Capturing Complex Microenvironments for Directed Stem Cell Differentiation

Loss of vascular function associated with cardiovascular disease, such as arthrosclerosis, represents the leading medical epidemic in the United States and typically requires surgical intervention through synthetic or autologous vascular grafts. To overcome the limitations associated with adult cell sources, which are often restricted by supply or compromised by disease, mesenchymal stem cells (MSCs) have emerged as potential candidates for vascular tissue engineering. While evidence suggests the roles of several factors influencing MSC differentiation into vascular phenotypes, including matrix rigidity, geometry and chemistry, the phenomena associated with these events are still largely unknown. Further, the development of mature vascular phenotypes, such as vascular smooth muscle cells (vSMCs), with functional behavior remains elusive to the research community. This thesis proposed to engineer and direct specific and mature vascular differentiation from MSCs by way of highly tailored matrices mimicking the vascular niche environment. Taking inspiration from natural organization, we contend that a biomimetic design approach to tissue scaffolds that display features of the natural cellular microenvironment whilst mimicking the bulk tissue properties may elicit highly specific differentiation of MSCs to vascular phenotypes. To validate our hypothesis, we employed a systemic approach incorporating physical and chemical microenvironmental cues, i.e. stiffness, biological ligands and chemical factors, with the aim to augment vascular phenotype expression, functionality, and final incorporation into a tailored biomaterial scaffolds. First, we present a novel technique for the preparation of silk hydrogels directly from high pressure CO2 environments without the need for crosslinking agents or additional additives such as surfactants or co-solvents. Through this novel method, we demonstrate the utility of CO2 as a volatile electrolyte, capable of sufficiently influencing the sol-gel transition of silk proteins, resulting in the formation of stable hydrogels with properties suitable for biomedical applications. Second, we hypothesized that suitable soluble factor regimen and matrix rigidity can instruct MSC differentiation towards more mature, functional vSMCs. To address this, we investigated cellular differentiation on tunable SF hydrogels prepared using a solvent-free CO2 processing method. The focus of this portion of the thesis is on exploiting the combined use of substrate stiffness and growth factor (TGF- β1) on SF matrices, with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells (hMSCs). Our data reveal that hMSC differentiation into mature SMCs can be achieved within modest culture periods (72 h) by combining appropriate SF hydrogel stiffness (33 kPa) with growth factor (TGF-β1). These findings advance our understanding of how complex multicomponent biomaterials, whereby mimicking the intricacy of natural tissue environments, can play a significant role in developing optimal stem cell differentiation protocols. Third, we postulated that the presentation of ECM proteins on 3D matrices with tunable stiffness will augment the differentiation of MSCs to vascular lineages. To address this, we established a high-throughput ECM platform based on soft, fibrous PEG hydrogels meanwhile highly-tunable in stiffness and 3-dimensional geometry. Using this technique, we identified several microenvironments supporting MSC adhesion, spreading and differentiation toward early vascular lineages. This portion of the thesis supports the hypothesis that a complex milieu exists coupling protein functional behavior with substrate rigidity and that this phenomenon may potentially be exploited through proper application of high-throughput screening methodologies. In the final work of this thesis, we explored the integration of ECM-derived small engineered peptides with 3D soft matrices to refine the differentiation of MSCs to vascular phenotypes, and further successfully recapitulate the complex vascular niche necessary for specific and efficient MSC differentiation into vascular lineages. In line with this, we report the development of a microarray platform based on electrospun nanofibrous hydrogels of photoclickable thiol-ene poly(ethylene glycol) (PEG) hydrogels. Here, we demonstrate the ability to control primary cell adhesion to soft, fibrous hydrogels functionalized with RGD peptide. However, future work will be focused on designing combinatorial peptide studies, whereby, the integration of several biological ligands of interest with tunable physical properties can instruct stem cell differentiation in a highly specific manner. This thesis has provided fundamental insights into the effects of physiological stimuli on vascular differentiation of MSC in terms of the specificity and maturity of the final differentiated cells. Better understanding of such mechanisms will prove paramount in the sequential stages of MSC differentiation to mature vascular cells. Additionally, the findings of this thesis will help to better define the process of regenerating functional healthy vascular tissue from MSCs. Altogether, a combinatorial approach investigating the effects of matrix elasticity, biological ligands and growth factors on MSC differentiation in a 3D nanofiber culture will be critical towards understanding and recapitulating MSC differentiation in the in vivo vascular environment.