Carol M. Warner (1946-)
Erin J. Cram, Max Diem, Ann A. Kiessling, Badrinath Roysam
Date of Award
Doctor of Philosophy
Department or Academic Unit
College of Arts and Sciences. Department of Biology.
biology, cell, Apoptosis, Mitochondria, Pluripotent stem cells
Mitochondria - Formation, Stem cells - Research
Embryonic stem (ES) cells are pluripotent and therefore they have the capacity to form any tissue type in the body. ES cells are also capable of unlimited self-renewal. Mouse ES cells are isolated and grown from blastocyst stage preimplantation embryos on a Primary Mouse Embryonic Fibroblast (PMEF) feeder layer of cells in the presence of Leukemia Inhibitory Factor (LIF). Pluripotent stem cells may also be derived from adult cells by using retroviral vectors to introduce the appropriate pluripotency genes. The resulting cells are termed induced pluripotent stem (iPS) cells, and they are thought to be equivalent to their ES cell counterparts. The potential for stem cell therapy using pluripotent stem cells relies on the ability to selectively direct the growth of particular types of cells. Pluripotent stem cells provide a unique model system for studying early development and tissue differentiation, creating new disease models, and identifying novel drug targets for therapeutic application. Mitochondria are responsible for generating the energy for the cell, and they are also master regulators of apoptosis (programmed cell death). They have a mitochondrial membrane potential associated with them which reflects their level of activity. Active mitochondria localize to regions which require larger amounts of energy. Loss of mitochondrial membrane potential in cells is indicative of apoptosis. The published literature pertaining to mitochondria and apoptosis in pluripotent and differentiating stem cells is scarce and often conflicting. The overall goal of this research was to study the role of mitochondrial activity and apoptosis in pluripotent stem cell growth and differentiation. To accomplish this goal we defined four specific aims: 1) create and characterize an ES cell line from mtGFP-tg mice that have their mitochondria endogenously labeled with GFP, and then use the mtGFP-tg ES cell line to evaluate mitochondrial localization; 2) compare cell division, pluripotency markers, and differentiation markers in undifferentiated and differentiating mouse pluripotent stem cells by comparing the mtGFP-tg ES cell line, a conventional C57BL/6 ES cell line, and an iPS cell line; 3) analyze mitochondrial activity and localization in undifferentiated and differentiating mouse pluripotent stem cells by comparing the mtGFP-tg ES cell line, a conventional C57BL/6 ES cell line, and an iPS cell line; 4) analyze early, intermediate, and late stage apoptosis markers in undifferentiated and differentiating mouse pluripotent stem cells by comparing a conventional C57BL/6 ES cell line and an iPS cell line. We successfully created an ES cell line from mtGFP-tg mice. The mtGFP-tg ES cells had a normal karyotype, and they demonstrated characteristics of pluripotency and differentiation capabilities comparable to conventional ES cells. Their mitochondrial GFP fluorescence was extremely bright and stable, allowing us to image, for the first time, mitochondria in undifferentiated and differentiating cells without the use of externally applied mitochondrial stains. Our imaging data represent the first 3D sectioning through an EB, made possible by using endogenous mitochondrial fluorescence. Using our mtGFP-tg ES cell line in conjunction with a C57BL/6 ES and an iPS cell line, we confirmed that all three pluripotent stem cell lines had high levels of pluripotency markers in their undifferentiated state. Upon differentiation, the pluripotency markers decreased whereas markers for the three developing germ layers increased throughout differentiation. These data confirm the known characteristics of robust pluripotent stem cells. We analyzed mitochondrial activity in all three pluripotent stem cell lines by using JC-1 and TMRE staining. We found that pluripotent stem cells had high levels of mitochondrial activity in their undifferentiated state. Mitochondrial activity was confined to the perimeters of undifferentiated pluripotent stem cell colonies, an observation which has not been published previously. Upon differentiation, mitochondrial activity significantly decreased. Early, intermediate, and late apoptosis were assessed in C57BL/6 ES and iPS cells. The two pluripotent stem cell lines showed low levels of apoptosis in their undifferentiated state. Upon differentiation, apoptosis significantly increased. These data represent the first full analysis of mitochondrial activity and apoptosis throughout the differentiation process. Taken together, these results support a role for mitochondrial activity and apoptosis in pluripotent stem cell growth and differentiation. In particular, high mitochondrial activity is associated with pluripotency whereas apoptosis is associated with loss of pluripotency. To the best of our knowledge, these data provide the first analyses of mitochondrial activity and apoptosis in iPS cells. iPS cells were equivalent to conventional C57BL/6 ES cells in terms of pluripotency, mitochondrial activity, and apoptosis in their undifferentiated state. However, upon differentiation or long-term culture we found several significant differences in pluripotency, mitochondrial activity, and apoptosis thus bringing into question the complete equivalency of iPS cells to conventional ES cells. All together, we conclude from our analyses that there are significant differences in pluripotency, mitochondrial activity, and apoptosis during differentiation of pluripotent stem cells and that there are several significant differences among the mtGFP-tg, C57BL/6, and iPS pluripotent stem cell lines. These data increase our knowledge of the pluripotency and differentiation capabilities of pluripotent stem cells, which may impact the development of stem cell therapy for the treatment of human diseases.
Judith A. Newmark
Newmark, Judith A., "Mitochondrial and apoptotic dynamics in undifferentiated and differentiating pluripotent stem cells" (2009). Biology Dissertations. Paper 10. http://hdl.handle.net/2047/d20000051
Click button above to open, or right-click to save.