Wednesday, May 1, 2024
5/1/2024
DESCRIPTION (provided by applicant): Undifferentiated stem cells are present in the adult organism. In most cases it is unclear whether these cells are capable of differentiating along multiple pathways or programmed to develop along a single pathway. Furthermore, the origin of adult stem cells is unknown. The following studies focus on myogenic stem cells. The general hypotheses to be tested in these experiments are that stem cells committed to the skeletal muscle lineage: (1) are present in the epiblast of the early chick embryo, (2) become incorporated into organs lacking skeletal muscle during gastrulation, and (3) are able to induce multipotent stem cells to form muscle. These hypotheses were formulated as a result of previous studies in which small numbers of cells from the epiblast and a variety of fetal organs lacking skeletal muscle express the myogenic transcription factor MyoD in vivo and differentiate in vitro. Commitment to the skeletal muscle lineage will be tested by first analyzing whether transcription factors that regulate the development of other tissues are co-expressed with MyoD in epiblast cells. Second, MyoD positive epiblast cells will be isolated from the quail embryo using the G8 antibody, implanted into the developing heart fields and chondrogenic region of the limb bud, then analyzed for the expression of cardiac and cartilage specific molecules. To test whether the myogenic stem cells present in fetal organs originate from cells that express MyoD in the epiblast, the MyoD/G8 positive cells will be isolated from the quail epiblast, implanted into the chick epiblast, the embryos grown to fetal stages, and organs examined for the presence of quail cells. Organs from adult chickens and mice will be analyzed by RT-PCR and in situ hybridization for the expression of MyoD and Myf5. Finally, the ability of myogenic stem cells to induce multipotent stem cells to form muscle will be tested by co-culturing MyoD/G8 positive cells with chick epiblast cells and mouse embryonic stem cells. These experiments will begin to address the possibility that stably programmed cells could be used to enhance tissue regeneration in vivo when combined with multipotent stem cells.
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