Blastocyst Development after Fertilization with in vitro Spermatids Derived from Non-Human Primate Embryonic Stem Cells

Abstract: Objective To demonstrate that functional spermatids can be derived in vitro from non-human primate pluripotent stem cells. Design Green Fluorescent Protein (GFP)-labeled, rhesus macaque non-human primate Embryonic Stem Cells (nhpESCs) were differentiated into advanced male germ cell lineages using a modified serum-free spermatogonial stem cell culture medium. In vitro-derived round spermatid-like cells derived from differentiated nhpESCs were assessed for their ability to fertilize rhesus oocytes by intracytoplasmic sperm(atid) injection (ICSI). Setting Multiple academic laboratory settings Patients N/A Interventions ICSI of in vitro-derived spermatids from nhpESCs into rhesus macaque oocytes. Main Outcome Measures Differentiation into spermatogenic cell lineages was measured through multiple assessments including RNA sequencing and immunocytochemistry for various spermatogenic markers. In vitro spermatids were assessed for their ability to fertilize oocytes by ICSI by assessing early fertilization events such as spermatid DNA decondensation and pronucleus formation/apposition. Pre-implantation embryo development from the 1-cell zygote stage to the blastocyst stage was also assessed. Results nhpESCs can be differentiated into advanced germ cell lineages, including haploid round spermatid-like cells. These round spermatid-like cells (rSLCs) undergo DNA decondensation and pronucleus formation/apposition when microinjected into rhesus macaque mature oocytes, which, after artificial activation and co-injection of TET3 protein, undergo embryonic divisions with ∼12% developing successfully into expanded blastocysts. Conclusion This work demonstrates that rSLCs, generated in vitro from primate pluripotent stem cells, mimic many of the capabilities of in vivo round spermatids and perform events essential for preimplantation development. This work represents, for the first time, that functional spermatid-like cells can be derived in vitro from primate pluripotent stem cells.