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    • In summary these perivascular derivatives demonstrate an imp

    2018-11-06

    In summary, these perivascular derivatives demonstrate an important building block toward not only reconstructing physiologically relevant vasculature but also the study of developmental processes and diseases implicating these cell types. Important elements of our system are the several noted discrepancies between our in vitro results and in vivo phenotypes, alluding to the complexity of the field. A major challenge of correlating in vitro results to phenotypes observed in vivo is the presence of heterogeneous vSMC subpopulations of unknown origin that lie between the contractile and synthetic phenotypic spectrum in vivo. Unanswered questions, particularly those regarding the in vivo synthetic phenotype, still remain. For instance, ECM deposition of syn-vSMCs located in vastly different environments such as normal developing or remodeling vessels compared to diseased or injured vessels remains unknown. To further add to the complication, by utilizing SMMHC lineage tracing, Tang et al. reported that synthetic vSMCs actually arise from multipotent stem hoechst 33258 lying within vessels and not phenotypic switching by vSMCs in vivo (Drukker et al., 2012; Tang et al., 2012). The advantage of our study is the derivation and comparison of synthetic and contractile vSMCs from a known common early smooth muscle precursor. In fact, some of our study’s in vitro results actually yield a more useful phenotype for engineering blood vessels, such as increased ECM production from the hiPSC derivatives compared to control cell lines; however, other discrepancies, such as lower expression of fibronectin splice variant ED-A in derived syn-vSMCs compared to con-vSMCs, drive the need for continued study on the derivation of specialized cell types to rebuild tissue. Additionally, studies in a 3D environment would allow further investigation of morphological features such as nucleic size that may better match in vivo properties.
    Experimental Procedures Complete experimental procedures are available online (see the Supplemental Experimental Procedures).
    Acknowledgments
    Introduction Adult cardiac progenitor cells (aCPCs) are a rare subpopulation of cells first reported in the adult rat heart that are self-renewing, clonogenic, and multipotent with the capacity to give rise to cardiac myocytes (CMs), smooth muscle cells (SMCs), and endothelial cells (ECs) in vitro and in vivo (Beltrami et al., 2003). Their biology, particularly human aCPCs, remains poorly understood because techniques to study human aCPCs in vivo are very limited, and in vitro studies are challenging due to the rarity of this cell type and lack of reliable methods to propagate and expand these cells in their undifferentiated state. Cardiosphere (CS) formation, a 3D culture system, has been proposed as one method to expand aCPCs in vitro (Messina et al., 2004; Davis et al., 2009). Sphere systems have been used to culture many stem cell types, including neural and skin stem cells (Reynolds and Weiss, 1992; Fernandes et al., 2004; Gago et al., 2009). Rat CS-derived clones are multipotent (Davis et al., 2009), but whether these cells exist in human CS or their identity is unknown. CSs are a heterogeneous mix of nonmyocyte cells derived from the mononuclear fraction of dissociated heart tissue, which includes mesenchymal stem cells, SMCs, ECs, and cardiac fibroblasts. Although CSs have been reported to contain an aCPC, this has been questioned, and its identity or cell surface markers that can be used to isolate these cells are unknown (Masuda et al., 2012; Smith et al., 2007). Interestingly, there is no panel of cell surface markers that can directly identify endogenous CPCs. Numerous putative aCPCs have been reported in adult mouse heart, which were identified based on the expression of C-KIT, SCA1, or the ability to exclude Hoechst dye (Beltrami et al., 2003; Oh et al., 2004; Tomita et al., 2005). Much less data exist in human hearts, although a rare cardiac progenitor cell population of C-KIT and NKX2.5+ cells has been described (Mishra et al., 2011; Goumans et al., 2007; Bearzi et al., 2007; Smits et al., 2009). However, C-KIT by itself is not specific and cannot be used by itself to identify aCPCs (Bearzi et al., 2009; Sandstedt et al., 2010). A significant proportion of the C-KIT cells in human hearts coexpresses CD45, suggesting that they may be hematopoietic in origin (Kubo et al., 2008). The early cardiac transcription factor, NK2 homeobox 5 (NKX2.5), is often used in conjunction with c-KIT to identify aCPCs (Wu et al., 2006, 2008; Mishra et al., 2011), but because it is an intracellular marker, its use is restricted to fixed cells and cannot be used for purifying human live cells. At least a subset of cardiac C-KIT cells expressed NKX2.5, but whether this C-KIT+/NKX2.5+ subpopulation in adult human heart is an authentic aCPC and whether this C-KIT+/NKX2.5+ cell also represents the aCPCs within CS is unknown. No systematic analysis of cell surface markers of cloned human aCPCs has been performed, and no panel of cell surface markers allowing direct isolation of aCPCs is available.