iPSCs derived from infertile men carrying complex genetic abnormalities can generate primordial germ-like cells

Abstract

Despite increasing insight into the genetics of infertility, the developmental disease processes remain unclear due to the lack of adequate experimental models. The advent of induced pluripotent stem cell (iPSC) technology has provided a unique tool for in vitro disease modeling enabling major advances in our understanding of developmental disease processes. We report the full characterization of complex genetic abnormalities in two infertile patients with either azoospermia or XX male syndrome and we identify genes of potential interest implicated in their infertility. Using the erythroblasts of both patients, we generated primed iPSCs and converted them into a naive-like pluripotent state. Naive-iPSCs were then differentiated into primordial germ-like cells (PGC-LCs). The expression of early PGC marker genes SOX17, CD-38, NANOS3, c-KIT, TFAP2C, and D2-40, confirmed progression towards the early germline stage. Our results demonstrate that iPSCs from two infertile patients with significant genetic abnormalities are capable of efficient production of PGCs. Such in vitro model of infertility will certainly help identifying causative factors leading to early germ cells development failure and provide a valuable tool to explore novel therapeutic strategies.

Figure 1

Short-read Whole genome sequencing results of patient 1 and conventional and molecular cytogenetic analysis for patient 2. (a) The CCR of patient 1 involved 7 breakpoints: one on the short arm of the derivative chromosome 7 7p21.3 region along with two deletions (chr7:11,801,000–11,791,000 and chr7:10,257,000–10,656,000), one on the short arm of the derivative chromosome 12 12p13.31 region (chr12:9,015,000–9,015,001) and the remaining 5 breakpoints on the long arm of the derivative chromosome 12 (chr12:41,698,000–41,698,001 proximal 12q12 breakpoint; chr12:101,314,000–101,314,001, chr12:101,346,000–101,346,001, chr12:101,406,000–101,406,001, chr12:101,441,000–101,441,001 distal 12q23.2 breakpoints). Genomic positions are described according to the hg38 genome assembly. (b) Circos plot showing the 3 events of the CCR of patient 1: the insertion and the pericentric and paracentric inversions. (c) G-banded standard karyotype designated as 46,XX (patient 2). (d) FISH analysis on buccal swab using centromeric probe for chromosome 18 (blue, 2 signals), chromosomes X (green, 2 signals) and chromosome Y (red, no signal) (patient 2). (e) FISH analysis on buccal swab using centromeric probe for chromosome X (blue, 2 signals), chromosome Y heterochromatic region (green, no signal) and SRY gene locus on chromosome Y (red, no signal) (patient 2).

Figure 2

Generation and characterization of hiPSCs. (a and b) Endogenous expression of pluripotency-related markers by patient 2-derived iPSCs: (a) RT-PCR analysis for detection of the pluripotency markers SOX2, OCT4, NANOG and REX-1. Full length gels are showed in Supplementary Fig. S5; (b) Immunofluorescence staining of three stem cell proteins (SSEA4, TRA-1–60 and OCT3/4) in patient iPS clones 15 and 20. (c) Germ cell layer components within teratomas obtained with patient 2-derived iPSCs. The differentiation, at passage 8 (cl.15) and 7 (cl.20), of iPSCs into ectoderm, endoderm and mesoderm was evaluated on whole sections stained with hematoxylin and eosin, for iPS clones 15 (left panel) and 20 (right panel). Hematoxylin and eosin stain, X20. Images of immunofluorescence staining were analyzed using MetaMorph (Molecular Devices, https://fr.moleculardevices.com) and ImageJ 1.52 (National Institutes of Health, https://imagej.nih.gov) Softwares. C cartilage, EB embryoid body, G gut epithelial tissue, hiPSC human induced pluripotent stem cells, N neural tissue.

Figure 3

Induction of naive iPSCs state, EB differentiation and gene expression analysis. Timeline of in vitro conversion of primed-hiPSCs into naive-hiPSCs and PGC-LCs differentiation (a). RT-qPCR analysis for detection of the pluripotency marker NANOG (b); early PGC marker genes SOX17 and NANOS3 (c). Relative expression levels are shown with normalization to RPLPO gene. Histograms represent mean from independent biological replicates represented by each point. Mann–Whitney tests were used for PCR analysis. Mann–Whitney tests were performed to compare expression values. *p < 0.05. BMP2/4 bone morphogenetic protein 2/4, EB embryoid body, EB D4 embryoid body day 4, EGF epidermal growth factor, GSK3 glycogen synthase kinase-3, hiPSCs human induced pluripotent stem cells, iPSCs induced pluripotent stem cells, JNK c-Jun N-terminal kinase, LIF leukemia inhibitory factor, MAPK mitogen-activated protein kinases, MEK mitogen-activated protein kinase kinase, SCF stem cell factor, TGF-β1 transforming growth factor β1.

Figure 4

Immunohistochemical (IHC) expression of TFAP2C and D2-40 on EBs sections. The full protocol was repeated three times for the iPSCs of patient 1 and two times for the iPSCs of patient 2. (a) IHC pictures showing PGC-LCs positive for TFAP2C and D2-40 in EB D4 from patient 1 cl.32-hiPSCs and patient 2 cl.15-hiPSCs. (b) Percentage rate of PGC-LCs cells population differentiated within EBs from patient 1 cl.32-hiPSCs and patient 2 cl.15-hiPSCs as estimated after quantification. (c) Immunofluorescence (IF) showing localization of DAPI (blue), D2–40 (green) and 5-hydroxymethylcytosine (5hmC, red) in PGC-LCs and somatic cells in EBs at day 4. In order to clearly show the localization of PGC-LCs, we used the specific germ cell membranous marker D2-40 in combination with 5hmC for IF assay. Images show that, 5hmC is detectable in PGC-LCs in higher levels than in in somatic cells.

Figure 5

RT-qPCR analysis of candidate genes on embryoid bodies. (a) SYCP3 expression level in patient 1 compared to 46,XX control cells. (b) AMH, NUP107 and STAT5B expression levels in patient 2 compared to 46,XX control. Histograms represent mean from independent biological replicates represented by each point. Gene expression was normalized to RPLPO. Mann–Whitney tests were used for PCR analysis. AMH anti-Müllerian hormone, NUP107 nucleoporin 107, STAT5B signal transducer and activator of transcription 5B, SYCP3 synaptonemal complex protein 3.