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  • Introduction The vasculature is a multicellular system in wh

    2018-10-24

    Introduction The vasculature is a multicellular system in which each cell type plays an important and indispensible role in its function. The inner lining of endothelial cells (ECs), which are in direct contact with the blood, is surrounded and supported by perivascular cells—either vascular smooth muscle cells (vSMCs) or pericytes. vSMCs surround larger vessels such as arteries and veins, whereas pericytes typically surround smaller microvessels and capillaries (Alberts et al., 2002). The disparate vessel locations for each perivascular cell type suggest that further differences exist that should be investigated and better understood in vitro in order to appropriately rebuild blood vessels for therapeutic applications (Dar and Itskovitz-Eldor, 2013; Wanjare et al., 2013b). Along with the aforementioned functional similarities, perivascular cell types also exhibit overlapping marker expression. Adding to this complexity, neither perivascular cell type can be distinguished by one marker alone; instead, a combination of markers is needed for their identification. For example, both cell types have been demonstrated to express alpha smooth muscle pop over here (α-SMA). The expression of α-SMA and the transmembrane chondroitin sulfate proteoglycan neuron-glial 2 (NG2) help distinguish pericytes in different vessel types (Crisan et al., 2012); pericytes of the capillaries are NG2+α-SMA−, of the venules are NG2-α-SMA+, and of the arterioles are NG2+α-SMA+. When cultured in vitro, however, pericytes are positive for both of these markers. Other markers that are expressed on both perivascular cell types include calponin and platelet-derived growth factor receptor β (PDGFRβ) (Birukov et al., 1991; Dar et al., 2012). Examining differences in perivascular cell types is further complicated by added heterogeneities within the subtypes (Hedin and Thyberg, 1987; Kusuma and Gerecht, 2013). Two distinct vSMC phenotypes have been elucidated: synthetic and contractile (Beamish et al., 2010; Hedin and Thyberg, 1987; Wanjare et al., 2013a). Both participate in neovascularization, but synthetic vSMCs predominate in the embryo and in diseased or injured adult vessels while contractile vSMCs predominate in healthy adult vessels. Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced PSCs (hiPSCs), have been widely used to study somatic cell types due to their ability to obtain cell derivatives of identical genetic backgrounds. They are known for their ability to self-renew indefinitely in culture and to differentiate toward every cell type, including perivascular cells (Dar and Itskovitz-Eldor, 2013). hiPSCs are derived from a patient’s own cells and thus can yield derived cell populations that are patient specific, providing a clinically relevant pluripotent cell source for therapeutic use. Indeed, we and others have examined the derivation of both vSMCs (Drukker et al., 2012; Ferreira et al., 2007; Wanjare et al., 2013a) and pericytes (Dar et al., 2012; Kusuma et al., 2013; Orlova et al., 2014). Using a stepwise differentiation protocol, we have demonstrated the maturation of smooth muscle-like cells (SMLCs) (Vo et al., 2010) to synthetic vSMCs (syn-vSMCs) and contractile vSMCs (con-vSMCs) from both hESCs and hiPSCs (Wanjare et al., 2013a). Using a similar but distinct stepwise differentiation protocol, we have also demonstrated the derivation of pericytes from various hPSC lines (Kusuma et al., 2013). Building off of our previous studies, we sought to comprehensively define differences among con-vSMCs, syn-vSMCs, and pericytes derived from a common hPSC source in order to uncover cellular and functional differences in vitro, toward the long-term goal of rebuilding vasculature for therapeutic applications. For example, the quality of tissue-engineered blood vessels is dependent on the characteristics of the in vitro perivascular cells used. Current challenges of engineering blood vessels include precise mechanical requirements and tissue-specific cell types (Kumar et al., 2011). The in vitro characterization of our hPSC-derived perivascular cells may mediate the production of tissue-engineered blood vessels that have the patency and mechanical responsiveness equivalent to the native tissue (Chan-Park et al., 2009). Of clinical relevance, the hiPSC-BC1 line is used as the hPSC source for our studies. BC1 is derived without viral integration and has been fully genetically sequenced (Cheng et al., 2012; Chou et al., 2011). Here, we focus on differences in perivascular cells derived from BC1 and hESC H9 cells with respect to cellular characteristics, protein expression, ECM deposition, and remodeling, migration, invasion, and contractility.