ABSTRACT Skeletal muscle stem cell (MuSC) transplantation is emerging as a promising strategy for treating muscle- associated trauma and diseases. However, MuSCs spontaneously lose their “stemness” and engraftment potential in conventional 2D culture, critically limiting their applicability for cell-based therapy. This inability to culture MuSCs further limits the employment of cell engineering strategies, such as ex vivo gene editing patient derived cells. Thus, there is a critical need for a strategy to maintain and expand therapeutically potent MuSC ex vivo long-term. To date, MuSC culture systems that recapitulate the asymmetric MuSC niche, consisting of myofiber and basal lamina, required to establish cellular polarity, support physiologic cell division, and guide fate specification, do not exist. The contribution of active mechanical forces in modulating MuSC polarity, division, and fate specification also remains overlooked. We believe that establishing MuSC polarity is a requirement for supporting long-term symmetric expansion ex vivo through biochemical and biophysical means. To this end, the proposed research will engineer an asymmetric designer platform for expanding self-renewing MuSCs ex vivo long-term by directing cellular polarity and fate specification. In Aim 1, we will identify minimally essential asymmetric cues for establishing MuSC polarity by engineering an asymmetric hydrogel. In Aim 2, we will determine the effects of biochemically promoting symmetric cell division on long-term expansion of MuSCs using the asymmetric hydrogel. Biochemical strategies to inhibit asymmetric division and to promote symmetric division to achieve ex vivo MuSC expansion will be tested. In Aim 3, we will determine the effects of dynamic biomechanical stretch on regulation of symmetric MuSC expansion using the asymmetric hydrogel. Mechanisms by which externally applied forces align mitotic spindle orientation and regulate cell division of polarized MuSCs will be tested. Successful outcomes of this research will not only develop a platform for expanding MuSCs for cell therapies by directing stem cell polarity and fate specification but also provide establish a new paradigm for harnessing stem cell polarity in cell-instructive biomaterials for regenerative medicine.