Microenvironmental Regulation of Human Stem Cell Fate Via Integrin α6/β1–FAK signaling, Impact of Feeder-derived Mitochondrial Transfer as a Culture Confounder, and Development of Bioengineered Substrates for Xeno Free Culture

Abstract

Human pluripotent stem cells (hPSCs) and mesenchymal stem/stromal cells (hMSCs) are highly responsive to microenvironmental cues. Yet, the mechanisms linking extracellular matrix (ECM) signaling to nuclear gene regulation, fate stability, and culture reproducibility remain incompletely defined. In this dissertation, we investigate how microenvironmental regulation shapes stem cell behavior across three related contexts: ECM–cell interactions, cell–cell interactions, and ECM–soluble factor–cell interactions. To define ECM-dependent fate control in hPSCs, integrin β1 was activated using manganese and a β1-integrin–activating antibody, followed by analyses of OCT4, SOX2, NANOG, pFAK Y397, and FAK-associated nuclear complexes. Integrin β1 activation altered FAK localization, engaged the FAK–MDM2–p53 axis, increased p53 and p21, and induced transcriptional changes consistent with early lineage priming. To examine cell–cell interactions in feeder-based culture, feeder-derived mitochondrial transfer was assessed using complementary imaging, molecular, and bioenergetic analyses. Mitotically inactivated mouse embryonic fibroblasts transferred mitochondrial material to hPSCs at high frequency, primarily through extracellular vesicle–enriched pathways. Mouse COX2mtDNA persisted for three to four passages and was associated with altered membrane potential and reduced mitochondrial ATP output. To investigate ECM–soluble factor–cell interactions in hMSCs, defined synthetic substrates and glycosaminoglycan-mimetic sulfonated polymer brushes were evaluated using molecular and functional potency assays. These biomaterials promoted laminin remodeling, increased hMSC expansion by 30–40, preserved trilineage differentiation, and elevated FGFR1 and ITGA6 expression. Collectively, these findings establish a unified microenvironmental control framework in which integrin β1-dependent nuclear FAK signaling regulates stem cell fate, feeder-derived mitochondrial transfer acts as a major bioenergetic confounder, and defined biomaterial strategies improve reproducibility and support scalable stem cell expansion.