|Program and Abstracts
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Characterization And Transplantation Of Human Muscle Stem Cells For Reconstructive Applications.
Xiaoti Xu, M.D.1, Karlijn Wilschut, Ph. D1, Gayle Kouklis, B.S.1, Hua Tian, Ph.D.1, Robert Hesse, B.S.1, Jason H. Pomerantz, M.D.2.
1Department of Surgery, Division of Plastic and Reconstructive Surgery, University of California San Francisco, San Francisco, CA, USA, 2Departments of Surgery and Orofacial Sciences, Division of Plastic and Reconstructive Surgery, Craniofacial and Mesenchymal Biology Program, Eli and Edythe Broad Center of Regeneration Medicine, University of California San Francisco, San Francisco, CA, USA.
The purpose of this study is to enable translational applications of muscle stem cell biology. The goal is to advance treatment of muscle defects and disorders for which more sophisticated and precise approaches would overcome current reconstructive limitations. Muscle regeneration and repair is mediated by a population of stem cells called satellite cells, located within muscle tissue that act locally to repair muscle injury. In mice, transplantation of satellite cells into injured muscle can ameliorate histological and functional deficits, and satellite cells are therefore ideal targets for muscle regenerative therapy or muscle engineering. However, effective isolation and transplantation of similar cells from humans has not been reported. Efforts to do so have been hampered by difficulty isolating human satellite cells and challenges associated with developing transplantation protocols. Here we report isolation, transplantation and regeneration of human satellite cells that fulfill characteristics of bona-fide muscle stem cells, thus defining a potential new tool in the reconstructive armamentarium.
Fresh human muscle biopsies were collected during surgery and histological analysis for PAX7, a satellite cell specific transcription factor, was performed to determine satellite cell content of each sample. For transplantation, fresh human muscle samples were enzymatically digested and CD56+/CD29+ satellite cells were isolated using flow cytometry. These cells were transplanted into injured tibialis anterior muscles of immunodeficient mice. Recipient muscles were evaluated for engraftment as well as the ability to replenish the satellite cell niche using reagents specific for human muscle. Recipient muscles were also evaluated for the ability of transplanted human cells to expand after reinjury.
The number of PAX7 cells per length of muscle fiber was counted for 7 different human muscles from 43 patients. Satellite cells exist at a consistent frequency of 2-4 cells/mm of fiber in muscles of the human trunk, limbs and head. Xenotransplantation into mice of 1000-5000 FACS enriched CD56+/CD29+ human satellite cells led to stable engraftment and formation of human-derived myofibers. Human cells with characteristic
PAX7, CD56 and CD29 expression patterns populated the satellite cell niche beneath the basal lamina on the periphery of new human fibers. After additional injury, transplanted satellite cells robustly regenerated to form hundreds of human-derived fibers.
This is the first demonstration of isolation and transplantation of bona-fide human skeletal muscle stem cells. Our data show that CD56+/CD29+ marks human muscle satellite cells from a variety of muscles including expendable muscles used in reconstructive surgery. After transplantation CD56+/CD29+ cells proliferate, respond to injury by regenerating mature muscle, reoccupy the stem cell niche and self-renew, establishing their identity as muscle stem cells. The ability to isolate human muscle stem cells establishes a foundation for stem cell based muscle regenerative clinical applications that will lead to novel and improved reconstructive approaches.
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