UCLA

Infertility is caused by a multitude of genetic and environmental factors, and many who seek treatment are unresponsive to in vitro fertilization. Treatments such as these are costly, as well as physically and emotionally demanding, so it is critical to better understand the individual underlying causes of infertility to improve patient care. Germ cells give rise to the next generation and are responsible for passing along genetic and epigenetic information, and abnormalities in germ cell development can result in infertility. In addition, mutations in certain genes related to germ cell formation can contribute to their improper development, resulting in germ cell tumor formation or other developmental disorders in their future offspring. In these studies, we investigated the earliest cell type in the human germline, known as human Primordial Germ Cells (hPGCs). We developed models and techniques to represent hPGC formation and identified genes of interest to investigate their potential role in healthy hPGC identify and development. Two genes, KLF4 and TFCP2L1, are upregulated in hPGCs and are known to have a role in naive pluripotency. Given this, and other shared characteristics between the hPGCs and naive pluripotency, we hypothesized that KLF4 and TFCP2L1 have a role in hPGC development. We first used CRISPR/Cas9 gene editing technology at the human embryonic stem cell (hESC) stage of our PGC-like cell (hPGCLC) aggregate model, to demonstrate the requirement of certain genes including EOMES in PGCLC induction. Then, we developed an extended culture system to study hPGCLCs in vitro. In extended culture, hPGCLCs begin the process of reprogramming to represent the later hPGC stage, where we can study characteristics such as cell cycle and survival. We knocked out KLF4 and TFCP2L1 separately in hESC lines, and discovered that neither gene is required for PGCLC induction, but each has an anticipated role in re-acquiring naive pluripotency in the hESC state. Despite these functional null mutations, neither gene is required for progression of hPGCLC development in vitro including in progression through S phase as measured by Edu, or for PGCLC colony survival. Given this, we conclude that the naive pluripotent states observed in the pre-implantation and hPGC stages are uniquely controlled, and that these transcription factors may serve alternate roles in hPGC development. Possible roles include protection against germ cell tumors, or facilitation of later and advanced stage hPGC development. Further work will address these possibilities, using animal and stem cell models of gonadal development to study later stage progression.