The laboratory opossum is the only marsupial that is available in large numbers for biomedical research; it is a unique or specialized model for research on many human diseases and developmental processes, as well as for comparative biology and comparative genomics purposes. The long-term objective is to establish induced pluripotent stem cell (iPSC) lines from this species for use in those research areas, and particularly for developing iPSC therapies.
The specific aims are 1) to optimize iPSC reprogramming methodology and establish iPSC lines from two inbred strains, 2) to validate the method while reprogramming iPSCs from four partially inbred strains, and 3) to demonstrate the functional potential of reprogrammed iPSC lines. Using rapidly dividing skin fibroblasts derived from embryos, we will adapt a method developed by one of us in creating the first iPSC lines from any marsupial species, the Tasmanian devil. We will use lentivirus-based delivery of human reprogramming factors in the initial experiments, and we will determine the reprogramming efficiency in MEF feeder-based and feeder- free culture conditions. We will then optimize a transgene integration-free reprogramming protocol using OriP/EBNA episomal plasmid based delivery of the human iPSC reprogramming factors. In the third set of experiments, we will test various culture conditions by modulating growth factors and small molecules to improve reprogramming efficiency. The established iPSCs will be characterized by immunocytochemistry and differential gene expression analysis of the pluripotency markers.
For Aim 2, the developed methodology will be applied to fibroblasts of animals from four partially inbred strains.
For Aim 3, the established iPSC lines from all six animals will be differentiated into neurons, cardiomyocytes, and hepatocytes (terminally differentiated cells of all three germ layers). Fibroblasts from the six animals, their reprogrammed iPSCs (2 clones/animal), and differentiated cells (from 1 iPSC clone/animal) will undergo genome-wide RNA sequencing (RNASeq) to better understand the gene/transcript expression profiles and functional potential of the iPSCs and their differentiated cells. The genomic integrity of the generated cells will be determined from RNASeq data by performing ?location enrichment analysis? and ?CGH-PCF analysis.? In addition, iPSC expression profiles will be compared with existing human and mouse iPSC RNASeq data in a comparative approach to develop a better understanding of pluripotency. The established iPSC lines will be used by us (beyond the term of this project) for the development of iPSC therapies for hypercholesterolemia and non-alcoholic fatty liver disease. They also will be available to collaborators for research in diverse areas of biomedicine such as therapies for spinal cord injury, and prevention of birth defects caused by drugs such as thalidomide; as well as for the development of fundamental knowledge that relates to health and disease in fields such as epigenetics, embryogenesis and organogenesis, gene regulation and functional genomics, T-cell function and other aspects of immunology, cell motility, and X- chromosome inactivation (see letters of support).
Laboratory-bred gray short-tailed opossums are widely used in biomedical research on human diseases as well as research on early developmental processes and abnormalities. We propose to develop, from inbred strains of laboratory opossums, induced pluripotent stem cells which will have the capacity to differentiate into any cell type of the body. These stem cells will be used in future research to develop therapies in which diseased tissues of opossums are treated with the stem cells in an effort to alleviate conditions such as atherosclerosis, non- alcoholic fatty liver disease, and spinal cord injuries.