Biomechanical strain induces elastin and collagen production in human pluripotent stem cell-derived vascular smooth muscle cells.

Blood vessels are subjected to numerous biomechanical forces that work harmoniously but, when unbalanced because of vascular smooth muscle cell (vSMC) dysfunction, can trigger a wide range of ailments such as cerebrovascular, peripheral artery, and coronary artery diseases. Human pluripotent stem cells (hPSCs) serve as useful therapeutic tools that may help provide insight on the effect that such biomechanical stimuli have on vSMC function and differentiation. In this study, we aimed to examine the effect of biomechanical strain on vSMCs derived from hPSCs. The effects of two types of tensile strain on hPSC-vSMC derivatives at different stages of differentiation were examined. The derivatives included smooth muscle-like cells (SMLCs), mature SMLCs, and contractile vSMCs. All vSMC derivatives 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. Furthermore, long-term uniaxial strain deterred collagen type III gene expression, whereas long-term circumferential strain upregulated both collagen type III and elastin gene expression. Finally, long-term uniaxial strain downregulated extracellular matrix (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.

Vascularization of Engineered Spatially Patterned Myocardial Tissue Derived From Human Pluripotent Stem Cells in vivo.

Tissue engineering approaches to regenerate myocardial tissue after disease or injury is promising. Integration with the host vasculature is critical to the survival and therapeutic efficacy of engineered myocardial tissues. To create more physiologically oriented engineered myocardial tissue with organized cellular arrangements and endothelial interactions, randomly oriented or parallel-aligned microfibrous polycaprolactone scaffolds were seeded with human pluripotent stem cell-derived cardiomyocytes (iCMs) and/or endothelial cells (iECs). The resultant engineered myocardial tissues were assessed in a subcutaneous transplantation model and in a myocardial injury model to evaluate the effect of scaffold anisotropy and endothelial interactions on vascular integration of the engineered myocardial tissue. Here we demonstrated that engineered myocardial tissue composed of randomly oriented scaffolds seeded with iECs promoted the survival of iECs for up to 14 days. However, engineered myocardial tissue composed of aligned scaffolds preferentially guided the organization of host capillaries along the direction of the microfibers. In a myocardial injury model, epicardially transplanted engineered myocardial tissues composed of randomly oriented scaffolds seeded with iCMs augmented microvessel formation leading to a significantly higher arteriole density after 4 weeks, compared to engineered tissues derived from aligned scaffolds. These findings that the scaffold microtopography imparts differential effect on revascularization, in which randomly oriented scaffolds promote pro-survival and pro-angiogenic effects, and aligned scaffolds direct the formation of anisotropic vessels. These findings suggest a dominant role of scaffold topography over endothelial co-culture in modulating cellular survival, vascularization, and microvessel architecture.

Regulation of the microenvironment for cardiac tissue engineering.

The microenvironment of myocardium plays an important role in the fate and function of cardiomyocytes (CMs). Cardiovascular tissue engineering strategies commonly utilize stem cell sources in conjunction with microenvironmental cues that often include biochemical, electrical, spatial and biomechanical factors. Microenvironmental stimulation of CMs, in addition to the incorporation of intercellular interactions from non-CMs, results in the generation of engineered cardiac constructs. Current studies suggest that use of these factors when engineering cardiac constructs improve cardiac function when implanted in vivo. In this review, we summarize the approaches to modulate biochemical, electrical, biomechanical and spatial factors to induce CM differentiation and their subsequent organization for cardiac tissue engineering application.

Microfibrous Scaffolds Enhance Endothelial Differentiation and Organization of Induced Pluripotent Stem Cells.

INTRODUCTION: Human induced pluripotent stem cells (iPSCs) are a promising source of endothelial cells (iPSC-ECs) for engineering three-dimensional (3D) vascularized cardiac tissues. To mimic cardiac microvasculature, in which capillaries are oriented in parallel, we hypothesized that endothelial differentiation of iPSCs within topographically aligned 3D scaffolds would be a facile one-step approach to generate iPSC-ECs as well as induce aligned vascular organization. METHODS: Human iPSCs underwent endothelial differentiation within electrospun 3D polycaprolactone (PCL) scaffolds having either randomly oriented or parallel-aligned microfibers. Using transcriptional, protein, and endothelial functional assays, endothelial differentiation was compared between conventional two-dimensional (2D) films and 3D scaffolds having either randomly oriented or aligned microfibers. Furthermore, the role of parallel-aligned microfiber patterning on the organization of vessel-like networks was assessed. RESULTS: The cells in both the randomly oriented and aligned 3D scaffolds demonstrated an 11-fold upregulation in gene expression of the endothelial phenotypic marker, CD31, compared to cells on 2D films. This upregulation corresponded to >3-fold increase in CD31 protein expression in 3D scaffolds, compared to 2D films. Concomitantly, other endothelial phenotypic markers including CD144 and endothelial nitric oxide synthase also showed significant transcriptional upregulation in 3D scaffolds by >7-fold, compared to 2D films. Nitric oxide production, which is characteristic of endothelial function, was produced 4-fold more abundantly in 3D scaffolds, compared to on 2D PCL films. Within aligned scaffolds, the iPSC-ECs displayed parallel-aligned vascular-like networks with 70% longer branch length, compared to cells in randomly oriented scaffolds, suggesting that fiber topography modulates vascular network-like formation and patterning. CONCLUSION: Together, these results demonstrate that 3D scaffold structure promotes endothelial differentiation, compared to 2D substrates, and that aligned topographical patterning induces anisotropic vascular network organization.

Combinatorial Extracellular Matrix Microenvironments for Probing Endothelial Differentiation of Human Pluripotent Stem Cells.

Endothelial cells derived from human pluripotent stem cells are a promising cell type for enhancing angiogenesis in ischemic cardiovascular tissues. However, our understanding of microenvironmental factors that modulate the process of endothelial differentiation is limited. We examined the role of combinatorial extracellular matrix (ECM) proteins on endothelial differentiation systematically using an arrayed microscale platform. Human pluripotent stem cells were differentiated on the arrayed ECM microenvironments for 5 days. Combinatorial ECMs composed of collagen IV + heparan sulfate + laminin (CHL) or collagen IV + gelatin + heparan sulfate (CGH) demonstrated significantly higher expression of CD31, compared to single-factor ECMs. These results were corroborated by fluorescence activated cell sorting showing a 48% yield of CD31+/VE-cadherin+ cells on CHL, compared to 27% on matrigel. To elucidate the signaling mechanism, a gene expression time course revealed that VE-cadherin and FLK1 were upregulated in a dynamically similar manner as integrin subunit ß3 (>50 fold). To demonstrate the functional importance of integrin ß3 in promoting endothelial differentiation, the addition of neutralization antibody inhibited endothelial differentiation on CHL-modified dishes by >50%. These data suggest that optimal combinatorial ECMs enhance endothelial differentiation, compared to many single-factor ECMs, in part through an integrin ß3-mediated pathway.

Anisotropic microfibrous scaffolds enhance the organization and function of cardiomyocytes derived from induced pluripotent stem cells.

Engineering of myocardial tissue constructs is a promising approach for treatment of coronary heart disease. To engineer myocardial tissues that better mimic the highly ordered physiological arrangement and function of native cardiomyocytes, we generated electrospun microfibrous polycaprolactone scaffolds with either randomly oriented (14 µm fiber diameter) or parallel-aligned (7 µm fiber diameter) microfiber arrangement and co-seeded the scaffolds with human induced pluripotent stem cell-derived cardiomyocytes (iCMs) and endothelial cells (iECs) for up to 12 days after iCM seeding. Here we demonstrated that aligned microfibrous scaffolds induced iCM alignment along the direction of the aligned microfibers after 2 days of iCM seeding, as well as promoted greater iCM maturation by increasing the sarcomeric length and gene expression of myosin heavy chain adult isoform (MYH7), in comparison to randomly oriented scaffolds. Furthermore, the benefit of scaffold anisotropy was evident in the significantly higher maximum contraction velocity of iCMs on the aligned scaffolds, compared to randomly oriented scaffolds, at 12 days of culture. Co-seeding of iCMs with iECs led to reduced contractility, compared to when iCMs were seeded alone. These findings demonstrate a dominant role of scaffold anisotropy in engineering cardiovascular tissues that maintain iCM organization and contractile function.

Defining differences among perivascular cells derived from human pluripotent stem cells.

Distinguishing between perivascular cell types remains a hurdle in vascular biology due to overlapping marker expressions and similar functionalities. 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. We studied contractile vascular smooth muscle cells (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 define 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.

Derivation and maturation of synthetic and contractile vascular smooth muscle cells from human pluripotent stem cells.

AIMS: Embryonic vascular smooth muscle cells (vSMCs) have a synthetic phenotype; in adults, they commit to the mature contractile phenotype. Research shows that human pluripotent stem cells (hPSCs) differentiate into vSMCs, but nobody has yet documented their maturation into the synthetic or contractile phenotypes. This study sought to control the fate decisions of hPSC derivatives to guide their maturation towards a desired phenotype. METHODS AND RESULTS: 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-ß1, 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 fibres 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. CONCLUSION: We control hPSC differentiation into synthetic or contractile phenotypes by using appropriate concentrations of relevant factors. Deriving Con-vSMCs from an integration-free hiPSC line may prove useful for regenerative therapy involving blood vessel differentiation and stabilization.

Perivascular cells in blood vessel regeneration.

Vascular engineering seeks to design and construct functional blood vessels comprising endothelial cells (ECs) and perivascular cells (PCs), with the ultimate goal of clinical translation. While EC behavior has been extensively investigated, PCs play an equally significant role in the development of novel regenerative strategies, providing functionality and stability to vessels. The two major classes of PCs are vascular smooth muscle cells (vSMCs) and pericytes; vSMCs can be further sub-classified as either contractile or synthetic. The inclusion of these cell types is crucial for successful regeneration of blood vessels. Furthermore, understanding distinctions between vSMCs and pericytes will enable improved therapeutics in a tissue-specific manner. Here we focus on the approaches and challenges facing the use of PCs in vascular regeneration, including their characteristics, stem cell sources, and interactions with ECs. Finally, we discuss biochemical and microRNA (miR) regulators of PC behavior and engineering approaches that mimic various cues affecting PC function.