When hHpSTCs were plated onto culture plastic and in KM supplemented with 5% fetal bovine serum, they were activated into the cells with a myofibroblast phenotype emerging within 3 to 5 days
of culture (Supporting Information Fig. 3). The cells were even longer (up to 50 μm or longer), had a centrally located nucleus, and expressed the highest levels DAPT ic50 observed for ICAM1, ASMA, and desmin. We surveyed the biological activities of numerous mesenchymal cell lines and primary cultures of mesenchymal cells as feeders (Supporting Information Table 4). We eventually realized that even transient exposure to serum resulted in muting of the distinctions in paracrine signals produced by the different mesenchymal cell subpopulations and their skewing toward biological activity typical for fibrosis or cirrhosis. Therefore, a serum-free medium for mesenchymal cells (MCM) was developed (Supporting Information Fig. 1) that enabled us to define feeder effects and the paracrine signals produced with freshly immunoselected mesenchymal cell subpopulations from fetal human livers versus adult human livers under serum-free conditions in short-term cultures (up to 2 weeks). Using this strategy, we determined that all the mesenchymal subpopulations produced multiple types of collagens, basal adhesion molecules, selleck proteoglycans, and elastin but at quite different levels
(Fig. 1E and Table 1). The angioblasts (CD117+/KDR+ or CD133+/KDR+) from fetal livers produced less matrix than any of the tested mesenchymal cell subpopulations, produced low levels of type III, IV, and V collagens (only type III was detectable by immunohistochemistry), laminin A4 but not the other laminins or fibronectin, chondroitin sulfate proteoglycans (CS-PGs) and low levels of syndecan (only
CS-PGs were detected by immunohistochemistry), and HAs. Those from adult livers produced higher levels of syndecan, laminin A4, fibronectin, and type IV collagen. Fetal liver–derived endothelial (CD-31+) cells made all of the forms of heparan sulfate proteoglycans (HS-PGs), type I, III, and V collagens (but not type IV collagen), low levels of laminin B2 and fibronectin, and elastin. Adult liver–derived endothelial cells (CD-31++) made high levels of HS-PG2 and syndecan, type I and IV collagens, laminins A4 and B3, fibronectin, MCE and elastin. In summary, the matrix chemistry in angioblast and endothelial subpopulations was dominated by HS-PGs and some but not all laminin forms, and there was a significant increase in elastin with development. The stellate cell subpopulations produced the highest amounts of most of the analyzed matrix components, expressed low or negligible levels of laminins, strongly expressed fibronectin and elastin, and were high producers of all the collagens (especially type I) and the CS-PGs. As for the angioblast and endothelial cell subpopulations, the levels were highest in those from adult livers. Supporting Information Fig.