Statement of Benefit to California:
Year 1The original goal of this project was to generate oocytes (eggs) from human embryonic stem (hES) cells in cell culture dishes in the laboratory. Such oocytes could be of use as vehicles to reprogram the DNA from cells of patients with life-threatening or debilitating conditions, thereby allowing generation of new lines of hES cells that are immune matched to the patient. The paucity of donated human oocytes precludes research using such material, and production of human oocytes from hES cells in the laboratory would in theory provide a limitless source of material.
Since our last progress report another CIRM-funded group, Dr. Renee Reijo Pera’s lab at Stanford University, has published exciting results demonstrating successful production of primordial oocytes from mouse ES (mES) and human ES (hES) cells. Consequently, during the remaining period of the award we propose to use the Reijo Pera methods for production of female germ line cells in our lab using H9 and HUES-9 female hES cells. After accomplishing this, we will introduce human mtDNA containing mutations that cause either a severe or mild reduction in oxidative phosphorylation (energy) production into H9 and HUES-9 hES cells and investigate the impact of the different mtDNA mutations on the ability of hES cells to form cells with characteristics of PGCs, then primordial oocytes in vitro and in vivo. An important related goal of this research is to investigate whether development of oocytes from ES cells could be used as a method to remove deleterious mtDNA mutations from the hES cell population, thereby improving the utility and possibly safety of derived cell types for therapeutic purposes.
Year 2During this reporting period we have focused on our approved revised research plan. We continued our efforts to generate embryonic female germ cells from human embryonic stem (hES) cells in vitro using methods reported in the literature at the end of 2009. Our revised plan included the new goal of using in vitro developed female embryonic germ cells (oocytes) as a resource to investigate how mitochondrial genomic DNA containing deleterious mutations is segregated during female germ cell development. As well as providing novel information about the biology of germ cell development, this research may provide important information relevant to development of safe methods for therapeutic cloning. We began by using two different female hES cell lines (HUES-6 and H9) to investigate whether we could use the reported methods to develop female germ cells from hES in our lab. A third female hES cell line (HUES-9) we originally intended to use was found to have a high propensity to gain an extra chromosome (become aneuploid) and was therefore not used. Despite following the reported methods that demonstrated in vitro differentiation of hES cells into female germ cells, we were unable to reproduce the previously reported results in our own lab. The reasons for this are currently unclear but may involve subtle, but important, differences in the methodology or materials we used.