Title: Engineering microenvironmental cues for guiding stem cell fate
Description: Injury, aging, and congenital disabilities of the muscular and neural systems impose a significant burden on patients and their families. Due to the tissue’s limited regenerative capacity, effective treatment interventions for restoring progressive damage is still lacking. Cell replacement therapy is primarily limited by the restricted supply of viable donor cells and variable graft survival. For addressing these limitations, we propose new strategies to obtain a target cell of interest from an autologous cell source. Herein, we engineer cell fate decisions by 1) harnessing host microenvironment and the CRISPR/dCas9-mediated transcriptional activation system to promote myogenesis of human endothelial progenitor cells (EPCs); and 2) employing substrate-mediated biophysical cues with soluble factors (biochemical cues) to drive cell commitment to neuronal lineages. For the first strategy, we hypothesized that therapeutic cells could be obtained in situ by employing the CRISPR/dCas9 system to engineer cell fate in the host tissue.

Using this system, we transactivated MYOD1, a master regulator for myogenesis, to directly reprogram primary EPCs to skeletal myoblasts (SkMs). EPCs were chosen as a cell source for their easy accessibility, high proliferation, and potential contribution to regenerate vasculature and musculature tissue. The early myogenic commitment of EPCs was confirmed in vitro by MYOD1 expression, which yielded a 230-fold higher induction than the original EPCs. These cells were then transplanted for assessing their therapeutic efficacy in myotoxin-induced muscle injury model in immunodeficient mice. A one-month post-injury study resulted in the integration of induced SkMs to the injured host tissue, promotion of neoangiogenesis, and reduction in fibrotic scar formation. These findings indicate that CRISPR/Cas9-mediated target gene activation can be achieved in situ to accelerate muscle regeneration after myotoxin-induced damage. For the second strategy, we utilized both soluble and insoluble factors to convert the cell fate of neural stem/progenitor and somatic cells to various neuronal lineages, including motor neurons (MNs) and dopaminergic (DA) neurons.

For soluble factors, cells were exposed to various biochemical factors, inspired by the neuronal niche environs during the natural developmental process. For insoluble factors, the conductive graphene substrate was used to support the endogenous electrical signal between neurons for enhancing the neuronal phenotypes and their functionality. We postulated that exposing the cells to these collective stimuli in vitro can alter their intrinsic signaling pathway to tailor their fate to neuronal lineages. To test the hypothesis, neural stem/progenitor and somatic cells were cultured on various substrates with or without electroactive graphene and aligned patterns. After two weeks to one month of cell fate induction in the chemically defined conditions, our results implied that cell adhesion, survival, neurite outgrowth, and maturation were facilitated on the electroactive substrates with aligned patterns compared to the control platforms. Taken together, our results in this dissertation demonstrate the feasibility of tailoring the donor cell fates within or across the germ layers.

We achieved this by employing a transcriptional gene activation system and tunable microenvironmental cues elicited by soluble (chemical and growth factors) and insoluble (physical cues from the substrate) factors. Utilizing such strategies hold great promise for elucidating the optimal conditions to guide cell fate to target lineages. This work provides a rational basis for establishing a robust protocol and an in vitro culture platform to module cell fate decisions that could help realize the autologous cell-based therapy for muscular and neurodegenerative diseases.