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A cKIT for Studying Primordial Germ Cells

“The ontogeny of cKIT+ human primordial germ cells proves to be a resource for human germ line reprogramming, imprint erasure and in vitro differentiation”

Research into embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) has allowed a greater understanding of the development and differentiation of specific cellular lineages and the molecular mechanisms which control them. However, our lack of understanding of human foetal development translates to a lack of details to validate, guide and quality control differentiation in vitro. Germ cell development from pluripotent sources could aid research and therapeutics in human fertility (Hayashi et al, Ohinata et al and Reijo et al). Development is understood to begin in primordial germ cells expressing the tyrosine protein kinase receptor cKIT (see paper for extended references), but how these cells progress is relatively less understood, although widespread changes to epigenetic modifications are key to this process (Hajkova et al 2008, Hajkova et al 2010, Popp et al and Seki et al). Now, in a study from researchers from the laboratory of Amander T. Clark from the University of California, Los Angeles, USA, cKIT-positive PGCs have been tracked from 6 to 20 developmental weeks through the analysis of human embryonic and fetal samples allowing the identification of the temporal nature of alterations in multiple epigenetic modifications and, additionally, they show that established hESC lines are not equivalent to human PGCs (Gkountela et al).

cKIT and VASA were evaluated using immunofluorescence microscopy, finding that all VASA+ cells had cKIT on their surface in the testes (7 to 11 weeks) and ovaries (7 to 9.5 weeks), with expression becoming uncoupled at 12.5 weeks and 11 weeks respectively, with this stage signified as being the common PGC progenitor stage. Analysis of SSEA1 demonstrated its unsuitability for use as a PGC marker as it was expressed on cells positive and negative for cKIT and VASA. OCT4A and TRA-1-81 expression localised to cKIT cells from 7 to 10.5 weeks (testes) and 6-8.5 weeks (ovaries). After this stage, VASA is repressed in the ovaries and while cKIT positive PGCs express OCT4A, some cKIT positive cells lack OCT4A expression. This establishes the existence of an early PGC progenitor which expresses cKIT, OCT4A and VASA and, after 11 weeks in testes and 9.5 weeks in ovaries, two cell populations arise; cKIT+OCT4A+ cells and VASA+ cells.

Individual cKIT+ cells were then sorted from testes and ovaries from 8 to 20 developmental weeks of age, and germ line identity was inferred from PCR analysis in the fraction of cells expressing high levels of cKIT as opposed to those expressing low levels. cKIT+ cell number decreased in testes (2.83% to 0.9% cKIT+ cells per testis) and increased in ovaries (2.45% to 4.75% cKIT+ cells per ovary) from 8–11 weeks to 9.6–16.5 weeks. At the common PGC progenitor stage, 14/16 cKIT+ cells in the testis and 13/20 cKIT+ cells in the ovary expressed 5 PGC signature genes (OCT4, BLIMP1, DAZL, VASA and NANOS3) and, additionally, SYCP3, used to indicate meiotic potential, was expressed in every cell at 14 and 16.5 weeks.

Epigenetic analysis studied DNA methylation, histone methylation and histone replacement of these cells. 5-methyl cytosine (5mC) was below detection levels throughout all stages of PGC development by immunofluorescence microscopy, however bisulphite sequencing at paternally (H19 and MEG3) and maternally (PEG3 and KCNQ1) methylated differentially methylated regions (DMRs) allowed a more detailed analysis. At all ages in male cKIT+ PGCs, CpG methylation was found at H19 and MEG3 DMRs, but not PEG3 and KCNQ1 DMRs which showed a drastic decrease between 16 and 17 weeks, while in female cKIT+, PGCs demonstrated a significant reduction of CpG methylation by 16.5 weeks at all loci. DNA methylation was further analysed through immunofluorescence microscopy for 5-hydroxy-mC (5hmC). This modification exhibited punctate nuclear staining in the common PGC progenitor stage and was lost in male OCT4A+ PGCs at 13.5 to 16 weeks then re-established at the beginning of week 17, being heterogeneous at 11–19 weeks in female OCT4A+ PGCs. Tri-methylation of lysine 27 histone H3 (H3K27me3), a repressive histone mark was enriched in common male PGC progenitors from 7 to 10.5 weeks, whereafter it is below the detection level in OCT4A+ and VASA+ PGCs, until week 17 when H3K27me3 appears heterogeneously. In female common PGC progenitors at 6–8.5 weeks, H3K27me3 is absent in 50–60% of cells, after which it is lost completely, similar to levels of H2A.Z, except that H2A.Z reappears in the nucleus of a few VASA+ cells in both sexes at week 17.

RNA-seq was next used to analyse male cKIT+ PGCs sorted at 16–16.5 weeks (initiating imprint erasure), female cKIT+ PGCs at 16–16.5 weeks (some imprinted loci show near complete demethylation) and H1 ESCs. PGCs were enriched for RNAs associated with gene ontology terms including negative transcription regulation, sex differentiation, and in females, meiosis and germ plasm, whereas hESCs in comparison were enriched in GO terms related to macromolecule biosynthetic processing, RNA processing/splicing and mitosis. Comparing male and female PGCs revealed 433 differentially expressed genes, with GO terms such as meiosis, oocyte development and DNA repair, and, in females, included enrichment in DAZL, VASA, ZP3 and STRA8, and in males, NANOS2 and NANOS3. Analysis of genes which have the potential to alter DNA methylation (ten-eleven translocation (TET) genes and DNA methyltransferases (DNMTs)), found overexpression of TET2 and repression of TET1 in PGCs compared to hESCs, while DNMT3A and DNMT3B were reduced in comparison to hESCs.

Finally, in vitro differentiation of PGCs from hESCs was attempted using H1 (XY) and UCLA1 (XX) hESCs (Diaz Perez et al and Thomson). hESCs were sorted by cKIT+ and TRA-1-81 expression (TRA 1-81 expression is correlated to high OCT4 correlation in human gonad) and separately by TRA-1-60. Sorted cells expressed PGC-specific genes as well as OCT4; however BLIMP1, a major PGC determinant (Bao et al), was rarely expressed and further analysis found little evidence of co-expression of any of the PGC determinants. For this reason, hESCs were then differentiated by a number of differing methods followed by sorting on TRA-1-81+/cKIT+ cells. Cells differentiated as embryoid bodies demonstrated an increase in BLIMP1 expression at day 9, with enrichment for OCT4 or OCT4 and NANOS3 with no co-expression of DAZL or VASA to levels higher than observed in hESCs. The authors suggest that these cells correspond to human PGCs before gonadal colonization and loss of 5mC.

Thus, the authors have delineated PGC development, and propose a “roadmap” for in vitro germ cell development from hESCs. 16 days of differentiation using their method creates a cKIT+/TRA-1-81+/OCT4A+ PGC population equivalent to a pre-gonadal PG, which then loses DNA methylation and gains H3K27me3, followed by DAZL and VASA expression, giving rise to the cKIT+/OCT4A+/VASA+ common gonadal PGC progenitors. Erasure of cytosine methylation from DMRs of imprinted genes and the loss of H3K27me3 and H2A.Z then occurs, followed by the uncoupling of cKIT expression from VASA, leading to separate populations.



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Study originally appeared in Nature Cell Biology.

Stem Cell Correspondent Stuart P. Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.