Approximately 1 in 10 couples of reproductive age in America are diagnosed with infertility. Infertility does not bias by race, gender or ethnicity. Underlying causes of human infertility are often unknown. However, abnormal formation, or irreversible damage to the lineage responsible for creating egg and sperm can cause infertility as an adult. We propose that one of the best models to understand mechanism of human egg and sperm differentiation (also known as germline differentiation) involve differentiating germline cells from pluripotent stem cells in vitro. Currently, the field of germline differentiation in vito is hindered by a lack of knowledge of human germline development in the embryo, low yield following in vitro differentiation, unknown variability in germline differentiation potential betwen lines, and failure to use the germline xenotransplantation model as a functional assay. To overcome these bottlenecks we propose to develop a comprehensive transcriptome and DNA methylome map of in vivo human germline cells during gestation using RNA- sequencing and whole genome bisulfite-sequencing. This will be used to transcriptionally and epigenetically stage germline cells acquired in vitro. Next, we propose to induce ectopic expression of PRDM14, NANOS3 and DAZL in a panel of nine well-characterized hESC lines by incorporating the genes into a safe harbor locus using genome-editing technology. We will measure germline identity using a next generation single-cell gene expression panel, and demethylation at loci that stably demethylate in early human germline development. Finally, we propose to use the germline xenotransplantation assay to transplant in vitro male germline cells into the testes of mice rendered infertile by chemotherapy. The endpoint of this assay will involve colony formation, proliferation and expression of mature germline markers. Xenotransplantation outcomes will be compared to control xenotransplantation of human spermatogonia and progenitor human germline cells from gestational stage testes. Results from this project will have a long lasting impact on the field of germline differentiation in vitro as it provides a measure of differentiation efficiency across multiple well characterized hESC lines, as well as incorporation of state-of-the art gene editing technology and functional assays to improve and confirm germline identity in vitro.

Public Health Relevance

This project seeks to determine whether progenitor egg and sperm (germ line) differentiation can be achieved from nine well characterized human embryonic stem cell lines, as well as testing germ cell quality in a xenotransplantation assay of male infertility. Results from this work will have a long lasting impact on the field because it wil determine whether our approach for germ line differentiation yields germ line cells that are transcriptionally, epigenetically and functionally equivalent to germ line found in the human embryo.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Ravindranath, Neelakanta
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University of California Los Angeles
Schools of Arts and Sciences
Los Angeles
United States
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Pastor, William A; Liu, Wanlu; Chen, Di et al. (2018) TFAP2C regulates transcription in human naive pluripotency by opening enhancers. Nat Cell Biol 20:553-564
Tao, Yu; Yen, Ming-Ren; Chitiashvili, Tsotne et al. (2018) TRIM28-Regulated Transposon Repression Is Required for Human Germline Competency and Not Primed or Naive Human Pluripotency. Stem Cell Reports 10:243-256
Gell, Joanna J; Zhao, Jasmine; Chen, Di et al. (2018) PRDM14 is expressed in germ cell tumors with constitutive overexpression altering human germline differentiation and proliferation. Stem Cell Res 27:46-56
Sahakyan, Anna; Kim, Rachel; Chronis, Constantinos et al. (2017) Human Naive Pluripotent Stem Cells Model X Chromosome Dampening and X Inactivation. Cell Stem Cell 20:87-101
O'Brien, Carmel M; Chy, Hun S; Zhou, Qi et al. (2017) New Monoclonal Antibodies to Defined Cell Surface Proteins on Human Pluripotent Stem Cells. Stem Cells 35:626-640
Chen, Di; Liu, Wanlu; Lukianchikov, Anastasia et al. (2017) Germline competency of human embryonic stem cells depends on eomesodermin. Biol Reprod 97:850-861
Clark, Amander T; Gkountela, Sofia; Chen, Di et al. (2017) Primate Primordial Germ Cells Acquire Transplantation Potential by Carnegie Stage 23. Stem Cell Reports 9:329-341
Chen, Di; Gell, Joanna J; Tao, Yu et al. (2017) Modeling human infertility with pluripotent stem cells. Stem Cell Res 21:187-192
Patel, Sanjeet; Bonora, Giancarlo; Sahakyan, Anna et al. (2017) Human Embryonic Stem Cells Do Not Change Their X Inactivation Status during Differentiation. Cell Rep 18:54-67
Pastor, William A; Chen, Di; Liu, Wanlu et al. (2016) Naive Human Pluripotent Cells Feature a Methylation Landscape Devoid of Blastocyst or Germline Memory. Cell Stem Cell 18:323-329

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