysed wells contained proliferating clonal cells developed from one single stem cell. These clonal cell lines were further propagated and characterized.
Cultivation of 3D cellular aggregates from single-cell clones and generation of ‘tissue bodies’
In order to test for spontaneous differentiation, PSCs were cultured following a modified method, which has been well established for ES cells (Wobus et al., 1988). Six hundred cells were cultured in hanging drops composed of 20 µl of DMEM medium containing 20% heat-inactivated FCS, 0.1 mM non-essential amino acids (Gibco), 2 mM L-glutamine (PAA Laboratories), 1 U/ml penicillin, 10 mg/ml of streptomycin and 0.0007% ß-mercaptoethanol (Sigma, Taufkirchen, Germany). After 2 days of culture in hanging drops, the cells aggregated and formed ‘organoid bodies’ (OBs), which were maintained in suspension culture in bacteriological dishes for another 3–9 days. OBs were then seeded in chamber slides for microscopic analysis. Further cultivation of the aggregates in bacteriological culture dishes led to accumulation of generated OBs. In long-term cultures, they formed 3D tissue-like structures with sizes of >1.5 cm.
Immunocytochemistry
Rat PSCs were transferred onto 2-well chamber slides (Becton Dickinson GmbH, Heidelberg, Germany) and were further cultured until they reached confluence. Cells were fixed for 5 min with methanol/acetone (7:3) containing 1 g/ml of DAPI (Roche Molecular Biochemicals, Mannheim, Germany) at –20° C and washed three times in phosphate-buffered saline (PBS). After incubation in 1.65% normal goat serum at room temperature for 15 min, the specimens were incubated with primary antibodies for 1 h at 37°C in a humid chamber. Primary antisera were directed against octamer-4 (Oct-4, mouse monoclonal, 1:50, Chemicon, Hofheim, Germany), stage-specific embryonic antigen 1 (SSEA-1, mouse monoclonal, 1:50, Chemicon), nestin (mouse monoclonal, 1:5000, Chemicon), synaptonemal complex protein 3 (SCP3, rabbit polyclonal, 1:200, Acris, Hiddenhausen, Germany) and disrupted meiotic cDNA 1 homologue (DMC1, mouse monoclonal, 1:100, Acris).
After rinsing three times with PBS, slides were incubated for 1 h at 37°C with either Cy3-labelled anti-mouse immunoglobulin G (IgG) or fluorescein isothiocyanate (FITC)-labelled anti-rabbit IgG (Dianova, Hamburg, Germany) diluted 1:400 and 1:200, respectively. Slides were washed three times in PBS, covered in Vectashield mounting medium (Axxora, Grünberg, Germany) and analysed with a fluorescence microscope (Axioskop Zeiss, Göttingen, Germany). Images were captured with the axiovision software (Zeiss, Göttingen, Germany) and fluorescence intensity was normalized to a black background. All immunocytochemical results showed typical morphologic features corresponding to the stained cellular structures, indicating a specific staining of the antibodies. The negative controls carried out with the secondary antibodies alone showed only an unspecific, diffuse, faint staining (data not shown).
Cytospin preparation
To immobilize the floating oocyte-like cells (OLCs) for immunocytochemistry, we centrifuged cells onto microscope slides. Single cells released from the ‘tissue bodies’ were harvested by centrifugation of the growth medium (5 min, 180 x g), and the pellet was resuspended in 2 ml of PBS. The cells were immobilized on SuperFrost Plus-Slides (Menzel, Braunschweig, Germany) by centrifugation with the Shandon Cytospin II (Thermo Electron, Waltham, USA). For each slide, 500 µl of cell suspension was loaded on Shandon Cytofunnel chambers and was spun down for 3 min at 34 x g. Cytospin preparations were air-dried for 5 min and then fixed in ice-cold methanol/acetone (7:3) with DAPI for 5 min. Immunocytochemistry was performed as described for cells in chamber slides.
To compare immunocytochemical staining patterns of analysed cells, we used natural rat oocytes as positive controls. Therefore, rat ovaries were minced with small scissors, and then tissue was digested with digestion medium including collagenase according to Kruse et al. (2004) at 37°C for 30 min. The cell suspension was then centrifuged onto microscope slides with a cytospin as described above, and immunocytochemical analysis was performed.
Time-lapse microscopy
Time-lapse digital microscopy provides an important tool for studying cell movement. We used the microscope IX-71 with incubation chamber (Olympus, Hamburg, Germany) to show release of OLCs from TBs generated from the rat clonal cell line. Image sequences were taken during cultivation of TBs in a 60 x 15-mm culture dish with a camera Orca-ER, C4742-95-12ERG (Hamamatsu, Garching, Germany). The temperature in the box was maintained at 37°C. Time-lapse images were captured with a 10-fold objective with relief contrast and a camera field of 1344 x 1024 pixel. Sequence capture software Openlab (Improvision, Coventry, UK) was used for automated time-lapse recordings. Recording time was 600 ms per picture taken with time intervals of 5 s.
RT–PCR analysis
Total cellular RNA was isolated using NucleoSpin®RNA II-Kit (Macherey-Nagel, Düren, Germany). With Superscript II Reverse Transcriptase RNase H– (RT, Invitrogen, Karlsruhe, Germany) and oligo dT-Primers (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions, 0.5 µg of total RNA was reverse transcribed into cDNA. The PCRs were performed in 50 µl reaction volume using Taq DNA Polymerase (Fermentas, St Leon-Rot, Germany) and intron-spanning primers if possible. The reactions were performed for 35–42 cycles. Control runs without reverse transcriptions were performed to test RNA preparations for contamination with genomic DNA and showed no amplification products. To normalize cDNA concentration in different RT probes, we measured relative expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as representative for an internal housekeeping gene control.
Primers used were GAPDH (f) 5'-TGATGCTGGTGCTGAGTATG-3', GAPDH (r) 5'-GGGGTAGGAACACGGAAGG-3'; Vasa (f) 5'-CTGTGCTGTCCTGATGTTCC-3', Vasa (r) 5'-AACGCTCTCCTGACCCTTTC-3'; SCP3 (f) 5'-AGAGCCAGAGAATGAAAGCAA-3', SCP3 (r) 5'-CAAACAAACAA ACCCCAGAAA-3'; GDF-9 (f) 5'-CCAAAGAGGGGGTTCCTAAA-3', GDF-9 (r) 5'-CCTGTGTGTGTGACCTTGTG-3'; CD9 (f) 5'-GGTTTCCTGGGCTGCTGT-3', CD9 (r) 5'-GGATGGCTTTGAGTGTTTCC-3'; nestin (f) 5'-GAGTGGGGTAGATGGGGATT-3', nestin (r) 5'-CAGGGAGGAAGAGAGGAACA-3'; Oct-4 (f) 5'-AGGGACCGAGTAGAGTGTGG-3', Oct-4 (r) 5'-GAGAACCGTGTGAGGTGGAA-3'; DMC1 (f) 5'-CAGCGAAAAGAGGAAGATGG-3', DMC1 (r) 5'-CACACAGAGGGTATGAGACAGC-3'.
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Results |
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Morphological analysis of primary and clonal cell lines from rat PSCs
Rat PSCs derived from isolated exocrine acini could be propagated in long-term cultures at least for 140 passages. The cells grew adherent and exhibited spindle-like or stellate shapes and resembled the morphologic features of undifferentiated fibroblast-like cells (Figure 1A and B). The generated clonal cell line showed similar morphologic features, but a few cells with a different shape were dispersed between the uniformly growing cell layer having a remarkable size (Figure 1C and D).
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Induction and activation of differentiation were performed through ‘organoid body’ formation. Therefore, clonal cells were cultured in hanging drops, which led to the formation of cellular aggregates. These generated OBs adhered to the culture dish after plating and could be subcultivated. The developed OBs produced new cells at the outside margin, which had again the same morphologic features as illustrated in primary cell cultures (Figure 2A).
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Further cultivation of OBs from clonal cells led to complex cellular aggregates, which failed to attach to the culture dish’s surface and grew in suspension (Figure 2B). Long-term culture of aggregated OBs resulted in large ‘tissue bodies’, which permanently released new round-shaped cells at the surface (Figure 2C and D). A total of three ‘tissue bodies’ were developed from the aggregated OBs all displaying the same characteristics.
Properties of the TBs generated from the clonal cell line
The formed ‘tissue bodies’ from the clonal cell line grew in culture and expanded to a size of 1.5 cm diameter. Subcultivation of TBs was possible at least up to 1 year by changing the media twice a week. Microscopical analysis of the TB margin revealed loose and floating cell aggregates, from which single cells were released. Depending on the size of the TB, 20–50 cells per day were produced. Time-lapse sequences over a time frame of 60 s demonstrating single-cell release are shown in Figure 3 (Supplementary video online). These generated cells accumulated in the culture media and were carefully analysed. They proliferated also in suspension and had properties completely different from the started adherent growing cultures (Figure 2 in comparison with Figure 1). Released cells showed sphere-like shape, and some of these floating suspension cells grew in culture to an extended size reminiscent of oocytes (Figure 2E–H). These OLCs tended to form aggregates with some smaller cells over time and resembled follicle-like structures (Figure 2H). A small number of OLCs reached up to 100 µm in diameter and formed an apparent zona pellucida-like membrane (Figure 2F–H).
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Comparative expression analysis of stem cell markers with cells from primary culture, clonal cell culture and OLCs
To characterize the formed OLCs of the clonal cell line, we tested different markers typical of stem cells and specific stages of oocyte maturation by immunocytochemical and RT–PCR analyses (Figures 4, 5 and 6). It was shown that in OLCs, all markers indicating differentiation into oocytes, namely Oct-4, SSEA-1, SCP3, DMC1, Vasa, CD9 and growth differentiation marker 9 (GDF-9), could be detected.
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Immunocytochemical staining for the transcription factor Oct-4 is present in the clonal cell population and in the OLCs, whereas only faint staining could be detected in the primary cells. Whereas nuclear localization of Oct-4 could be detected in a few OLCs, which also had high staining of cytoplasm, this protein could only be located in the cytoplasm in cells of the clonal cell line.
Analysis of SSEA-1 expression showed distinct differences in the analysed cell populations. In primary cell cultures, staining for this surface marker could hardly be demonstrated, but in the clonal cell line and the OLCs, we could find prominent staining of the cells with best results in the latter ones. A comparable expression pattern was confirmed for the SCP3, a meiosis-specific protein (Schalk et al., 1998; Yuan et al., 2002). SCP3 is predominantly expressed in the clonal cell population and the OLCs with mainly cytoplasmic localization, but OLCs show additional nuclear filamentous staining of chromosomes in the nuclei. In contrast to these results, the cells from the primary cell population present only faint staining for SCP3. Single cells from isolated natural rat ovaries present strong cytoplasmic staining of the SCP3 protein and faint filamentous nuclear staining.
The second meiosis marker DMC1 (Bishop, 1994; Pittman et al., 1998; Pelttari et al., 2001) was also detected in immunocytochemical staining, and the spotted nuclear localization could be shown in some large OLCs, indicating correct functionality of this protein. Weaker DMC1 expression could be detected in cells of the primary and clonal cell population, but instead of showing the distinct spotted pattern, a more diffuse protein staining was positive in the nuclei. Single DMC1-positive cells from rat ovaries showed strong cytoplasmic and a pearl-necklet-like nuclear staining.
A more comprehensive expression analysis was performed by RT–PCR comparing expression patterns of primary cultures in passages 8 and 75, clonal cell line and OLCs.
For positive controls in RT–PCR analysis, we used RNA from the rat ovary and rat embryonic RNA from day 19 showing functionality of the various primers. GAPDH, a housekeeping gene, was used as control and to normalize RNA amount. Therefore, it is possible to estimate an expression tendency in the results with this actually non-quantitative method.
To test cell populations for their stem cell characteristics, the expression of the stem cell markers Oct-4, Nestin and CD9 was analysed. Expression of these markers could be observed in all analysed cell populations. Additionally, we examined the expression of common germ cell markers in these cell populations. The meiosis-specific SCP3 transcript was detected in all cell populations just like the oocyte-specific transcript for the Vasa protein, which is known to be expressed in post-migratory primordial germ cells until the post-meiotic stage of oocytes (Toyooka et al., 2000; Noce et al., 2001). The transcript of an additional meiosis marker DMC1 was present in primary cell populations of passage 8, the clonal cell population and in the OLCs. The expression of the oocyte marker GDF-9, which is required for normal folliculogenesis, was also determined (Jaatinen et al., 1999; Su et al., 2004). Interestingly, the expression of this mRNA is only detectable in the primary cell cultures from passage 75, the clonal cell population and the OLCs. No expression could be seen in the primary cell cultures of passage 8.
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Discussion |
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In recent publications, it was shown that adult stem cells exhibit a broader differentiation potential, as previously assumed. It was documented that stem cells from e.g. bone marrow (Jiang et al., 2002
; Ortiz-Gonzalez et al., 2004), umbilical cord blood (Kogler et al., 2004), testes (Guan et al., 2006) or pancreas (Choi et al., 2004; Kruse et al., 2004; Seaberg et al., 2004; Seeberger et al., 2006) are able to differentiate into cell types of all three germ layers. Nevertheless, the generation of germ cells seemed to be exclusively possible for embryonic stem cells (Hubner et al., 2003; Toyooka et al., 2003; Clark et al., 2004; Geijsen et al., 2004). A current publication, however, demonstrates that OLCs could be generated in vitro from fetal porcine skin cells (Dyce et al., 2006). Very recently, Nayernia et al. (2006) demonstrated the generation of male germ cells from bone marrow stem cells. These findings confirmed the controversies of suggestions that stem cells found in the bone marrow of adult mice can restock ovaries with new oocytes (Johnson et al., 2004; Byskov et al., 2005; Johnson et al., 2005; Eggan et al., 2006).
Here, we demonstrate first results of stem cells derived from exocrine rat pancreas, which are also able to generate cells with typical oocyte-like properties in vitro. These OLCs were formed from a rat clonal cell line established from passage 75. By creation of this clone, special attributes regarding cell morphology arose in this cell line. Cells from primary cultures grew adherent with a fibroblast-like shape as described previously (Kruse et al., 2004), whereas cell populations from the clonal cell line analysed herein are more heterogeneous in cell dimension with some cells having a different round shape and extended size.
As we previously showed, these PSCs have the ability to differentiate into various cell lineages especially after culture in hanging drops with formation of ‘organoid bodies’ (Kruse et al., 2004). Here, we tested the features of the generated cell clone after ‘organoid body’ generation. These bodies tended to aggregate after being cultured in suspension and grew up to a size of >1.5 cm. Remarkably, these large tissue aggregates produced permanently new cells at the outside margin which were released and proliferated in suspension culture without adhering to the culture dish. As shown in the time-lapse sequence (Figure 3 and Supplemented video), the released spheroid cells had different morphological appearance resembling oocyte-like shape. A close morphological observation of OLCs revealed striking similarities to in vitro generated OLCs from embryonic stem cells (Hubner et al., 2003; Geijsen et al., 2004) and fetal porcine skin cells (Dyce et al., 2006) as illustrated in Figure 2. So far, we have not determined the locational origin of the released OLCs, because we forbear from destroying the TBs for sectioning. Nevertheless, whether the cells originate from the surface of the TBs or were produced from certain inner structures will be part of further investigations.
To prove putative differentiation of PSCs into OLCs, we performed a comprehensive expression analysis of stem cell markers as well as germ cell markers comparing primary, clonal and OLC populations. Immunocytochemical analysis of the stem cell markers Oct-4 (Schöler et al., 1989; Fujiwara et al., 1994; Yeom et al., 1996) and SSEA-1 (Solter and Knowles, 1978; Ruhnke et al., 2003), which are in addition potent germ cell markers (Wu and Chow, 2005), could be clearly detected in OLCs, whereas the primary cell populations from passage 75 showed only faint staining for these markers. Regarding Oct-4 expression, we found increased protein staining in the clonal cell population. Interestingly, in primary as well as in clonal cells, the highest amount of Oct-4 was localized in the cytoplasm, which was also demonstrated for human oocytes and cleavage stage embryos (Cauffman et al., 2005) and artificial generated blastocysts originating from the OLCs of fetal porcine skin cells (Dyce et al., 2006). Nevertheless, there were some single cells among the OLC population with appropriate Oct-4 expression in the nuclei (Figure 4C). Similarly, expression of SSEA-1 was present in the clonal cells and could be strongly detected in the OLCs.
In addition, OLCs expressed the germ cell markers SCP3 and DMC1, which are specifically expressed during meiosis indicating the ability for reduction divisions. In agreement with Johnson et al. (2004), we also found a distinct immunostaining of SCP3 in control experiments with histological sections from rat ovaries showing single immunopositive cells proximal to the surface of the ovary (data not shown). However, for better comparison, we singularize all cells from the ovary tissue to analyse immunocytochemical stainings also in cytospin preparations. Immunocytochemistry indicated that the staining pattern of these proteins in OLCs closely resembles that observed in primordial germ cells (Di Carlo et al., 2000) and in single cells from adult rat ovaries (Johnson et al., 2004), which was also confirmed in our investigations. These meiosis markers could not be determined in primary cells and only in moderate to low amounts in the clonal cells. The detected cytoplasmic SCP3 and DMC1 expression may represent the newly synthesized protein that migrates to the nucleus later on. This unexpected pathway may be because of the artificial in vitro culture system, in which such a cellular scenario may be possible. Following immunocytochemical analysis, the expression of different stem and germ cell markers was also confirmed by RT–PCR analyses. Moreover, we here compared expression patterns of the cell populations from primary cell cultures in passages 8 and 75, the clonal cell line and from the OLCs. In addition to Oct-4, we tested the presence of some transcripts of other stem cell markers, namely nestin (Wiese et al., 2004; Amoh et al., 2005) and CD9 (Wulf et al., 2004; Nagano et al., 2005) and could detect expression in all cell populations. Expression of SCP3 transcripts was also present in all tested cell populations, although SCP3 protein expression could not be confirmed in immunocytochemical staining of cells from the primary cell population. This discrepancy may exist because of the RT–PCR technique being more sensitive in detecting small amounts of transcripts. Alternatively, the transcript may be present earlier than the protein because of possible post-transcriptional regulation of this transcript, as shown for many genes important for differentiation (Kleene, 2003; Mattick and Makunin, 2005; Graindorge et al., 2006). Therefore, starting SCP3 expression in primary cultures could be shown in RT–PCR analysis but not in immunocytochemistry. For the second meiosis marker DMC1, which could be detected in OLCs via immunocytochemistry, transcript expression could also be confirmed in RT–PCR analyses.
To analyse further germ cell characteristics of OLCs, we examined the expression of two additional germ cell markers. The oocyte-specific Vasa transcript is expressed in the post-migratory primordial germ cells until the post-meiotic stage of oocytes (Toyooka et al., 2000; Noce et al., 2001). The oocyte-secreted GDF-9 is expressed in mature oocytes and is known to be essential for folliculogenesis (Jaatinen et al., 1999; Su et al., 2004). Vasa expression could be demonstrated in considerable amounts for all analysed cell populations. In contrast to the overall expression of germ cell markers in the primary, clonal and OLC populations, we could not detect expression of GDF-9 in the early primary cultures of passage 8. GDF-9 expression could be demonstrated in the late primary cultures of passage 75, and transcript amounts could also be shown in the clonal cells and the OLCs.
As we investigated a mixture of different stages of oocyte development (Figure 2E–H), probably including primordial germ cells up to various follicle stages, we also found different markers typical for distinct oogenesis phases. So far, we cannot prove that OLCs go through proper meiosis, which was negated in a recent study for formed follicle-like structures from mouse embryonic stem cells that also express Oct-4, SSEA-1 and SCP3 (Novak etal., 2006), but despite SCP3, we found one further meiosis marker DMC1 and could additionally confirm the expression of various oocyte-specific genes in RT–PCR.
These data suggest that PSCs can develop into OLCs with ovoid morphology, forming of follicle-like structures and typical expression of various germ cell markers. Nevertheless, all cell populations independent of the clonal or non-clonal origin seemed to be a heterogeneous mixture of PSCs, various progenitor cells, differentiated cells and OLCs as demonstrated by expression of stem cell marker (e.g. Nestin), germ cell marker (e.g. Vasa and SCP3) or marker for differentiated cells (e.g. 
-SMA, data not shown). After all, an additional analysis with specific induction of PSCs towards female germ cell differentiation needs to be carried out to test formation of real oocytes.
Whether our PSC-derived OLCs can be spontaneously activated and have the ability to develop into blastocysts, as shown in recent publications for in vitro generated oocytes (Hubner et al., 2003; Dyce et al., 2006) needs to be further investigated.
Besides the results presented herein, an in vitro germ line potential of adult stem cells was published very recently for stem cells derived from fetal porcine skin. With our studies, we support these findings and demonstrate that somatic stem cells have the intrinsic ability to develop into OLCs in vitro.
To interpret these surprisingly new properties of adult stem cells, some theories had to be updated. According to published schema explaining the developmental mechanism of adult stem cell derived oocytes (Schöler and Wu, 2006), we favour the following theory (Figure 7). In mammalian development, primordial germ cells migrate from the posterior wall of the yolk sac through the hindgut to the gonads (Tam and Zhou, 1996). It is possible that some of the migrating primordial germ cells get lost on their way and reside in the adult pancreas, which forms at the same time from the foregut. Maybe, they retain their germ cell potential and could be reactivated by in vitro culture conditions. Our findings that some of the germ cell markers (e.g. SCP3) were found already in the early primary cell populations are in line with this assumption.
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Alternatively, adult stem cells may have a greater differentiation potential than previously thought. As already shown for PSCs, differentiation into various cell types such as osteocytes, adipocytes, chondrocytes, hepatocytes (Seeberger et al., 2006) as well as ß-cells and neuronal or glial cells (Seaberg et al., 2004) is possible. On the basis of this developmental plasticity, it is not implausible that PSCs also give rise to germ cells. This approach is supported by results with adult stem cells from bone marrow, which express different molecular markers associated with germ cells (Johnson et al., 2005) and exhibit the ability to differentiate into male germ cells (Nayernia et al., 2006). Our data may confirm the existence of pluripotent adult stem cells such as pancreatic-derived stem cells being able to differentiate into cells of all three germ layers (Kruse et al., 2004) and possibly female germ cells.
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Supplementary material |
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Supplementary data are available at http://molehr.oxfordjournals.org/
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Acknowledgements |
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This study was supported by a grant from the European Union (CellPROM), from the government of Schleswig-Holstein and from the Research Focus on Regenerative Medicine, Faculty of Medicine, University of Lübeck. We thank Dr S. Blanke and A. Göpel for preparation of pancreatic tissue and generation of clonal cell lines. Furthermore, we are grateful to A. Lankenau and U. Joos for skilful help with the time-lapse studies. We also thank Dr J. Rohwedel for fruitful discussion about the fate of primordial germ cells.
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References |
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Submitted on June 26, 2006; resubmitted on October 6, 2006; accepted on October 9, 2006.
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