Browsing by Author "Orwig, Kyle E."

Now showing 1 - 2 of 2

Over Expression of NANOS3 and DAZL in Human Embryonic Stem Cells
(2016-10) Panula, Sarita; Reda, Ahmed; Stukenborg, Jan-Bernd; Ramathal, Cyril; Sukhwani, Meena; Albalushi, Halima; Edsgard, Daniel; Nakamura, Michiko; Soder, Olle; Orwig, Kyle E.; Yamanaka, Shinya; Reijo Pera, Renee A.
The mechanisms underlying human germ cell development are largely unknown, partly due to the scarcity of primordial germ cells and the inaccessibility of the human germline to genetic analysis. Human embryonic stem cells can differentiate to germ cells in vitro and can be genetically modified to study the genetic requirements for germ cell development. Here, we studied NANOS3 and DAZL, which have critical roles in germ cell development in several species, via their over expression in human embryonic stem cells using global transcriptional analysis, in vitro germ cell differentiation, and in vivo germ cell formation assay by xenotransplantation. We found that NANOS3 over expression prolonged pluripotency and delayed differentiation. In addition, we observed a possible connection of NANOS3 with inhibition of apoptosis. For DAZL, our results suggest a post-transcriptional regulation mechanism in hES cells. In addition, we found that DAZL suppressed the translation of OCT4, and affected the transcription of several genes associated with germ cells, cell cycle arrest, and cell migration. Furthermore, DAZL over expressed cells formed spermatogonia-like colonies in a rare instance upon xenotransplantation. These data can be used to further elucidate the role of NANOS3 and DAZL in germ cell development both in vitro and in vivo.
A PAX5-OCT4-PRDM1 developmental switch specifies human primordial germ cells
(2018-04) Fang, Fang; Angulo, Benjamin; Xia, Ninuo; Sukhwani, Meena; Wang, Zhengyuan; Carey, Charles C.; Mazurie, Aurélien J.; Cui, Jun; Wilkinson, Royce A.; Wiedenheft, Blake A.; Irie, Naoko; Surani, M. Azim; Orwig, Kyle E.; Reijo Pera, Renee A.
Dysregulation of genetic pathways during human germ cell development leads to infertility. Here, we analysed bona fide human primordial germ cells (hPGCs) to probe the developmental genetics of human germ cell specification and differentiation. We examined the distribution of OCT4 occupancy in hPGCs relative to human embryonic stem cells (hESCs). We demonstrated that development, from pluripotent stem cells to germ cells, is driven by switching partners with OCT4 from SOX2 to PAX5 and PRDM1. Gain- and loss-of-function studies revealed that PAX5 encodes a critical regulator of hPGC development. Moreover, an epistasis analysis indicated that PAX5 acts upstream of OCT4 and PRDM1. The PAX5-OCT4-PRDM1 proteins form a core transcriptional network that activates germline and represses somatic programmes during human germ cell differentiation. These findings illustrate the power of combined genome editing, cell differentiation and engraftment for probing human developmental genetics that have historically been difficult to study.