Substratum-induced differentiation of human pluripotent stem cells reveals the coactivator YAP is a potent regulator of neuronal specification

Abstract

Physical stimuli can act in either a synergistic or antagonistic manner to regulate cell fate decisions, but it is less clear whether insoluble signals alone can direct human pluripotent stem (hPS) cell differentiation into specialized cell types. We previously reported that stiff materials promote nuclear localization of the Yes-associated protein (YAP) transcriptional coactivator and support long-term self-renewal of hPS cells. Here, we show that even in the presence of soluble pluripotency factors, compliant substrata inhibit the nuclear localization of YAP and promote highly efficient differentiation of hPS cells into postmitotic neurons. In the absence of neurogenic factors, the effective substrata produce neurons rapidly (2 wk) and more efficiently (>75%) than conventional differentiation methods. The neurons derived from substrate induction express mature markers and possess action potentials. The hPS differentiation observed on compliant surfaces could be recapitulated on stiff surfaces by adding small-molecule inhibitors of F-actin polymerization or by depleting YAP. These studies reveal that the matrix alone can mediate differentiation of hPS cells into a mature cell type, independent of soluble inductive factors. That mechanical cues can override soluble signals suggests that their contributions to early tissue development and lineage commitment are profound.H uman pluripotent stem (hPS) cells, which include human embryonic (hES) and human induced pluripotent stem cells, possess the remarkable capacity to self-renew indefinitely and differentiate into almost any specialized cell type (1, 2). They represent a potentially unlimited supply of cells for regenerative medicine, drug screening, and studies of human development. These applications require efficient and reproducible conditions to direct hPS cell differentiation into specialized cell types, including neuronal cells. To date, the focus has been on identifying soluble factors, such as growth factors and small molecules, that can influence hPS cell differentiation. The ability of insoluble signals to promote hPS cell-lineage specification remains less clear.Studies in murine ES cells (3, 4) and tissue-specific stem cells (5-10) indicate that the adhesive and mechanical properties of the substratum used can influence cell fate decisions (11). For example, human mesenchymal stem (hMS) cells are sensitive to changes in substrate elasticity and respond by differentiating toward distinct cell lineages depending on the stiffness of the matrix (5). These hMS cells, however, tend to exist in heterogeneous cell populations and lack a specific and unique cell characterization marker (12). Their differentiation capacity is restricted to a few tissues that arise from the mesoderm lineage, such as bone, fat, and cartilage. Indeed, there are questions about whether these cells undergo transdifferentiation to cell types, such as neurons (12)(13)(14). With the unique ability to differentiate into almost any cell type, hPS cells serve as an excellent model for und...

Advances in Reprogramming-Based Study of Neurologic Disorders

“…Murphy et al recently reviewed the significance of the mechanical properties of ECM in determining stem cell fate [173]. composition in hiPSC-derived neural cultures has also been shown [174]. Altogether, one could recreate the necessary mechanical properties (elasticity and stiffness) [175,176], relative cellular positioning, vascular innervations, and morphogen gradients characteristic of in vivo neural development [177].…”

The potential of induced pluripotent stem cells in models of neurological disorders: implications on future therapy

“…In spite of the above mentioned challenges, improvements for iPSC differentiation to neural cells and tissues are being made through the development of better defined, optimised and efficient protocols[30, [99][100][101][102][103], bolstered by increased availability of superior stem cells attributable to improved somatic cell reprogramming, stem cell culture, banking and distribution [104][105][106][107]. A major advance from traditional differentiation methods is the circumvention of embryoid body (EB) formation for more efficient and direct induction of neural progenitor cells (NPCs) and expansion of neurospheres [99][100][101][102].…”

“…A fourth and most recent method by Musah et al represents a different approach to neuronal induction by using substratum mechanics rather than soluble signalling factors to regulate neuronal specification from iPSCs [103]. Consistent with advances in biomaterials based cell support and tissue engineering (see below section), whereby physical and other non-chemical stimuli are increasingly being applied to regulate cell fate, Musah and colleagues use hydrogels with elasticity similar to brain tissue to rapidly and efficiently differentiate iPSCs to neurons.…”

Wnt-YAP interactions in the neural fate of human pluripotent stem cells and the implications for neural organoid formation

“…37 Similarly, neuronal specification (i.e., GABAergic interneurons) of hPSCs was enhanced in the absence of neurogenic factors on soft substrates (0.7 kPa vs. 3-10 kPa) that inhibited the nuclear localization of YAP. 38 While these studies were performed in 2-D cultures, inhibition of nuclear YAP for neural differentiation of hPSCs based on 3-D cultures remains to be revealed.…”

“…Musah et al, 2014 [38] (Continued on next page) YAP/TAZ is found to be related to the interactions between Hippo signaling and the Rho GTPase signaling. 40 Stress fibers reduce YAP phosphorylation and promote nuclear YAP accumulation, 41 so disruption of stress fibers using cytochalasin D reduces nuclear YAP.…”