Materials-Directed Differentiation of Mesenchymal Stem Cells for Tissue Engineering and Regeneration

Cell-based therapies are a promising alternative to grafts and organ transplantation for treating tissue loss or damage due to trauma, malfunction, or disease. Over the past two decades, mesenchymal stem cells (MSCs) have attracted much attention as a potential cell population for use in regenerative medicine. While the proliferative capacity and multilineage potential of MSCs provide an opportunity to generate clinically relevant numbers of transplantable cells, their use in tissue regenerative applications has met with relatively limited success to date apart from secreting paracrine-acting factors to modulate the defect microenvironment. Presently, there is significant effort to engineer the biophysical properties of biomaterials to direct MSC differentiation and further expand on the potential of MSCs in tissue engineering, regeneration, and repair. Biomaterials can dictate MSC differentiation by modulating features of the substrate including composition, mechanical properties, porosity, and topography. The purpose of this review is to highlight recent approaches for guiding MSC fate using biomaterials and provide a description of the underlying characteristics that promote differentiation toward a desired phenotype

Morphogen Delivery by Osteoconductive Nanoparticles Instructs Stromal Cell Spheroid Phenotype

Mesenchymal stem/stromal cells (MSCs) exhibit a rapid loss in osteogenic phenotype upon removal of osteoinductive cues, as commonly occurs during transplantation. Osteogenic differentiation can be more effectively but not fully maintained by aggregating MSCs into spheroids. Therefore, the development of effective strategies that prolong the efficacy of inductive growth factors would be advantageous for advancing cell-based therapies. To address this challenge, osteoinductive bone morphogenetic protein-2 (BMP-2) was adsorbed to osteoconductive hydroxyapatite (HA) nanoparticles for incorporation into MSC spheroids. MSC induction was evaluated in osteogenic conditions and retention of the osteogenic phenotype in the absence of other osteogenic cues. HA was more uniformly incorporated into spheroids at lower concentrations, while BMP-2 dosage was dependent upon initial morphogen concentration. MSC spheroids containing BMP-2-loaded HA nanoparticles exhibited greater alkaline phosphatase (ALP) activity and more uniform spatial expression of osteocalcin compared to spheroids with uncoated HA nanoparticles. Spheroids cultured in media containing soluble BMP-2 demonstrated differentiation only at the spheroid periphery. Furthermore, the osteogenic phenotype of MSC spheroids was better retained with BMP-2-laden HA upon the removal of soluble osteogenic cues. These findings represent a promising strategy for simultaneous delivery of osteoconductive and osteoinductive signals for enhancing MSC participation in bone formation

High-Throughput Formation of Mesenchymal Stem Cell Spheroids and Entrapment in Alginate Hydrogels

Mesenchymal stem cells (MSCs) are a promising cell source for tissue repair and regeneration due to their multilineage capacity, potential for autologous use, and secretion of potent bioactive factors to catalyze the endogenous repair program. However, a major limitation to current cell-based tissue engineering approaches is the drastic loss of cells upon transplantation. The causation of this loss, whether due to apoptosis following a dramatic change in the microenvironment or migration away from the defect site, has yet to be determined. MSCs formed into aggregates, known as spheroids, possess a strong therapeutic advantage compared to the more commonly used dissociated cells due to their improved resistance to apoptosis and increased secretion of endogenous trophic factors. Furthermore, the use of biomaterials such as alginate hydrogels to transplant cells in situ improves cell survival, localizes payloads at the defect site, and facilitates continued instruction of cells by manipulating the biophysical properties of the biomaterial. Transplantation of MSC spheroids without a vehicle into tissue defects comprises the majority of studies to date, ceding control of spheroid function due to the cell's interaction with the native tissue extracellular matrix and abrogating the established benefits of spheroid formation. Thus, there is a significant need to consider the role of biomaterials in transplanting MSC spheroids using an appropriate carrier. In this chapter, we describe high-throughput formation of spheroids, steps for further characterization, and encapsulation in alginate hydrogels with an eye toward localizing MSC spheroids at the target site

Engineering fibrin hydrogels to promote the wound healing potential of mesenchymal stem cell spheroids

Mesenchymal stem cells (MSCs) secrete endogenous factors such as vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2) that promote angiogenesis, modulate the inflammatory microenvironment, and stimulate wound repair, and MSC spheroids secrete more trophic factors than dissociated, individual MSCs. Compared to injection of cells alone, transplantation of MSCs in a biomaterial can enhance their wound healing potential by localizing cells at the defect site and upregulating trophic factor secretion. To capitalize on the therapeutic potential of spheroids, we engineered a fibrin gel delivery vehicle to simultaneously enhance the proangiogenic and anti-inflammatory potential of entrapped human MSC spheroids. We used multifactorial statistical analysis to determine the interaction between four input variables derived from fibrin gel synthesis on four output variables (gel stiffness, gel contraction, and secretion of VEGF and PGE2). Manipulation of the four input variables tuned fibrin gel biophysical properties to promote the simultaneous secretion of VEGF and PGE2 by entrapped MSC spheroids while maintaining overall gel integrity. MSC spheroids in stiffer gels secreted the most VEGF, while PGE2 secretion was highest in more compliant gels. Simultaneous VEGF and PGE2 secretion was greatest using hydrogels with intermediate mechanical properties, as small increases in stiffness increased VEGF secretion while maintaining PGE2 secretion by entrapped spheroids. The fibrin gel formulation predicted to simultaneously increase VEGF and PGE2 secretion stimulated endothelial cell proliferation, enhanced macrophage polarization, and promoted angiogenesis when used to treat a wounded three-dimensional human skin equivalent. These data demonstrate that a statistical approach is an effective strategy to formulate fibrin gel formulations that enhance the wound healing potential of human MSCs.Statement of significanceMesenchymal stem cells (MSCs) are under investigation for wound healing applications due to their secretion of bioactive factors that enhance granulation tissue formation, blood vessel ingrowth, and reduce inflammation. However, the effectiveness of cell-based therapies is reduced due to poor engraftment and high rates of cell death when transplanted into harsh environments characteristic of large wounds. Compared to dissociated cells, MSCs exhibit increased overall function when aggregated into three-dimensional spheroids, and transplantation of cells using biomaterials is one strategy for guiding cell function in the defect site. The present study demonstrates that the biophysical properties of fibrin hydrogels, designed for use as a cell carrier, can be engineered to dictate the secretion of bioactive factors by entrapped MSC spheroids. This strategy enables MSCs to contribute to wound healing by synergistically promoting neovascularization and modulating the inflammatory milieu

Direct Observation of Tunneling Nanotubes within Human Mesenchymal Stem Cell Spheroids

Tunneling nanotubes (TNTs) play an important role in cell-cell communication. TNTs have been predominantly reported among cells in monolayer culture. Using various imaging modalities, including scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM), this work reports the finding of TNTs between cells within human mesenchymal stem cell (MSC) spheroids. TNTs visualized by SEM are consistent in size and geometry with those observed in cellular monolayer culture. LSCM imaging of living spheroids confirms the presence of F-actin filaments within the TNTs, which are known to maintain nanotube integrity. In addition, LSCM revealed the distribution of F-actin fibers across the entire spheroid body instead of being confined within individual cells. Intracellular material transport by TNTs was tested in MSC spheroids treated with cytochalasin D (CytoD), a known actin polymerization inhibitor for disrupting TNT formation. CytoD treatment decreased the transport of cytosolic material by at least four-fold compared to untreated spheroids. To the best of our knowledge, this work represents the first direct observation of TNTs within MSC spheroids. These findings offer new physical insight into cellular interactions within spheroids, providing structural information for increasing interests in spheroid-based cell therapy

Tunneling nanotubes mediate the expression of senescence markers in mesenchymal stem/stromal cell spheroids

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is limited by acquired senescence following prolonged culture expansion and high-passage numbers. However, the degree of cell senescence is dynamic, and cell-cell communication is critical to promote cell survival. MSC spheroids exhibit improved viability compared with monodispersed cells, and actin-rich tunneling nanotubes (TNTs) may mediate cell survival and other functions through the exchange of cytoplasmic components. Building upon our previous demonstration of TNTs bridging MSCs within these cell aggregates, we hypothesized that TNTs would influence the expression of senescence markers in MSC spheroids. We confirmed the existence of functional TNTs in MSC spheroids formed from low-passage, high-passage, and mixtures of low- and high-passage cells using scanning electron microscopy, confocal microscopy, and flow cytometry. The contribution of TNTs toward the expression of senescence markers was investigated by blocking TNT formation with cytochalasin D (CytoD), an inhibitor of actin polymerization. CytoD-treated spheroids exhibited decreases in cytosol transfer. Compared with spheroids formed solely of high-passage MSCs, the addition of low-passage MSCs reduced p16 expression, a known genetic marker of senescence. We observed a significant increase in p16 expression in high-passage cells when TNT formation was inhibited, establishing the importance of TNTs in MSC spheroids. These data confirm the restorative role of TNTs within MSC spheroids formed with low- and high-passage cells and represent an exciting approach to use higher-passage cells in cell-based therapies