Perivascular Cells Derived from Human Pluripotent Stem Cells Using Biochemical and Biomechanical Stimuli
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2014-09-17
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Johns Hopkins University
Abstract
Blood vessels play a vital role in the body because they deliver oxygen and nutrients to all organs and tissues. Vascular smooth muscle cells (vSMCs) and pericytes are the two major classes of perivascular cells (PCs) that encircle blood vessels in order to allow proper vascular function within the body. However, injury or disease may alter the function of these cells, which often compromises the stability of blood vessels. Vascular engineering using human pluripotent stem cells (hPSCs) represents a potential therapy that seeks to derive functional PCs, with the ultimate goal of clinical translation. The ability direct hPSCs to PCs relies on the utilization of different in vitro biochemical and biomechanical approaches that recapitulate in vivo environments.
We first sought to understand the development and differentiation of vSMCs derived from hPSCs by guiding the maturation of vSMC derivatives towards either a contractile phenotype or synthetic phenotype using biochemical cues. While the synthetic phenotype is usually associated with embryonic vSMCs, in healthy adult vessels vSMCs commit to the mature contractile phenotype. The long-term differentiation of hPSCs, including the integration-free-induced PSC line, in high serum with platelet derived growth factor-BB (PDGF-BB) and transforming growth factor-1, allowed us to induce the synthetic vSMC (Syn-vSMC) phenotype with increased extracellular matrix (ECM) protein expression and reduced expression of contractile proteins. By monitoring the expression of two contractile proteins, smooth muscle myosin heavy chain (SMMHC) and elastin, we show that serum starvation and PDGF-BB deprivation caused maturation towards the contractile vSMC (Con-vSMC) phenotype. Con-vSMCs differ distinctively from Syn-vSMC derivatives in their condensed morphology, prominent filamentous arrangement of cytoskeleton proteins, production and assembly of elastin, low proliferation, numerous and active caveolae, enlarged endoplasmic reticulum, and ample stress fibers and bundles, as well as their high contractility. When transplanted subcutaneously into nude mice, the human Con-vSMCs aligned next to the host’s growing functional vasculature, with occasional circumferential wrapping and vascular tube narrowing. We controlled hPSC differentiation into synthetic or contractile phenotypes by using appropriate concentrations of relevant factors. Deriving Con-vSMCs from an integration-free human induced pluripotent stem cell (hiPSC) line may prove useful for regenerative therapy involving blood vessel differentiation and stabilization.
Next, we studied human perivascular development and functionality by performing direct comparisons between perivascular cell derivatives with the same genetic background. Distinguishing between perivascular cell types remains a hurdle in vascular biology due to overlapping marker expressions and similar functionalities. We studied contractile vSMCs, synthetic vSMCs, and pericytes derived from a common human pluripotent stem cell source. Using in vitro cultures, we show unique cell morphology, subcellular organelle organization (namely endoplasmic reticulum, mitochondria, and stress fibers), and expression of smooth muscle myosin heavy chain and elastin for each cell type. While differences in extracellular matrix deposition and remodeling were less pronounced, the multipotency, in vivo, migratory, invasion, and contractile functionalities are distinctive for each cell type. Overall, we defined a repertoire of functional phenotypes in vitro specific for each of the human perivascular cell types, enabling their study and use in basic and translational research. Clarifying and defining heterogeneities in vitro among perivascular cells could lead to improved cell-based tissue regeneration strategies and a better understanding of human developmental processes.
Finally, we studied the effect of biomechanical strain on the ECM expression of vSMCs derived from hPSCs. The effects of two types of tensile strain on hPSC vSMC derivatives at different stages of development were examined. The derivatives included smooth muscle-like cells (SMLCs), mature SMLCs (mSMLCs), and contractile vSMCs (Con-vSMCs). All vSMC derivatives were exposed to transforming growth factor (TGF-1) and cyclic uniaxial strain at 1Hz and 7% elongation using a deformable silicone substrate. Additionally, a custom engineered bioreactor was used to propel pulsatile flow through silicone tubing with an inner diameter of 300m in order to generate cyclic circumferential strain on the hPSC derivatives. Stimulated hPSC- derivatives were analyzed for cell alignment and the expression of extracellular matrix (ECM) genes. All vSMC derivatives including SMLCs, mSMLCs, and Con-vSMCs aligned perpendicularly to the direction of cyclic uniaxial strain. Serum deprivation and short-term uniaxial strain had a synergistic effect in enhancing collagen type I, fibronectin, and elastin gene expression of derivatives. Furthermore, long-term uniaxial strain deterred collagen type III gene expression while long-term circumferential strain upregulated both collagen type III and elastin gene expression. Long-term uniaxial strain downregulated ECM expression in more mature vSMC derivatives while upregulating elastin in less mature vSMC derivatives. Overall our findings suggest that in vitro application of both cyclic uniaxial and circumferential tensile strain on hPSC- vSMC derivatives induces cell alignment and affects ECM gene expression. Therefore, mechanical stimulation of hPSC- vSMC derivatives using tensile strain may be important in modulating the phenotype and thus the function of vSMCs in tissue engineered vessels.
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Perivascular