Human oocyte development for genetic, pharmacological and reprogramming applications

The specific aims of our research grant # RC1-00137 entitled “Human Oocyte Development for Genetic, Pharmacological and Reprogramming Applications” are as follows: Aim 1) Assess and compare the potential of multiple nonfederal 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. Historically, it has not been possible to examine the differentiation of human germ cells (the cells that give rise to mature eggs and sperm); however, with the derivation of hESC lines, we sought to determine whether hESCs might provide a system to differentiate human germ cells. Our proposed research plan is based on our initial studies indicating that hESCs provide a useful system. However, it was not clear whether hESC lines differed from each other in terms of differentiation at both the quantitative and qualitative levels. In addition, although a number of labs have reported the differentiation of both mouse and human germ cells, in general only immature germ cells were produced. Finally, we note that a common problem in IVF (in vitro fertilization), is the retrieval of immature oocytes and an inability to mature these oocytes in the clinic. Against this backdrop, in the last funding cycle, we have succeeded in extending our analysis of hESC lines and differentiation of the germ cell lineage to eight independently-derived lines, we have demonstrated that we can modulate germ cell differentiation in vitro to increase numbers of germ cells formed via external and internal cell-based induction, we have succeeded in differentiating germ cells that enter and progress through meiosis (a critical step in the production of mature eggs and sperm), and we have developed the methods to mature human oocytes in vitro and derive hESC lines from single blastomeres. A subset of these findings has been submitted for publication (or is in press) and additional publications are in preparation. These results will shed light on human development and the cell-based decisions to form germ cells versus other cell types of the body (somatic cells) and will potentially contribute to alleviating a common health problem that afflicts 10-15% of couples in California (infertility), as well as forming a foundation for understanding reprogramming in early human development (a process required for normal development that is a basis for development of novel strategies to produce pluripotent stem cell lines).

Our aims for RC1-00137 entitled “Human Oocyte Development for Genetic, Pharmacological and Reprogramming Applications” are as follows: Aim 1) Assess and compare the potential of multiple nonfederal 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. In the last funding cycle, we have succeeded in extending our analysis of human embryonic stem cell lines and differentiation of the germ cell lineage (that gives rise ultimately to oocytes or eggs) to 11 lines (initially, we proposed 12 lines in total). We have demonstrated that we can control or regulate germ cell differentiation in vitro to increase numbers of germ cells via external and internal induction, and we have succeeded in differentiating germ cells that enter and progress through meiosis, in multiple lines. We have developed the ability to use transplantation to promote mouse oocyte (egg) differentiation (in parallel studies); these studies form the foundation for applying our methods to human oocyte formation. Finally, we have published the results of our studies aimed at development of methods to mature human oocytes in vitro in a dish and derive hESC lines from single blastomeres. The later have also included analysis of epigenetic modifications during human embryo development to potentially enhance the ability to derive optimal lines from single cells via SCNT (somatic cell nuclear transfer). As the field of human pluripotent stem cell research has advanced, it has become clear that there is a need to consider the potential importance of SCNT to complement other methods of derivation of isogenic stem cell lines, in addition to generation of induced pluripotent stem cell lines (iPSCs). In addition, we have the ability to learn much about basic and clinical properties of human germ cells through differentiation and maturation and we can apply methods to help men and women with reproductive health problems. Our publications are: 1) McElroy SL, Byrne JA, Chavez SL, Behr B, Hsueh AJ, Westphal LM, Reijo Pera RA. (2010) Parthenogenic blastocysts derived from cumulus-free in vitro matured human oocytes. PLoS One 5: e10979. This publication reports the first blastocyst derived from parthenogenic activation of immature oocytes (from the GV stage) to reach maturity (McElroy, Byrne et al. 2010). 2) Kee K, Angeles VT, Flores M, Nguyen HN, Reijo Pera RA (2009) Human DAZL, DAZ and BOULE genes modulate primordial germ cell and haploid gamete formation. Nature 462, 222-5. Note that the research reported in this publication was supported in part by CIRM (development of methods for human germ cell differentiation)(Kee, Angeles et al. 2009). In addition, we reported this publication in the last report as in press. It has now been published.

Our aims for RC1-00137 entitled “Human Oocyte Development for Genetic, Pharmacological and Reprogramming Applications” are as follows: Aim 1) Assess and compare the potential of multiple nonfederal 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. In the last funding cycle, we have succeeded in extending our analysis of hESC lines and finished the first aim to compare the potential of multiple lines. In the second aim, we have made significant progress towards differentiating mature human germ cells (oocytes) and understanding the fundamentals of germ cell development that will contribute to futher success. Finally, as the field of human pluripotent stem cell research has advanced, it has become clear that there is a need to consider the potential importance of SCNT and the use of additional factors that function in the oocyte to embryo transition to improve line generation. In addressing our aims, we have made tremendous progress towards understanding basic and clinical properties of human germ cells through differentiation and maturation. The information will inform stem cell biology and as well as human reproductive health. We have published three manuscripts on this project, have several others in various stages of preparation/submission and have generated one application for intellectual property (patent).

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.