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Differentiation of early germ cells from human skin-derived stem cells without exogenous gene integration.

Ge W, Ma HG, Cheng SF, Sun YC, Sun LL, Sun XF, Li L, Dyce P, Li J, Shi QH, Shen W - Sci Rep (2015)

Bottom Line: Here we successfully isolated human fetus skin-derived stem cells (hfSDSCs) from fetus skin tissue and demonstrated that hfSDSCs can be differentiated into early human germ cell-like cells (hGCLCs).Ploidy analysis and fluorescent in situ hybridization (FISH) analysis indicated that a small percentage of putative 1N populations formed from hfSDSCs when compared with positive controls.In conclusion, our data here, for the first time, demonstrated that hfSDSCs possess the differentiation potential into germ lines, and they may differentiate both male and female hGCLCs in vitro under appropriate conditions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao, Shandong 266109, China.

ABSTRACT
Infertility has long been a difficult issue for many couples. The successful differentiation of germ cells and live progeny from pluripotent stem cells brings new hope to the couples suffering with infertility. Here we successfully isolated human fetus skin-derived stem cells (hfSDSCs) from fetus skin tissue and demonstrated that hfSDSCs can be differentiated into early human germ cell-like cells (hGCLCs). These cells express human germ cell markers DAZL and VASA. Moreover, these pluripotent stem cell-derived hGCLCs are free of exogenous gene integration. When hfSDSCs were differentiated in porcine follicle fluid (PFF) conditioned media, which has been shown to promote the differentiation of mouse and porcine SDSCs into oocyte-like cells (OLCs), we observed some vesicular structures formed from hfSDSCs. Moreover, when hfSDSCs were cultured with specific conditioned media, we observed punctate and elongated SCP3 staining foci, indicating the initiation of meiosis. Ploidy analysis and fluorescent in situ hybridization (FISH) analysis indicated that a small percentage of putative 1N populations formed from hfSDSCs when compared with positive controls. In conclusion, our data here, for the first time, demonstrated that hfSDSCs possess the differentiation potential into germ lines, and they may differentiate both male and female hGCLCs in vitro under appropriate conditions.

No MeSH data available.


Related in: MedlinePlus

Putative haploids formation from hfSDSCs.(A) The spreading of synaptonemal complexs from hfSDSCs. both punctate and elongated staining patterns were observed. (B) Comparison of SCP3 staining intensity between differentiated and undifferentiated hfSDSCs. (C) Analysis of DNA content in differentiated and undifferentiated hfSDSCs. 1N cells indicate the formation of putative haploids. (D) Fluorescent in situ hybridization for chromosome 16 and 18 indicates that putative 1N populations formed from hfSDSCs.
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f3: Putative haploids formation from hfSDSCs.(A) The spreading of synaptonemal complexs from hfSDSCs. both punctate and elongated staining patterns were observed. (B) Comparison of SCP3 staining intensity between differentiated and undifferentiated hfSDSCs. (C) Analysis of DNA content in differentiated and undifferentiated hfSDSCs. 1N cells indicate the formation of putative haploids. (D) Fluorescent in situ hybridization for chromosome 16 and 18 indicates that putative 1N populations formed from hfSDSCs.

Mentions: The majority of previous studies focused on the differentiation potential of SDSCs into female GCLCs, here, we also explored whether these SDSCs (both XX and XY) have the intrinsic ability to differentiate into male GCLCs. We have compared several methods that have been successfully used in human embryonic stem cells (ESCs) differentiation into putative haploid cells. All of these methods are free of exogenous gene integration so no worry regarding iPS cells is involved here. Interestingly, after 18 days differentiation, one of differentiated groups showed a little higher percentage of putative 1N cells (Supplementary Table S1). The dynamics of the putative 1N population were tracked for a month via flow cytometry, and we observed the highest putative 1N population after 25 days of differentiation (Supplementary Fig. S2B). In our further investigation, the spreading of synaptonemal complexes was analyzed to determine the meiotic progression of hfSDSCs differentiation as previously described2, and both punctate and elongated staining patterns were observed (Fig. 3A). This result suggests that the initiation of meiosis may start but fail to fully elongate. Similar to human ESCs, undifferentiated hfSDSCs also showed punctate SCP3 foci, however, our further investigation indicated that differentiated hfSDSCs possessed more SCP3 foci compared with undifferentiated hfSDSCs (Fig. 3B). Furthermore, we observed a 1.79% of putative 1N population via flow cytometry after 25 days differentiation (Fig. 3C). It is worth mentioning that the differentiation of hfSDSCs into putative haploid cells did not show a rise in spermatogonial stem cell (SSC) conditioned media (Diff Media 1) while a higher percentage of putative haploid cell formation was observed in a differentiation media containing LIF (Leukemia Inhibitory Factor), FSH (Follicle-stimulating Hormone), EGF (epidermal growth factor), and ITS (Insulin Transferring Selenium) (Diff Media 2) (Supplementary Table S1), which is different from previous observations using human ESCs and mouse SSCs1617. To further verify the putative 1N populations as indicated by FACs analyze, FISH probes against human chromosome 16 and 18 were used to analyze putative 1N populations and normal hfSDSCs (Fig. 3D), FISH data also indicates that putative haploids formed from hfSDSCs after differentiation. Ploidy analysis together with FISH analysis both demonstrated that putative haploids populations formed from hfSDSCs.


Differentiation of early germ cells from human skin-derived stem cells without exogenous gene integration.

Ge W, Ma HG, Cheng SF, Sun YC, Sun LL, Sun XF, Li L, Dyce P, Li J, Shi QH, Shen W - Sci Rep (2015)

Putative haploids formation from hfSDSCs.(A) The spreading of synaptonemal complexs from hfSDSCs. both punctate and elongated staining patterns were observed. (B) Comparison of SCP3 staining intensity between differentiated and undifferentiated hfSDSCs. (C) Analysis of DNA content in differentiated and undifferentiated hfSDSCs. 1N cells indicate the formation of putative haploids. (D) Fluorescent in situ hybridization for chromosome 16 and 18 indicates that putative 1N populations formed from hfSDSCs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561906&req=5

f3: Putative haploids formation from hfSDSCs.(A) The spreading of synaptonemal complexs from hfSDSCs. both punctate and elongated staining patterns were observed. (B) Comparison of SCP3 staining intensity between differentiated and undifferentiated hfSDSCs. (C) Analysis of DNA content in differentiated and undifferentiated hfSDSCs. 1N cells indicate the formation of putative haploids. (D) Fluorescent in situ hybridization for chromosome 16 and 18 indicates that putative 1N populations formed from hfSDSCs.
Mentions: The majority of previous studies focused on the differentiation potential of SDSCs into female GCLCs, here, we also explored whether these SDSCs (both XX and XY) have the intrinsic ability to differentiate into male GCLCs. We have compared several methods that have been successfully used in human embryonic stem cells (ESCs) differentiation into putative haploid cells. All of these methods are free of exogenous gene integration so no worry regarding iPS cells is involved here. Interestingly, after 18 days differentiation, one of differentiated groups showed a little higher percentage of putative 1N cells (Supplementary Table S1). The dynamics of the putative 1N population were tracked for a month via flow cytometry, and we observed the highest putative 1N population after 25 days of differentiation (Supplementary Fig. S2B). In our further investigation, the spreading of synaptonemal complexes was analyzed to determine the meiotic progression of hfSDSCs differentiation as previously described2, and both punctate and elongated staining patterns were observed (Fig. 3A). This result suggests that the initiation of meiosis may start but fail to fully elongate. Similar to human ESCs, undifferentiated hfSDSCs also showed punctate SCP3 foci, however, our further investigation indicated that differentiated hfSDSCs possessed more SCP3 foci compared with undifferentiated hfSDSCs (Fig. 3B). Furthermore, we observed a 1.79% of putative 1N population via flow cytometry after 25 days differentiation (Fig. 3C). It is worth mentioning that the differentiation of hfSDSCs into putative haploid cells did not show a rise in spermatogonial stem cell (SSC) conditioned media (Diff Media 1) while a higher percentage of putative haploid cell formation was observed in a differentiation media containing LIF (Leukemia Inhibitory Factor), FSH (Follicle-stimulating Hormone), EGF (epidermal growth factor), and ITS (Insulin Transferring Selenium) (Diff Media 2) (Supplementary Table S1), which is different from previous observations using human ESCs and mouse SSCs1617. To further verify the putative 1N populations as indicated by FACs analyze, FISH probes against human chromosome 16 and 18 were used to analyze putative 1N populations and normal hfSDSCs (Fig. 3D), FISH data also indicates that putative haploids formed from hfSDSCs after differentiation. Ploidy analysis together with FISH analysis both demonstrated that putative haploids populations formed from hfSDSCs.

Bottom Line: Here we successfully isolated human fetus skin-derived stem cells (hfSDSCs) from fetus skin tissue and demonstrated that hfSDSCs can be differentiated into early human germ cell-like cells (hGCLCs).Ploidy analysis and fluorescent in situ hybridization (FISH) analysis indicated that a small percentage of putative 1N populations formed from hfSDSCs when compared with positive controls.In conclusion, our data here, for the first time, demonstrated that hfSDSCs possess the differentiation potential into germ lines, and they may differentiate both male and female hGCLCs in vitro under appropriate conditions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao, Shandong 266109, China.

ABSTRACT
Infertility has long been a difficult issue for many couples. The successful differentiation of germ cells and live progeny from pluripotent stem cells brings new hope to the couples suffering with infertility. Here we successfully isolated human fetus skin-derived stem cells (hfSDSCs) from fetus skin tissue and demonstrated that hfSDSCs can be differentiated into early human germ cell-like cells (hGCLCs). These cells express human germ cell markers DAZL and VASA. Moreover, these pluripotent stem cell-derived hGCLCs are free of exogenous gene integration. When hfSDSCs were differentiated in porcine follicle fluid (PFF) conditioned media, which has been shown to promote the differentiation of mouse and porcine SDSCs into oocyte-like cells (OLCs), we observed some vesicular structures formed from hfSDSCs. Moreover, when hfSDSCs were cultured with specific conditioned media, we observed punctate and elongated SCP3 staining foci, indicating the initiation of meiosis. Ploidy analysis and fluorescent in situ hybridization (FISH) analysis indicated that a small percentage of putative 1N populations formed from hfSDSCs when compared with positive controls. In conclusion, our data here, for the first time, demonstrated that hfSDSCs possess the differentiation potential into germ lines, and they may differentiate both male and female hGCLCs in vitro under appropriate conditions.

No MeSH data available.


Related in: MedlinePlus