A systems approach toward human primordial germ cell differentiation

Differentiation of human primordial germ cells (hPGCs) from human pluripotent stem cells is the first step toward in vitro gametogenesis, which would enable mechanistic studies and treatment of infertility due to loss of gametes. The mechanisms of hPGC differentiation are poorly understood. As a result, current protocols produce heterogeneous mixtures of hPGC-like cells (hPGCLCs) and other cell types. Our long-term goal is to obtain a systems-level understanding of the signaling and gene regulatory network that control primordial germ cell differentiation in vitro and use this to improve directed differentiation of hPGCLCs for potential therapeutic purposes. The objective of this proposal is to quantitatively determine cell signaling requirements of hPGCLC differentiation with unprecedented spatial and temporal resolution. The central hypothesis is that heterogeneous differentiation and maturation of hPGCLCs is due to heterogeneous cell signaling activity. The rationale is that if we identify what distinguishes more mature hPGCLCs from less mature ones and other cell types that arise at the same time, we can understand defects in their differentiation in vivo and in vitro and create better hPGCLCs. Our hypothesis will be tested through two specific aims: 1) Determine the combinatorial signaling dynamics responsible for hPGCLC induction by single cell tracking of signaling and fate markers in live cells combined with highly multiplexed immunofluorescence. 2) Determine if there are distinct phases of induction and maintenance and elucidate the signaling logic and gene regulatory network that controls hPGCLC maintenance and maturation. Our approach is conceptually innovative by testing if a unique history of signaling determines hPGCLC fate, by accounting for the interplay between exogenous and endogenous signaling in vitro, and by delineating clearly distinct phases of differentiation that start with induction. More generally we are breaking new ground by taking a systems-level approach to human germ line biology that tests hypotheses rigorously and quantitatively by representing them as mathematical models. The proposed work is also experimentally innovative as we use substrate micropatterning to achieve reproducible hPGCLC differentiation and are the first lab to establish long-term high throughput tracking of differentiating pluripotent stem cells as well as the first to adapt multiplexed immunofluorescence to stem cell models of early development. The expected outcome of our work is better fundamental understanding of human germ line differentiation and improved methods to produce hPGCLCs for research of in vitro gametogenesis. Project Number: 1R01HD120309-01 | Fiscal Year: 2026 | NIH Institute/Center: Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) | Principal Investigator: Idse Heemskerk | Institution: UNIVERSITY OF MICHIGAN AT ANN ARBOR, ANN ARBOR, MI | Award Amount: $390,504 | Activity Code: R01 | Study Section: Cellular, Molecular and Integrative Reproduction Study Section[CMIR] View on NIH RePORTER: https://reporter.nih.gov/project-details/11272218