P-50: Elongating and Elongated Spermatids Manufactured In Vitro from Non-Human Primate Pluripotent Stem Cells

Background: We have recently shown that human embryonic (hESCs) and induced pluripotent stem cells (hiPSCs) can differentiate into advanced spermatogenic cells including round spermatids by in vitro culture (Easley et al., Direct differentiation of human pluripotent stem cells into haploid spermatogenic cells. Cell Reports 2, 440-446 2012) and also, in collaboration, that rhesus spermatogonial stem cells (SSCs) are reproductively viable after cryopreservation, thawing and transplantation into unrelated males (Hermann et al. Spermatogonial stem cell transplantation into rhesus testes regenerates spermatogenesis producing functional sperm. Notwithstanding the importance of investigating human male meiosis in vitro, serious bioethical limitations preclude the determination of the reproductive utility and/or constraints on these artificially manufactured gametes. While human stem cells differentiate in vitro into round spermatids, in humans and non-human primates, round spermatids after microinjection to mature oocytes are unable to initiate development and result in pregnancies; this is unlike the situation in mice. Materials and Methods: To address whether in vitro haploid sperm-like cells have reproductive potentials, we present here significant progress in generating elongated and elongating spermatids from rhesus. Here, we confirm and extend these findings showing a non-human primate, rhesus embryonic (nhpESCs), somatic cell nuclear transfer-derived embryonic (nhpNT-ESCs), and induced pluripotent stem cells (nhpiPSCs) can be differentiated into spermatocyte stem cell (SSC) - like cells that express PLZF, a marker for stem and progenitor spermatogonia, and VASA, a known germ cell marker expressed by several germ cell types. Results: Differentiating nhp pluripotent stem cells in SSC culture conditions also produces haploid spermatids. Most of these in vitro haploid cells resemble round spermatids as assessed by protamine 1, acrosin, Golgin3, and VAMP1 immunostaining as well as microtubule and centrosome marker analyses. However, round spermatids do not fertilize human or non-human primate oocytes. We have observed a small population of haploid spermatids derived in vitro that structurally resemble elongated spermatids, a spermatid population capable of fertilizing primate oocytes by intracytoplasmic sperm(atid) injection (ICSI). Conclusion: Importantly, we have now generated haploid elongated and elongating spermatids from primates; this is a vital threshold for the establishment of reproductively viable male gametes in vitro. We are currently assessing whether these in vitro “elongating” haploid spermatids are capable of fertilizing rhesus oocytes. If successful, this model will provide the first evidence that functional gametes can be manufactured in vitro from non-human primate pluripotent stem cells and may provide insight into potentially novel methods for diagnosing and even treating human male infertility.