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Case Reports
. 2017 Jan 3:7:39760.
doi: 10.1038/srep39760.

Induced pluripotent stem cell generation from a man carrying a complex chromosomal rearrangement as a genetic model for infertility studies

Aurélie Mouka  1   2 , Vincent Izard  3 , Gérard Tachdjian  1   2 , Sophie Brisset  1   2 , Frank Yates  4 , Anne Mayeur  1 , Loïc Drévillon  1 , Rafika Jarray  4 , Philippe Leboulch  5 , Leila Maouche-Chrétien  5 , Lucie Tosca  1   2
Affiliations
Case Reports

Induced pluripotent stem cell generation from a man carrying a complex chromosomal rearrangement as a genetic model for infertility studies

Aurélie Mouka et al. Sci Rep. .

Abstract

Despite progress in human reproductive biology, the cause of male infertility often remains unknown, due to the lack of appropriate and convenient in vitro models of meiosis. Induced pluripotent stem cells (iPSCs) derived from the cells of infertile patients could provide a gold standard model for generating primordial germ cells and studying their development and the process of spermatogenesis. We report the characterization of a complex chromosomal rearrangement (CCR) in an azoospermic patient, and the successful generation of specific-iPSCs from PBMC-derived erythroblasts. The CCR was characterized by karyotype, fluorescence in situ hybridization and oligonucleotide-based array-comparative genomic hybridization. The CCR included five breakpoints and was caused by the inverted insertion of a chromosome 12 segment into the short arm of one chromosome 7 and a pericentric inversion of the structurally rearranged chromosome 12. Gene mapping of the breakpoints led to the identification of a candidate gene, SYCP3. Erythroblasts from the patient were reprogrammed with Sendai virus vectors to generate iPSCs. We assessed iPSC pluripotency by RT-PCR, immunofluorescence staining and teratoma induction. The generation of specific-iPSCs from patients with a CCR provides a valuable in vitro genetic model for studying the mechanisms by which chromosomal abnormalities alter meiosis and germ cell development.

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Figures

Figure 1
Figure 1. Conventional cytogenetic analysis.
(A) Chromosome 7 and 12 G-banded karyotype and ideogram representation, showing structural abnormalities on chromosomes 7 and 12 [der(7) and der(12), respectively]. (B) Whole-chromosome painting probes for chromosomes 7 (green) and 12 (red) demonstrated the insertion of part of chromosome 12 into the short arm of chromosome 7. (C) Insertion event: FISH with BAC probes specific for the 12p13.31 (RP4-751H1; red) and 12q13.13 (RP11-1136G11; green) regions. (D) Inversion event: FISH with BAC probes specific for the 12p13.1 (RP11-180M15; red) and 12q24.11 (RP11-457O10; green) regions. (E,F) The fifth breakpoint: FISH with BAC probes specific for the 12p13.31 (RP11-444J21; red) and 12q23.2 (RP11-321F8; green) regions.
Figure 2
Figure 2. Location of the complex chromosomal rearrangement breakpoints.
Ideograms for chromosome 7 (A) and chromosome 12 (B), showing the five breakpoints identified by successive FISH hybridizations (see Supplementary Table 1). BP5 contained the SYCP3 gene. BP: breakpoint.
Figure 3
Figure 3. Timeline and result of in vitro generation of human induced pluripotent stem cells on MEFs and Matrigel.
C-Myc: v-myc avian myelocytomatosis viral oncogene homolog; D: day; iPSCs: induced pluripotent stem cells; KLF4: Kruppel-like factor 4 (gut); MEF: mouse embryonic fibroblast; OCT4 (also called POU5F1): POU class 5 homeobox 1; SOX2: SRY-box 2. The figure was produced, in part, by using Servier Medical Art, (www.servier.com/Powerpoint-image-bank).
Figure 4
Figure 4. Endogenous expression of pluripotency-related markers by iPSCs.
(A) RT-PCR analysis for detection of the pluripotency markers SOX2, OCT4, NANOG and REX-1. All the patient iPSC clones tested expressed these genes. See full-length gels in Supplementary Figure 2. (B) Immunofluorescence staining of three stem cell proteins (SSEA4, TRA-1-60 and OCT3/4) in patient iPS clones 12 and 32. Both clones 12 and 32, at passages 5 and 4, respectively, expressed the three pluripotency markers used.
Figure 5
Figure 5. Germ cell layer components within teratomas.
The differentiation, at passage 6, of iPSCs into ectoderm, endoderm and mesoderm was evaluated on whole sections stained with hematoxylin and eosin, for iPS clones 12 (A,C,E) and 32 (B,D,F). Both clones displayed structures representing the three lineages: (A,B) endoderm. (C,D) ectoderm. (E,F) mesoderm. Hematoxylin and eosin stain, ×20. AT, adipose tissue; BV, blood vessel; C, cartilage; G, gut epithelial tissue; Ep, keratin-containing epidermal tissue; M, striated muscle; N, neural tissue.
Figure 6
Figure 6. Two hypothetical chromosomal mechanisms accounting for the complex chromosomal rearrangement in the patient.
(A) With the inversion event occurring first. (B) With the insertion event occurring first. BP: breakpoint.
See this image and copyright information in PMC

References

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