Efficient Induction and Isolation of Human Primordial Germ Cell-Like Cells from Competent Human Pluripotent Stem Cells

2016 ◽  
pp. 217-226 ◽  
Author(s):  
Naoko Irie ◽  
M. Azim Surani
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cell ◽  
Human Pluripotent Stem Cells ◽  
Efficient Induction

scholarly journals Tissue of Origin, but Not XCI State, Influences Germ Cell Differentiation from Human Pluripotent Stem Cells

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2400
Author(s):  
Yolanda W. Chang ◽  
Arend W. Overeem ◽  
Celine M. Roelse ◽  
Xueying Fan ◽  
Christian Freund ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
X Chromosome ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cell ◽  
Human Pluripotent Stem Cells ◽  
Xist Expression ◽  
Germ Cell Differentiation ◽  
Differentiation Efficiency

Human pluripotent stem cells (hPSCs) are not only a promising tool to investigate differentiation to many cell types, including the germline, but are also a potential source of cells to use for regenerative medicine purposes in the future. However, current in vitro models to generate human primordial germ cell-like cells (hPGCLCs) have revealed high variability regarding differentiation efficiency depending on the hPSC lines used. Here, we investigated whether differences in X chromosome inactivation (XCI) in female hPSCs could contribute to the variability of hPGCLC differentiation efficiency during embryoid body (EB) formation. For this, we first characterized the XCI state in different hPSC lines by investigating the expression of XIST and H3K27me3, followed by differentiation and quantification of hPGCLCs. We observed that the XCI state did not influence the efficiency to differentiate to hPGCLCs; rather, hPSCs derived from cells isolated from urine showed an increased trend towards hPGCLCs differentiation compared to skin-derived hPSCs. In addition, we also characterized the XCI state in the generated hPGCLCs. Interestingly, we observed that independent of the XCI state of the hPSCs used, both hPGCLCs and soma cells in the EBs acquired XIST expression, indicative of an inactive X chromosome. In fact, culture conditions for EB formation seemed to promote XIST expression. Together, our results contribute to understanding how epigenetic properties of hPSCs influence differentiation and to optimize differentiation methods to obtain higher numbers of hPGCLCs, the first step to achieve human in vitro gametogenesis.

scholarly journals A multiplexed gRNA piggyBac transposon system facilitates efficient induction of CRISPRi and CRISPRa in human pluripotent stem cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Dane Z. Hazelbaker ◽  
Amanda Beccard ◽  
Gabriella Angelini ◽  
Patrizia Mazzucato ◽  
Angelica Messana ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Piggybac Transposon ◽  
Efficient Induction

scholarly journals Expression of primordial germ cell markers in human inducible pluripotent stem cells

2009 ◽  
Vol 92 (3) ◽  
pp. S65
Author(s):  
S.M. Salih ◽  
M.A. Garthwaite ◽  
A. Kapur ◽  
S. Albayrak ◽  
M. Giakoumopoulos ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cell ◽  
Cell Markers

219 Invitro culture environment influences the ability to generate porcine primordial germ cell-like from induced pluripotent stem cells

2020 ◽  
Vol 32 (2) ◽  
pp. 237
Author(s):  
N. Pieri ◽  
R. Botigelli ◽  
A. de Souza ◽  
K. Recchia ◽  
R. de Castro ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Growth Factor ◽  
Germ Cell ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Culture Condition ◽  
Primordial Germ Cell ◽  
Serum Replacement ◽  
Induced Pluripotent

The ability to generate primordial germ cell-like (PGCLs) from induced pluripotent stem cells (iPSCs) in swine could greatly contribute to regenerative medicine. Herein, we aimed to generate porcine PGCLs (ipPGCLs) from iPSCs derived from different culture systems. Porcine (p)iPSCs from fibroblasts of stillborn animals (n=3) were transduced with lentiviral vectors containing murine OCT4, SOX2, c-MYC, and KLF4 cDNAs and maintained in iPSC medium on mouse embryonic fibroblasts (MEFs). The cells were divided into three groups: (1) supplemented with 10ngmL−1 basic fibroblast growth factor (bFGF) and murine leukemia inhibitory factor (LIF), (2) only bFGF, or (3) only LIF. The piPSC colonies were generated and characterised for pluripotency. To induce piPSCs into ipPGCLs, three or more cell lines from each culture condition (after passage 20) were differentiated into epiblast stem cell-like cells (EpiLCs) by culture with 20ngmL−1 Activin A, 12ngmL−1 bFGF, and 1% knockout serum replacement (KSR) for 2 days. Then, cells were further induced to differentiate by nonadherent culture and supplementation with 500ngmL−1 bone morphogenetic protein (BMP)4, 500ngmL−1 BMP8a, LIF, 100ngmL−1 stem cell factor (SCF), and 50ngmL−1 epidermal growth factor for 4 days. The ipPGCLs were characterised by cell morphology and detection of germ cell markers by immunofluorescence and gene expression. Statistical analysis was determined by one-way ANOVA (Prism Software). Co-location quantification was determined using the plugin Colocalization Threshold in Image J software (National Institutes of Health). On average, the efficiency rate of iPSC generation was 71% for the iPSCs-bFGF group, 17% for the LIF group, and 85% for the bFGF+LIF group. All iPSCs colonies were positive for alkaline phosphatase and OCT4, SOX2, NANOG, TRA1-60, TRA1-81, SSEA1, and SSEA4 by immunofluorescence. Embryoid body assay revealed that the piPSCs were able to differentiate into three germ layers. The culture condition did not influence the expression of OCT4, NANOG, and KLF4 based on qRT-PCR, however; SOX2 was upregulated in the LIF group (P<0.05). The ipPGCLs generated showed a round morphology. Analysis of endogenous pluripotent genes OCT4, SOX2, and NANOG throughout differentiation (fibroblasts, iPSCs, EpiLCs, and PGCLs) revealed a mild upregulation in ipPGCLs, while OCT4 was slightly downregulated in ipPGCLs from iPSCs-LIF group. PRDM14 and STELLA were not observed in ipPGCLs, although BLIMP1 was present; DAZL and VASA were mildly upregulated. The STELLA, VASA, OCT4, and SOX2 proteins were detected in ipPGCLs, and DAZL was detected only in ipPGCLs from the iPSCs-FGF group. Protein co-localization analysis showed that ipPGCLs from the iPSCs-FGF group were 100% OCT4+STELLA-positive, 55% positive for DAZL+SOX2, and 66% positive for VASA+NANOG; for the LIF group: 99.3% were OCT4+STELLA positive, DAZL was not detected, 95.2% were positive for SOX2 and 85.6% for VASA+NANOG. In the bFGF+LIF group, 95.8% were positive for OCT4+STELLA, DAZL and SOX2 were not observed, and 70% were positive for VASA+NANOG. Exogenous reprogramming factors were still expressed and did not differ between groups. These results indicate that, under our conditions, the iPSCs-FGF group may represent the best culture condition for induction into ipPGCLs. Financial support for this study was provided by FAPESP (2015/25564-0 and 2015/26818-5).

scholarly journals Author Correction: Rapid and efficient induction of functional astrocytes from human pluripotent stem cells

2018 ◽  
Vol 16 (1) ◽  
pp. 134-134 ◽  
Author(s):  
Isaac Canals ◽  
Aurélie Ginisty ◽  
Ella Quist ◽  
Raissa Timmerman ◽  
Jonas Fritze ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Efficient Induction
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