Year 2

Our research is demonstrating that normal mitochondrial energy production is absolutely essential for the successful growth and differentiation of embryonic stem (ES) cells. Moreover, dysfunctional mitochondrial may also increase the potential to premature aging and cancerous growth of ES cells derived tissue cells. However, the biology of mitochondrial energy production in ES cells has been virtually ignored in ES cell studies. This oversight could have major negative consequences for the use of human ES cell-derived tissue therapeutics.

Development involves the utilization of energy to build new cell and tissue structures. The genetic information to make structures is encoded by the nuclear DNA (nDNA) genes, but the information to generate energy by the mitochondria, the cellular power plants, is encoded by the maternally-inherited mitochondrial DNA (mtDNA) plus genes in the nDNA. The mtDNA is located in the cytoplasm within the mitochondia. Each cells contains hundreds of mitochondrial and thousands of mtDNAs. Therefore, to fully understand the biology and developmental potential of ES cells, we must understand the genetics and energetics of the ir mitochondria.

To clarify the role of mitochondrial genes in ES cell biology, we have published a review summarizing all studies in which mouse ES (mES) cells which harbored deleterious nDNA or mtDNA mitochondrial gene mutations were used to generate mice. This revealed that mitochondrial defects had multiple deleterious consequences for both development and adult tissue function. This conclusion was confirmed by our creation of mice that harbored two mtDNAs, one with a severely deleterious mtDNA ND6 gene mutation linked to a milder COI mutation and the second mtDNA with only the COI mutation. Analysis of these mice revealed that the proto-oocytes of the female germline that harbored the severe mtDNA ND6 mutation were selectively and directionally eliminated from the female germline. Mice retaining the milded COI mutation developed a severe myopathy and cardiomyopathy. We also prepared and analyzed a mouse harboring two different but normal mtDNAs. Within these mice, one or the other mtDNA was selectively eliminated as he mice aged. Furthermore, animals with both mtDNAs showed a disturbance in circadian rhythms. Therefore, mtDNA variation is of paramount importance for normal development and even mild mtDNA mutations can have severe negative consequences in adults and their tissues.

To extend these studies to human ES cells (hES), we have attempted to transfer hES cell mtDNAs into human somatic cells. These cell lines are being characterized. We have also attempted to introduce mutant mtDNAs into hES cells to evaluate the effects on their developmental potential. This requires elimination of the hES cell mtDNAs, for which we are evaluating different procedures. The mtDNAs that we hope to transfer to hES cells are chloramphenicol-resistance (CAPR) and the pathogenic tRNALeu(UUR) MELAS mutation. In addition, we have fused human cells harboring the CAPR to hES cells. We will continue the preparation and characterization of cell lines in which hES cell mtDNAs are transferred to somatic cells and vis-versa.

To investigate the role of mitochondrial physiology on ES development, we prepared an extensive literature review on the mitochondrial regulation of the cellular biochemical pathways. We have developed a micro-chamber for assessing hES cell mitochondrial function.

To assess whether mtDNA mutations would increase the tumorigenicity of ES cells, we have characterized the biochemical defect associated with a prostate cancer COI mtDNA mutation. We are also evaluating the cancer potential of our mice harboring the mtDNA COI mutation.

To develop procedures for manipulating ES cell development by altering mitochondrial function, we have prepared a review on the role of mitochondrial physiology in regulating nDNA gene expression through modification of histones using mitochondrial high energy intermediates. To evaluate the effect of mitochondrial defects of differentiation, we have prepared a series of mouse muscle stem cell lines harboring related nDNA mitochondrial gene mutations and found marked changes in their developmental potential and physiology. We are also evaluating the physiological consequences of a human mtDNA variant found in Asian Americans in preparation for introducing it into hES cells. All of these studies have confirmed that energy biology has a profound effect of the developmental potential of ES cells.