Year 5 NCE

Our aims for RC1-00137 entitled “Human Oocyte Development for Genetic, Pharmacological and Reprogramming Applications” were as follows: Aim 1) Assess and compare the potential of multiple nonfederal human embryonic stem cell (hESC) lines to contribute to the germ cell lineage. Aim 2) Differentiate hESCs to oocytes. Aim 3) Assay the ability of differentiated germ cells to reprogram a somatic nucleus. We have now completed the project; in previous reports, we described our progress in completing Aim 1 with the analysis of more than 12 lines; we subsequently focused on Aims 2 and 3 with modification of these aims in light of recent developments in reprogramming of somatic cells. We have demonstrated over the period of funding that diverse human embryonic stem cell lines and induced pluripotent stem cell lines contribute differentially to germ cell and somatic differentiation. Furthermore, we have found that we can optimize the differentiation via external growth factors and exogenous overexpression of key translational factors. Further improvements in germ cell differentiation are also observed via transplantation of putative germ cells in aggregates with somatic cells as demonstrated in the mouse model. Finally, we have profiled gene expression during the mouse and human oocyte to embryo transition in order to optimize reprogramming of somatic cells. We observe that the patterns of gene expression are distinct in human versus mouse embryos; patterns of gene expression are also distinct between individual cells/blastomeres of the embryo. These studies are being further used to inform a non-integrative approach to germ cell production and differentiation. In addition, we can now explore specifics of human meiosis, a process that is remarkably susceptible to errors that lead to different infertility related diseases with the systems we generated. Results have led directly to the generation of funds from other sources in order to continue to pursue the research goals with the necessary personnel and material support.