Background: Human mesenchymal stem cells (hMSCs) are utilized preclinically and clinically as a candidate cell therapy for a wide range of inflammatory and degenerative diseases. Despite promising results in early clinical trials, consistent outcomes with hMSC-based therapies have proven elusive in many of these applications. In this work, we attempt to address this limitation through the design of a stem cell therapy to enrich hMSCs for desired electrical and ionic properties with enhanced stemness and immunomodulatory/regenerative capacity. Materials and Methods: In this study, we sought to develop initial protocols to achieve electrically enriched hMSCs (EE-hMSCs) with distinct electrical states and assess the potential relationship with respect to hMSC state and function. We sorted hMSCs based on fluorescence intensity of tetramethylrhodamine ethyl ester (TMRE) and investigated phenotypic differences between the sorted populations. Results: Subpopulations of EE-hMSCs exhibit differential expression of genes associated with senescence, stemness, immunomodulation, and autophagy. EE-hMSCs with low levels of TMRE, indicative of depolarized membrane potential, have reduced mRNA expression of senescence-associated markers, and increased mRNA expression of autophagy and immunomodulatory markers relative to EE-hMSCs with high levels of TMRE (hyperpolarized). Conclusions: This work suggests that the utilization of EE-hMSCs may provide a novel strategy for cell therapies, enabling live cell enrichment for distinct phenotypes that can be exploited for different therapeutic outcomes.

Clinical use of human embryonic stem cells (hESCs) in bone regeneration applications requires that their osteogenic differentiation be highly controllable as well as timeand cost-effective. The main goals of the current work were thus (a) to assess whether overexpression of pluripotency regulator Forkhead Box D3 (FOXD3) can enhance the osteogenic commitment of hESCs seeded in three-dimensional (3D) scaffolds and (b) to evaluate if the degree of FOXD3 overexpression regulates the strength and specificity of hESC osteogenic commitment. In conducting these studies, an interpenetrating hydrogel network consisting of poly(ethylene glycol) diacrylate and collagen I was utilized as a 3D culture platform. Expression of osteogenic, chondrogenic, pluripotency, and germ layer markers by encapsulated hESCs was measured after 2 weeks of culture in osteogenic medium in the presence or absence doxycycline-induced FOXD3 transgene expression. Towards the first goal, FOXD3 overexpression initiated 24 hr prior to hESC encapsulation, relative to unstimulated controls, resulted in upregulation of osteogenic markers and enhanced calcium deposition, without promoting off-target effects. However, when initiation of FOXD3overexpression was increased from 24 to 48 hr prior to encapsulation, hESC osteogenic commitment was not further enhanced and off-target effects were noted.Specifically, relative to 24-hr prestimulation, initiation of FOXD3 overexpression 48 hr prior to encapsulation yielded increased expression of pluripotency markers while reducing mesodermal but increasing endodermal germ layer marker expression.Combined, the current results indicate that the controlled overexpression of FOXD3 warrants further investigation as a mechanism to guide enhanced hESC osteogenic commitment.

Cell traction forces, biochemical signals, and cell metabolism are each known to regulate mesenchymal stem cell (MSC) differentiation. Biomaterials have therefore been designed to manipulate cell traction force (via their viscoelasticity and adhesion ligands) to guide MSC fate decisions. Similarly, the type and density of biochemical signals presented by a material have been tailored to influence MSC differentiation. However, the potential impact of biomaterial properties on regulating MSC differentiation through modulating cell metabolism is relatively unstudied. Here, we present data indicating that hydrogel elastic modulus and mesh size regulate MSC differentiation in part through modulating the activity of the metabolic enzyme glyceraldehyde-3phosphate-dehydrogenase (GAPDH). Toward this end, we first confirm that the differentiation profile of MSCs cultured in 2D on highly elastic, covalently crosslinked poly(ethylene glycol) diacrylate (PEGDA) hydrogels differs substantially from that of MSCs encapsulated within the same hydrogel formulations, indicating a dependence in 3D on a variable(s) beyond elastic modulus. Further results indicate that the GAPDH activity of MSCs in 3D hydrogels is a function of elastic modulus and mesh size, suggesting that GAPDH activity may be one of these variables. Studies in 2D supported a positive correlation between hydrogel elastic modulus and GAPDH activity. Additionally, inhibition of GAPDH activity on 2D surfaces induced alterations in the profiles of key differentiation markers, indicating that GAPDH activity can impact lineage progression. Cumulatively, these findings suggest that the potential impact of hydrogel properties on cell metabolism should be considered when evaluating biomaterial-driven MSC differentiation. Lay Summary Cell traction forces, biochemical signals, and cell metabolism are each known to regulate mesenchymal stem cell (MSC) differentiation. Biomaterials have therefore been designed to manipulate cell traction force (via their viscoelasticity and adhesion ligands) to guide MSC fate decisions. Similarly, the type and density of biochemical signals presented by a material have been tailored to influence MSC differentiation. However, the impact of biomaterial properties on regulating MSC differentiation through modulating cell metabolism is relatively unstudied. Here, we present data indicating that hydrogel elastic modulus and mesh size regulate MSC differentiation in part through modulating the activity of the metabolic enzyme glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

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