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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

Identification and embryoid body (EB) differentiation of human fetus skin-derived stem cells (hfSDSCs).(A) Identification of human SDSCs from 4 months female skin tissue by antibody against Beta1-integrin. (B) hfSDSCs formed floating spheres when cultured in vitro. (C) Immunofluorescent staining of Beta1-integrin and SSEA-1 in hfSDSCs. (D) The percentage of OCT-4-, SSEA-1- and Beta1-integrin-positive hfSDSCs. (E) Expression of pluripotent markers OCT4, SOX2 and NANOG in hfSDSCs by qRT-PCR. PC: primary culture, SC: sub-culture. (F) Typical EB-like colonies formed from hfSDSCs and these colonies are morphologically similar to EBs formed from human ESCs. (G) Germ cell markers VASA and Stella were found after EB induction for 4 days.
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f1: Identification and embryoid body (EB) differentiation of human fetus skin-derived stem cells (hfSDSCs).(A) Identification of human SDSCs from 4 months female skin tissue by antibody against Beta1-integrin. (B) hfSDSCs formed floating spheres when cultured in vitro. (C) Immunofluorescent staining of Beta1-integrin and SSEA-1 in hfSDSCs. (D) The percentage of OCT-4-, SSEA-1- and Beta1-integrin-positive hfSDSCs. (E) Expression of pluripotent markers OCT4, SOX2 and NANOG in hfSDSCs by qRT-PCR. PC: primary culture, SC: sub-culture. (F) Typical EB-like colonies formed from hfSDSCs and these colonies are morphologically similar to EBs formed from human ESCs. (G) Germ cell markers VASA and Stella were found after EB induction for 4 days.

Mentions: Human fetus skin tissues (3–5 months gestational age) were collected from a local hospital after elective abortion. The skin tissues were trypsinized with TypLE Express for 15–30 min at 37 °C according to the gestational age and hfSDSCs were harvested as previously described10. The location of hfSDSCs within the fetus skin tissue was illustrated by Hematoxylin/eosin (HE) staining and Beta1-integrin immunostaining (Fig. 1A). Obviously, hfSDSCs were located on the hair follicles structures and the surface of skin tissues. Similar to mouse and porcine SDSCs, hfSDSCs formed floating spheres when cultured in vitro (Fig. 1B). The positive signals of Βeta1-integrin and SSEA-1 (stage-specific embryonic antigen-1) were observed by immunofluorescence (Fig. 1C). When hfSDSCs were subcultured in vitro, the percentage of hfSDSCs that expressed the pluripotent markers was increased (Fig. 1D), and the mRNA expression of pluripotent markers including OCT4 (POU5F1, POU Class 5 Homeobox 1), NANOG (Nanog Homeobox) and SOX2 (Sex Determining Region Y-Box 2) was elevated (Fig. 1E). Karyotyping data revealed that in vitro-cultured hfSDSCs exhibited normal chromosome status (Supplementary Fig. S1A). Furthermore, the formation of embryoid body (EB) like structure was observed when hfSDSCs were cultured in EB induction media (Fig. 1F). Interestingly, EB-like colonies formed from hfSDSCs exhibited similar morphology as those formed from human ES cell lines. Immunofluorescence data also showed the expression of STELLA and VASA in the EB-like structures (Fig. 1G). Moreover, teratoma-like structures were observed when these EB-like colonies were further transplanted under the kidney capsule of SCID/Beige mice, which may explain their ability to give rise to GCLCs (Supplementary Fig. S1B).


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)

Identification and embryoid body (EB) differentiation of human fetus skin-derived stem cells (hfSDSCs).(A) Identification of human SDSCs from 4 months female skin tissue by antibody against Beta1-integrin. (B) hfSDSCs formed floating spheres when cultured in vitro. (C) Immunofluorescent staining of Beta1-integrin and SSEA-1 in hfSDSCs. (D) The percentage of OCT-4-, SSEA-1- and Beta1-integrin-positive hfSDSCs. (E) Expression of pluripotent markers OCT4, SOX2 and NANOG in hfSDSCs by qRT-PCR. PC: primary culture, SC: sub-culture. (F) Typical EB-like colonies formed from hfSDSCs and these colonies are morphologically similar to EBs formed from human ESCs. (G) Germ cell markers VASA and Stella were found after EB induction for 4 days.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Identification and embryoid body (EB) differentiation of human fetus skin-derived stem cells (hfSDSCs).(A) Identification of human SDSCs from 4 months female skin tissue by antibody against Beta1-integrin. (B) hfSDSCs formed floating spheres when cultured in vitro. (C) Immunofluorescent staining of Beta1-integrin and SSEA-1 in hfSDSCs. (D) The percentage of OCT-4-, SSEA-1- and Beta1-integrin-positive hfSDSCs. (E) Expression of pluripotent markers OCT4, SOX2 and NANOG in hfSDSCs by qRT-PCR. PC: primary culture, SC: sub-culture. (F) Typical EB-like colonies formed from hfSDSCs and these colonies are morphologically similar to EBs formed from human ESCs. (G) Germ cell markers VASA and Stella were found after EB induction for 4 days.
Mentions: Human fetus skin tissues (3–5 months gestational age) were collected from a local hospital after elective abortion. The skin tissues were trypsinized with TypLE Express for 15–30 min at 37 °C according to the gestational age and hfSDSCs were harvested as previously described10. The location of hfSDSCs within the fetus skin tissue was illustrated by Hematoxylin/eosin (HE) staining and Beta1-integrin immunostaining (Fig. 1A). Obviously, hfSDSCs were located on the hair follicles structures and the surface of skin tissues. Similar to mouse and porcine SDSCs, hfSDSCs formed floating spheres when cultured in vitro (Fig. 1B). The positive signals of Βeta1-integrin and SSEA-1 (stage-specific embryonic antigen-1) were observed by immunofluorescence (Fig. 1C). When hfSDSCs were subcultured in vitro, the percentage of hfSDSCs that expressed the pluripotent markers was increased (Fig. 1D), and the mRNA expression of pluripotent markers including OCT4 (POU5F1, POU Class 5 Homeobox 1), NANOG (Nanog Homeobox) and SOX2 (Sex Determining Region Y-Box 2) was elevated (Fig. 1E). Karyotyping data revealed that in vitro-cultured hfSDSCs exhibited normal chromosome status (Supplementary Fig. S1A). Furthermore, the formation of embryoid body (EB) like structure was observed when hfSDSCs were cultured in EB induction media (Fig. 1F). Interestingly, EB-like colonies formed from hfSDSCs exhibited similar morphology as those formed from human ES cell lines. Immunofluorescence data also showed the expression of STELLA and VASA in the EB-like structures (Fig. 1G). Moreover, teratoma-like structures were observed when these EB-like colonies were further transplanted under the kidney capsule of SCID/Beige mice, which may explain their ability to give rise to GCLCs (Supplementary Fig. S1B).

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