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Complete Meiosis from Human Induced Pluripotent Stem Cells

From the August Edition of Stem Cells
By Stuart P. Atkinson

Multiple studies have shown that mouse and human embryonic stem cells (ESCs) can differentiate in vitro into primordial germ cells (PGCs) and oocyte- or sperm-like cells (Marques-Mari et al). Therapeutic use of these cells would require patient specificity, so generation of such cells from individualised induced pluripotent stem cells (iPSC) would be required; a feat which has yet to be reported. However, in a study published in the August Edition of Stem Cells, Eguizabal et al from the laboratory of Juan Carlos Izpisúa Belmonte at the Center for Regenerative Medicine in Barcelona, Spain the complete differentiation of human iPSCs to post-meiotic cells has now been demonstrated, perhaps leading the way towards the ultimate goal of patient specific gamete formation.

Cord Blood-iPSCs and keratinocyte-iPSCs (male and female) were generated and characterized following previously reported protocols (Giogetti et al and Aasen et al) and all iPSC lines were negative for germ cell marker VASA (DDX4) at the RNA and protein level. Following an in vitro protocol aimed at recapitulating gamete development, hESCs (HS306 (female) and ES[6] (male)) and iPSCs were first allowed to differentiate for 3 weeks as a monolayer in absence of any growth cytokines followed by 3 weeks culture in the presence of retinoic acid (RA) (6 weeks total), followed by cell sorting (CD9+CD49f++CD90-SSEA4-) (spermatocyte/spermatogonia markers). RA signaling has been shown to stimulate both PGC division and entrance into meiosis (Trautmann et al and Pellegrini et al) and after RA treatment, a VASA+/SSEA1- population of cells was identified indicating possible progression toward pre-meiotic cells. Interestingly, there was evidence that this method of differentiation led to the in vitro reconstitution of a testicular niche as VASA+ cells were found surrounded by VIM+ (a marker of Sertoli cells), NES+ and HSD3B2+ (Leydig cell markers) cells. Further the expression of VASA was observed in all cell lines tested indicating the repeatability of the current differentiation protocol among cell lines with cell staining revealing that the CD9+CD49f++CD90-SSEA4- fraction comprised about 45% of all cells and contained a small VASA+ population (2%). In comparison, human testis gave a CD9+CD49f++CD90- population of about 29%, which comprised germ cells.

The sorted population from male ESCs and iPSCs were then treated with human LIF (hLIF), Forskolin, bFGF, and R115866, a CYP26 inhibitor for a further 4 weeks (10 weeks total) in an attempt to further advance differentiation toward meiosis. Cells displayed germline cells traits (round shape and a high nucleus to cytoplasm ratio) and also expressed the male germline marker ACR and did not express female germ markers (ZP1, ZP3, and FIG1a (IL4I1)). A fraction of cells expressed meiosis related markers (SCP3 and gH2AX), the pre-meiotic gene, STRA8 and meiotic prophase I were detected in chromosomal spreads suggesting meiotic competence, with the 9 week point identified as the point at which cells enter meiosis. However, although both hESC and hiPSC were shown to be able to initiate meiosis only hiPSCs were able to complete meiosis and generate haploid cells. Next the ability of the differentiation protocol to epigenetically reprogram the differentiating cells was studied by analysing six imprinted genes; 2 maternally expressed (PHLDA2 and CDKN1C), and four paternally expressed (MEST, IGF2, NNAT and SNRPN). The expression level of all genes after 10 weeks of differentiation was similar to that of mature spermatozoa, as was the expression of TERT and XIST. Finally these cells were tested for the presence of haploid cells by FACS and FISH using centromeric probes on chromosomes 18, X, and Y, and demonstrated the putative presence of haploid cells indicative of meiosis completion by 10 weeks (0.4% and 2.3% of haploid cells per sample) which were deemed masculine by the detection of ACR.

Overall this study demonstrates the competence of this differentiation protocol to produce haploid cells by mimicking in vivo gamete development, and importantly shows the establishment of appropriate imprinting, important for fetal and placental development. This work brings us closer to the production of personalized human gametes in vitro. However, it also raises an interesting point; the apparent bias of iPSCs to complete meiosis and generate haploid cells. Current thinking suggests that iPSCs have an epigenetic memory of their origin which inhibits development towards a fate different from their somatic cell of origin (Ohi et al, Kim et al and Polo et al),

although this may be erased by long term passaging in vitro (Polo et al). However in this case we observe that iPSCs from different origins (cord blood and keratinocytes) can develop in a similar way using this differentiation protocol, and more effectively than hESC which, one could argue, should be more competent for haploid cell development. Perhaps genomic/epigenomic/ proteomic analysis of ESC and iPSC throughout the differentiation time period could allow us to decipher important differences between the two and point towards novel genes and pathways involved in PGC and haploid cell development.



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