UC Berkeley

The central nervous system (CNS) controls crucial functions in mammals ranging from sensory processing and memory to hormonal regulation and motor function. Thus many diseases and injuries afflicting CNS cells, such as Alzheimer's and Parkinson's Diseases or stroke and traumatic injuries, result in devastating consequences. Stem cells serve as potential cell sources for cell transplantation therapies, but also model systems to study neural development. In both cases, it is crucial to understand how the hallmark properties of stem cells, self-renewal and potency, are regulated by their microenvironment. An important body of work has identified many biochemical factors, such as small molecules, growth factors, morphogens, and adhesive ligands that regulate stem cell behavior. However, more recently biophysical effects, such as microenvironmental stiffness, cell and tissue shape, and dynamic shear flow or cyclic strain, have been shown to affect diverse cellular processes, including proliferation, differentiation, and apoptosis. These dissertation studies aim to elucidate microenvironmental stiffness effects on stem cell systems, and how they can be harnessed to improve derivation of neural cell types.

A wide temporal range of development was studied using model systems with adult and embryonic origins. First, adult neural stem cells (aNSCs) from the rat hippocampus, in soluble conditions permissive of differentiation into neurons, astrocytes, and oligodendrocytes, were found to differentiate preferentially into neurons on soft substrates with stiffness similar to brain tissue (100-1000 Pa), while stiffer substrates promoted increased astrocytic differentiation. This bias in lineage commitment due to microenvironmental stiffness cues was transduced by a cellular mechanoadaptation mechanism in which aNSCs stiffened and increased cellular contractility in response to increasing substrate stiffness. This mechanotransduction was dependent on RhoA and Cdc42 activity and occurred within only 2 days after induction of

differentiation. Downstream neuronal maturation and subtype specification was also investigated. In soluble conditions inducing primarily neuronal differentiation, intermediate microenvironmental stiffnesses around 700 Pa promoted neuronal maturation and subtype specification of GABA and glutamatergic neurons. RhoA and Cdc42 activity increased neuronal maturation on softer substrates while inhibiting RhoA and Cdc42 activity abolished the stiffnessdependent differences in neuronal maturation.

Earlier periods of stem cell development were studied with human embryonic and induced pluripotent stem cells (hESCs and hiPSCs). While pluripotency marker expression and self-renewal were not affected by microenvironmental stiffness, neuronal differentiation was enhanced on softer substrates. Furthermore, softer substrates increased the percentage of dopaminergic neurons, the cell type lost in Parkinson's Disease. Interestingly, this increase in neuronal differentiation was due to the early increase in PAX6 and SOX1 positive neural ectoderm prior to neural patterning, demonstrating that microenvironmental stiffness may also be important at early periods of development and impact downstream lineage compositions. Finally, with the discovery of cellular reprogramming, not only can the developmental timeline can be reversed but trans-cell type reprogramming can be studied as well. Preliminary studies showed that microenvironmental stiffness has potentially interesting effects on both reprogramming somatic cells to hiPSCs, but also translineage reprogramming of somatic cells to neurons. Intermediate substrate stiffnesses around 700 Pa promoted hiPSC reprogramming. Interestingly, cell migration was greater on softer substrates but persistence or directionality was greater on stiffer substrates, suggesting that substrate stiffness effects on hiPSC colony formation is not likely due to significant differences in cell migration and collision events. Substrate stiffness also biased reprogramming of somatic cells directly into neurons with stiffer substrates promoting neuronal reprogramming.

The studies comprising this dissertation demonstrate that microenvironmental stiffness is important throughout a wide temporal range of neural development modeled by adult and embryonic/pluripotent stem cells, as well in neuronal reprogramming processes. They motivate the consideration of microenvironmental stiffness in fundamental biological studies but also as a design parameter for stem cell cultures and bioreactor systems. These studies motivate the future study of additional biophysical factors as potential regulatory cues for stem cells, especially neural stem cells.