First Description of Pluripotent Stem Cell-derived Functional Sperm

Review of “Complete Meiosis from Embryonic Stem Cell- Derived Germ Cells in Vitro” from Cell Stem Cell by Stuart P. Atkinson

The in vitro production of human gametes (eggs and sperm) has the potential to provide families with a unique chance to have children and scientists with a source of rare cells for study. One strategy to obtain gametes is to produce them from human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs), or better still, patient-specific (so parent-specific) induced pluripotent stem cells (iPSCs). So where are we with this technology?

Some studies have provided evidence of the production of germ-like cells from PSCs (both human and mouse) [1, 2]. However, neither study recapitulated all the key events of meiosis [3] or the production of haploid (one set of chromosomes) germ cells from a diploid (two sets of chromosomes) parental cell.

We now may have taken a step further forward with a report from the laboratory of Xiao-Yang Zhao, Jiahao Sha, and Qi Zhou (Chinese Academy of Sciences, Beijing, China) published in Cell Stem Cell. They demonstrate the first evidence of complete in vitro meiosis from mouse ESC-derived primordial germ cell-like cells (PGCLCs) and also the production of viable fertile offspring using the resulting spermatid-like cells (SLCs) [4].

The authors of the study generated mouse transgenic ESC lines harboring fluorescent reporters under the control of the promoters of genes expressed at key stages of germ cell development – Blimp1/Stella to identify cells resembling PGCs, Stra8 to identify early-stage spermatogonia and spermatocytes, and finally Prm1 to identify post-meiotic spermatids.

The authors applied the following step-wise protocol to produce the desired final product:

• Ground state ESCs differentiated towards epiblast-like cells (EpiLCs) using adherent culture and exposure to activin A and bFGF
• This mediated the downregulation of pluripotency and inner cell mass marker genes and the upregulation of epiblast marker genes (Fgf5, Wnt3, and Dnmt3b)
• EpiLCs then differentiated towards PGCLCs using floating culture in an N2/B27-based differentiation medium containing BMP4, BMP8a, EGF, SCF, and LIF
• Blimp1/Stella induction suggested the formation of PGCLCs
• Gene expression and histone modification analyses demonstrated that the PGCLCs resembled in vivo PGCs

But could the authors coax these PGC-like cells into functional sperm?

• Co-culture of PGCLCs with early postnatal testicular cells (to mimic the in vitro environment) and exposure to various morphogens (activin A, BMP-2/4/7 and retinoic acid) led to Stra8 expression, suggesting the initiation of meiosis 
• Removal of morphogens and replacement with sex hormones (FSH, BPE, and testosterone) allowed the formation of post-meiotic Prm1 -expressing SLCs
• Importantly, the authors observed the key hallmarks of meiosis – the erasure of genetic imprinting, chromosomal synapsis (homologous chromosomal pairing) and recombination, and single chromosomal DNA content of resultant cells confirmed the formation of male haploid SLCs

Excitingly, these SLCs also proved capable of producing offspring following intracytoplasmic sperm injection (ICSI) into donor eggs and subsequent embryo transfer into a recipient host. The pups born were healthy and fertile with a normal DNA methylation level, and so the authors suggest that this study is the first to conform to the “gold standards” proposed for in vitro-derived germ cells [3].

Now we need to propel this study into the human realm – can we produce haploid human spermatids from human ESCs, or even better, patient-specific human iPSCs, allowing us to treat male infertility? We all wait with baited breath.

References

1. Eguizabal C, Montserrat N, Vassena R, et al. Complete meiosis from human induced pluripotent stem cells. Stem Cells 2011;29:1186-1195.
2. Geijsen N, Horoschak M, Kim K, et al. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 2004;427:148-154.
3. Handel MA, Eppig JJ, and Schimenti JC Applying "gold standards" to in-vitro-derived germ cells. Cell 2014;157:1257-1261.
4. Zhou Q, Wang M, Yuan Y, et al. Complete Meiosis from Embryonic Stem Cell-Derived Germ Cells In Vitro. Cell Stem Cell 2016;18:330-340.