Robust In Vitro Induction of Human Germ Cell Fate from Pluripotent Stem Cells

Abstract

Mechanisms underlying human germ cell development are unclear, partly due to difficulties in studying human embryos and lack of suitable experimental systems. Here, we show that human induced pluripotent stem cells (hiPSCs) differentiate into incipient mesoderm-like cells (iMeLCs), which robustly generate human primordial germ cell-like cells (hPGCLCs) that can be purified using the surface markers EpCAM and INTEGRINα6. The transcriptomes of hPGCLCs and primordial germ cells (PGCs) isolated from non-human primates are similar, and although specification of hPGCLCs and mouse PGCs rely on similar signaling pathways, hPGCLC specification transcriptionally activates germline fate without transiently inducing eminent somatic programs. This includes genes important for naive pluripotency and repression of key epigenetic modifiers, concomitant with epigenetic reprogramming. Accordingly, BLIMP1, which represses somatic programs in mice, activates and stabilizes a germline transcriptional circuit and represses a default neuronal differentiation program. Together, these findings provide a foundation for understanding and reconstituting human germ cell development in vitro.

Functional Human Oocytes Generated by Transfer of Polar Body Genomes

“…However, the complexity of establishing an in vitro system for producing functional haploid human oocytes currently limits this application (Irie et al, 2015;Sasaki et al, 2015). Here, we investigate an alternative approach, namely de novo reconstruction of haploid human oocytes by recycling of PB1 genomes via nuclear transfer into donor cytoplasm (Gurdon, 2015).…”

Mechanism and Reconstitution In Vitro of Germ Cell Development in Mammals

“…The hPGCLCs induced by Sasaki et al (2015) also showed a transcriptome similar to those of human gonadal PGCs (except late PGC genes), and the TNAP/NANOS3-positive cells reported by Irie et al (2015). The cells defined by Sugawa et al (2015), on the other hand, exhibited remarkably different gene expression profiles from those described by Sasaki et al (2015) and Irie et al (2015), presumably because of the differences in the induction and/or sorting strategies.…”

“…In the study by Irie et al, SOX17 was shown to be critically required for induction of human PGC-like cells, and a "distinctive" pluripotency (suggested to correspond to the naïve pluripotency in mice) (Gafni et al 2013) was shown to play a key role in the high competence of hESCs for the germ cell fate . Sasaki et al (2015) demonstrated the robust induction of human PGC-like cells from human iPSCs (hiPSCs) in a primed pluripotent state maintained under a defined, feeder-free condition. Sasaki et al exploited hiPSCs bearing fluorescent reporters for BLIMP1 (BLIMP1-2A-tdTomato: BT) and TFAP2C (TFAP2C-2A-EGFP: AG) and induced hiPSCs first into a state similar to the incipient mesoderm (incipient mesoderm-like cells: iMeLCs) by stimulation with activin A and a WNT-signaling agonist (CHIR99021) for 2 d. iMeLCs showed expression of a number of early mesodermal markers such as EOMES, T, and MIXL1 at moderate levels and retained a high level of the key pluripotency factors POU5F1, SOX2, and NANOG.…”

“…Such knowledge would have been difficult to obtain using the in vivo materials, and thus the in vitro system will continue to be applied to answer many remaining questions in germ cell biology. More recently, based on this mouse system, we and others have achieved the induction of human PGCLCs from human pluripotent stem cells (hPSCs) Sasaki et al 2015), which creates an unprecedented opportunity for understanding the mechanism for human germ cell development. In this manuscript, we summarize recent advances in elucidation of the mechanisms underlying germ cell development in both mice and humans, as well as in their reconstitution in vitro.…”

Germline development in amniotes: A paradigm shift in primordial germ cell specification

“…As novel implantation models 44 and ‘gastrulation’ models using pluripotent stem cells are being developed 45, 46, these may prove to be useful assays to understand the molecular mechanisms that lead to PGC formation in humans. Moreover, directed PGC‐differentiation protocols applied to mouse and human pluripotent stem cells are starting to reveal species‐specific signals regulating specification of PGC‐like cells 47, 48, 49, even though in vivo data might be lacking.…”