Human germ cell formation in xenotransplants of induced pluripotent stem cells carrying X chromosome aneuploidies

Abstract STUDY QUESTION Does the X chromosome inactivation (XCI) of Klinefelter syndrome (KS)-derived human induced pluripotent stem cells (hiPSCs) correspond to female human pluripotent stem cells (hPSCs) and reflect the KS genotype? SUMMARY ANSWER Our results demonstrate for the first time that KS-derived hiPSCs show similar XCI behavior to female hPSCs in culture and show biological relevance to KS genotype-related clinical features. WHAT IS KNOWN ALREADY So far, assessment of XCI of KS-derived hiPSCs was based on H3K27me3 staining and X-inactive specific transcript gene expression disregarding the at least three XCI states (XaXi with XIST coating, XaXi lacking XIST coating, and XaXe (partially eroded XCI)) that female hPSCs display in culture. STUDY DESIGN, SIZE, DURATION The study used hiPSC lines generated from two azoospermic patients with KS and included two healthy male (HM) and one healthy female donor. PARTICIPANTS/MATERIALS, SETTING, METHODS In this study, we derived hiPSCs by reprograming fibroblasts with episomal plasmids and applying laminin 521 as culture substrate. hiPSCs were characterized by karyotyping, immunocytochemistry, immunohistochemistry, quantitative PCR, teratoma formation, and embryoid body differentiation. XCI and KS hiPSC relevance were assessed by whole genome transcriptomics analysis and immunocytochemistry plus FISH of KS, HM and female fibroblast, and their hiPSC derivatives. MAIN RESULTS AND THE ROLE OF CHANCE Applying whole genome transcriptomics analysis, we could identify differentially expressed genes (DEGs) between KS and HM donors with enrichment in gene ontology terms associated with fertility, cardiovascular development, ossification, and brain development, all associated with KS genotype-related clinical features. Furthermore, XCI analysis based on transcriptomics data, RNA FISH, and H3K27me3 staining revealed variable XCI states of KS hiPSCs similar to female hiPSCs, showing either normal (XaXi) or eroded (XaXe) XCI. KS hiPSCs with normal XCI showed nevertheless upregulated X-linked genes involved in nervous system development as well as synaptic transmission, supporting the potential use of KS-derived hiPSCs as an in vitro model for KS. LIMITATIONS, REASONS FOR CAUTION Detailed clinical information for patients included in this study was not available. Although a correlation between DEGs and the KS genotype could be observed, the biological relevance of these cells has to be confirmed with further experiments. In addition, karyotype analysis for two hiPSC lines was performed at passage 12 but not repeated at a later passage. Nevertheless, since all XCI experiments for those lines were performed between passage 11 and 15 the authors expect no karyotypic changes for those experiments. WIDER IMPLICATIONS OF THE FINDINGS As KS patients have variable clinical phenotypes that are influenced by the grade of aneuploidy, mosaicism, origin of the X chromosome, and XCI ‘escapee’ genes, which vary not only among individuals but also among different tissues within the same individual, differentiated KS hiPSCs could be used for a better understanding of KS pathogenesis. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Knut and Alice Wallenberg Foundation (2016.0121 and 2015.0096), Ming Wai Lau Centre for Reparative Medicine (2-343/2016), Ragnar Söderberg Foundation (M67/13), Swedish Research Council (2013-32485-100360-69), the Centre for Innovative Medicine (2–388/2016–40), Kronprinsessan Lovisas Förening För Barnasjukvård/Stiftelsen Axel Tielmans Minnesfond, Samariten Foundation, Jonasson Center at the Royal Institute of Technology, Sweden, and Initial Training Network Marie Curie Program ‘Growsperm’ (EU-FP7-PEOPLE-2013-ITN 603568). The authors declare no conflicts of interest.

Download Full-text

Corrigendum: Induced Pluripotent Stem Cells Derived From Two Idiopathic Azoospermia Patients Display Compromised Differentiation Potential for Primordial Germ Cell Fate

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.00718 ◽

2020 ◽

Vol 8 ◽

Author(s):

Fang Fang ◽

Zili Li ◽

Qian Zhao ◽

Zhen Ye ◽

Xiuli Gu ◽

...

Keyword(s):

Stem Cells ◽

Germ Cell ◽

Induced Pluripotent Stem Cells ◽

Cell Fate ◽

Pluripotent Stem Cells ◽

Primordial Germ Cell ◽

Differentiation Potential ◽

Induced Pluripotent

Download Full-text

Induced pluripotent stem cells (IPSCS) generated from patient derived cumulus cells undergoing in vitro fertilization (IVF) as a model to study germ cell biology

Fertility and Sterility ◽

10.1016/j.fertnstert.2013.07.477 ◽

2013 ◽

Vol 100 (3) ◽

pp. S454

Author(s):

A. Poelzl ◽

P.L. Yango ◽

E. Altman ◽

A.M. Zamah ◽

N.D. Tran

Keyword(s):

Stem Cells ◽

In Vitro Fertilization ◽

Germ Cell ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Cell Biology ◽

Cumulus Cells ◽

Vitro Fertilization ◽

Induced Pluripotent

Download Full-text

Embryonic germ cell extracts erase imprinted genes and improve the efficiency of induced pluripotent stem cells

Scientific Reports ◽

10.1038/s41598-018-29339-0 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 2

Author(s):

Jing Hu ◽

Qiaoshi Zhao ◽

Yukuan Feng ◽

Na Li ◽

Yanli Gu ◽

...

Keyword(s):

Stem Cells ◽

Germ Cell ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Imprinted Genes ◽

Cell Extracts ◽

Induced Pluripotent

Download Full-text

199 GERM-CELL-LIKE CELLS GENERATION FROM GOAT INDUCED PLURIPOTENT STEM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab199 ◽

2014 ◽

Vol 26 (1) ◽

pp. 214

Author(s):

D. K. Singhal ◽

H. N. Malik ◽

R. Singhal ◽

S. Saugandhika ◽

A. Dubey ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Germ Cell ◽

Induced Pluripotent Stem Cells ◽

Adult Female ◽

Germ Cells ◽

Pluripotent Stem Cells ◽

Fibroblast Cells ◽

Rt Pcr ◽

Induced Pluripotent

Primordial germ cells (PGCs) generated from embryonic stem (ES) cells in different species may be an alternative approach to dealing with the worldwide problem of increasing female infertility. Reprogramming of fibroblasts into induced pluripotent stem cells has been achieved by overexpression of different transcription factors. Here, we report the generation of female goat germ cells from goat induced pluripotent stems cells (giPSC). Goat induced pluripotent stem cells (giPSC) were produced by transduction of adult female goat fibroblast cells with Oct4, Sox2, and Nanog lentiviral particles and further sub-cultured on fibroblast feeder layers. GiPSC were characterised by different methods. These iPSC were found to express alkaline phosphatase, SSEA1, SSEA4, Tra-1–81, and Tra-1–60 surface markers. However, SSEA3 was not observed in giPSC. GiPSC also expressed Oct4, Nanog, and Sox2. Along with Oct4, Nanog, and Sox2, the expression of different transcription factors such as Cdx1, Dapp5, Dax1, Ecat, Eras, Fgf4, Gata6, Lin28, Rex1, and Utf1 was confirmed by RT-PCR. GiPSC were in vitro differentiated and three germ layers were characterised by immunostaining of Gata4 for endoderm, α-Actinin for mesoderm, and β-III tubulin for ectoderm and RT-PCR analysis of GATA4, α-Actinin and BMP4. IPSCs were directed differentiated into germ cells using retinoic acid and bone morphogenetic protein 4 without the inactivation of exogenous factors as these are also required for germ cells development. Differentiated germ cells were characterised by immunostaining against VASA and Dazl proteins. RT–PCR assay was performed for Dazl, Nanog, Nanos1, PUM8, SCP3, Stella, and VASA genes expression. Quantitative PCR was also performed for detection of VASA and Dazl expression during the course of germ cell differentiation. Flow-cytometric analysis of differentiated germ cells was confirmed the presence of germ cells in population of differentiated giPSC. Oocytes/ova-like structures, which were comparable to natural goat oocytes, were observed under scanning electron microscope (SEM). Cumulus–oocyte complex like structure was observed, which was further used for SEM. The study concluded that adult female goat fibroblast cells can be reprogrammed into induced pluripotent stem cells using ectopic expression of Oct4, Nanog, and Sox2 genes and the germ-cells-like cells generated from reprogrammed giPSC could be differentiated into goat oocytes/ova-like structure which have immense applications in human and animal reproduction.

Download Full-text

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

Reproduction Fertility and Development ◽

10.1071/rdv32n2ab219 ◽

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).

Download Full-text