l-alanine Supplier Our data are consistent with recent

Our data are consistent with recent studies showing that in vivo, transplants of BM l-alanine Supplier were able to form bone and support marrow formation, while white adipose-derived stromal cells (WAT), cells from umbilical cord (UC), and skin fibroblasts (SF) were not (Kaltz et al., 2008; Reinisch et al., 2015). Importantly, the “MSCs” derived from cord blood (CB) used in our study must be clearly distinguished from fibroblastic cells derived from UC “MSCs” in the Reinisch study; UC cells fail to differentiate in vitro and in vivo (Kaltz et al., 2008; Liedtke et al., 2016; Reinisch et al., 2015), differ in their typical HOX expression pattern (Liedtke et al., 2010, 2016), and have a different molecular transcriptomic signature lacking relevant integrin-binding sialoprotein (IBSP) expression (Bosch et al., 2012). Differences in our study include culture in 20% fetal bovine serum (FBS) and use of a ceramic carrier that favors direct osteogenesis (no evidence of endochondral bone formation). In Reinisch et al. (2015), BM cells were grown in 10% human platelet lysate and transplanted with a Matrigel equivalent (In Vitro Vasculogenesis Assay Kit; Millipore), and bone formed through an endochondral process. Another study investigated expression of μRNAs by BM cells, WAT cells, and CB cells at a multiclonal level (Ragni et al., 2013). While the μRNA patterns between the cells types were similar, a small number of highly differentially expressed μRNAs were identified, which could affect the expression of many genes. Of note, in vitro osteogenic, adipogenic, and chondrogenic differentiation assays were performed in both of these studies, and significant differences were noted between the cell types. Nonetheless, the authors of both papers concluded that an in vitro mesengenic process was at play in all cell types. However, in vitro analyses of osteogenic differentiation are not predictive of in vivo transplantation (e.g., Phillips et al., 2014). For chondrogenesis, 3D cultures are needed to clearly see bona fide chondrocytes lying in lacuna, surrounded by a matrix that stains purple with toluidine blue (metachromasia) as shown by Reinisch et al. (2015). Close examination of this study suggests that there was little or no chondrogenic differentiation of WAT, UC, or SFs compared with BM cells. In Ragni et al. (2013), chondrogenesis was difficult to assess based on Alcian blue-stained 2D cultures and expression of a limited number of markers, which is insufficient to confirm chondrogenic differentiation. Even if updated to equate “MSCs” with pericytes (Caplan, 2008; Crisan et al., 2008), the widespread concept of broadly and uniformly multipotent “MSCs” that are invariant in anatomical space and developmental time leaves the developmental origin of such cells unaddressed, and in fact collides with certain fundamental tenets of developmental biology, such as the early segregation of inherent osteogenic and myogenic potential into different lineages (Bianco and Robey, 2015). Our data now reveal a simple mechanism whereby assayable skeletogenic or myogenic progenitors can be established in different tissues. As previously postulated (Bianco et al., 2008, 2013), we have now directly shown that committed progenitors of different origin and differentiation potential can physically associate with nascent BV walls in an experimental assay, and be recruited to a mural cell fate. In an earlier study, the most robust contribution of grafted mesoderm progenitor cells in quail chick chimeras was, in fact, to the adventitia of BVs of any caliber (Minasi et al., 2002). In development, somite-derived cells associate with, and are incorporated into, the wall of the dorsal aorta (Esner et al., 2006). Pericytes originate from the recruitment of non-endothelial, mesenchymal cells to vascular walls (Armulik et al., 2011; Hellstrom et al., 1999; Hirschi and D\'Amore, 1996; Jain, 2003; Lindahl et al., 1997). Obviously, the same mechanism may operate in different tissues and at different developmental times. General mechanisms regulating mural cell recruitment have largely been elucidated, and provide the background against which to seek additional determinants operating in the retention of quiescent mesoderm progenitors in a vascular niche. CD146 itself may represent a player in this scenario, consistent with its nature as a cell-adhesion molecule (Shih, 1999), and with evidence provided here that its knockdown perturbs the establishment of properly structured BVs in vivo.