Regulation of Stem Cell Fate in Bioengineered Arrays of Hydrogel Microwells

Skeletal muscles provide necessary contractile forces to enable locomotion and breathing. Healthy skeletal muscle can recover from minor injuries, through a regeneration process involving skeletal muscle stem cells. However, there are several pathologic conditions (e.g. myopathies, aging-related sarcopenia) in which skeletal muscle is unable to sufficiently regenerate and ultimately atrophies. There is currently no cell-based therapy available to alleviate these disorders. Our objective is to develop innovative culture environments and investigate the mechanisms that could permit in vitro expansion of aged or diseased muscle stem cells. In the first and second aims, we have developed a new stiffness-adjustable artificial support material that allows preservation of muscle stem cell viability in vitro (Aim 1) and, more importantly, permits maintenance of muscle stem cell potential as observed after in vivo transplantation of the cultured muscle stem cells (Aim 2). We have demonstrated that the rigidity of the support material on which the cells are expanded is critical for maintaining their transplantation potential. We observed that standard rigid culture surfaces negatively affect muscle stem cell viability and are unfavorable for preserving the muscle stem cell function. We noticed that a soft, very elastic culture material can dramatically enhance the survival of muscle stem cells. We have analyzed the genes expressed by the muscle stem cells isolated from young and aged mouse skeletal muscles as we reasoned that the poor stem cell potential of older stem cells could be resulting from the lack of key stem cell genes. Here we show that gene expression patterns differ between the aged and young muscle stem cells, and that identification of these differentially expressed genes could help us to investigate their potential for rejuvenating the aged cells. We observed that aged muscle stem cells seeded in culture and observed by time lapse videos have diminished behaviors in vitro. Our goal is to exploit this altered behavior to test our ten selected regulators for expanding and rejuvenating aged muscle stem cells. In the third aim, we have developed a method to isolate muscle stem cells from human samples. Until recently, there has been a lack of information regarding the characterization of human stem muscle from skeletal tissue. By using flow cytometry, we have managed to identify a family of cell surface molecules that allows enrichment of the cells from a very heterogeneous population. These cells are highly myogenic, which means that in vitro they proliferate and generate muscle fibers, and we are now in the process of testing their behavior in culture on the artificial platform described above. In parallel, we are analyzing the genes expressed by these cells isolated from young or aged patients and we seek to compare these analyses with the mouse data that we have acquired.

We are pleased to report that we have been able to address all of the specific aims of the grant funded by CIRM. This progress goes a long way toward enabling therapies that will contribute to a substantially enhanced quality of life. The aging population is increasing in industrialized countries and aged-related muscular pathologies are becoming increasingly common leading to escalating medical costs and highly debilitating muscular weakness. It is well known that in young adults, skeletal muscle has a remarkable ability to heal itself and this capacity is solely due to the robust proliferative and differentiative potential of muscle stem cells. What is needed is a means of harnessing this stem cell potential by finding ways to increase stem cell numbers and function in cell culture for use in cell based therapies. This CIRM grant has enabled us to make progress that would otherwise not have been possible given the current NIH funding crisis. We are extremely grateful. Due to this grant we have succeeded in characterizing the functional differences between young and old muscle stem cells. Importantly, we have defined culture conditions that enable the maintenance, self-renewal, and expansion of muscle stem cells for the first time. (Aims 1 and 2). Finally, we have successfully applied our knowledge of adult mouse muscle stem cells to their human counterparts. We have identified markers for the prospective isolation of human muscle stem cells and growth conditions that allow their propagation in culture for cell based therapies (Aim3). Our findings have been well received with a seminal publication in Science this year and invitations to speak in symposia and at universities worldwide. Moreover our work was featured in the Wall Street Journal and on the front page of the New York Times in 2010. Another major advance in our laboratory, which was not part of this grant, was our development of the first mouse model that mimics the human genetic muscle wasting disease, Duchenne Muscular Dystrophy, which was published in Cell in 2010. Our future work will focus on testing muscle stem cell based therapies developed in the course of this grant in this muscle disease model. The findings will have relevance not only to DMD, but also more generally to sarcopenia and the profound muscle atrophy that accompanies aging.