In addition, the composition of serum-free, hormonally defined medium (HDM) for the different stages is shown, and it is the same composition established

in previous studies.9 Rigorous purification of parenchymal cells away from their native mesenchymal cell partners resulted in a loss of viability of the parenchymal cells (especially the stem cells NVP-LDE225 nmr and progenitors), as shown previously.2, 9 Cocultures of hHpSCs with different subpopulations of mesenchymal feeder cells elicited distinct biological responses. Those with angioblasts remained stem cells, and those with precursors to hepatic stellate cells and endothelia became hepatoblasts; this provided distinctive antigenic, biochemical, and ultrastructural features for both parenchymal and mesenchymal cell populations (Figs. 2 and 3). The hHpSC/angioblast partnership resulted in cells that were tightly bound to one another on their lateral borders through large numbers of tight junctions, desmosomes, and interdigitated microvilli. Efforts to disperse the angioblasts and hHpSCs into single cells were not successful with the customary enzymes (e.g., trypsin, chymotrypsin, dispase, and collagenases), and they resulted in a rapid loss of cell viability. Mechanical passaging, as used for human embryonic stem cells in culture, resulted Rapamycin datasheet in reasonably

efficient passaging of hHpSCs13 and was used for the studies reported here. The hHB/stellate cell/endothelial cell precursor partnership resulted in cells that were more loosely bound to one another, as evidenced by both light microscopy and ultrastructural analyses. Transmission electron microscopy (TEM) observations confirmed that hHBs were distinct from hHpSCs: there were striking increases in the number and size of the desmosomes and the intermediate filaments that terminated at the desmosomes

in the mesenchymal cells and in the appearance of bile canaliculi. In parallel with morphological changes, hHBs had an antigenic profile that overlapped medchemexpress with that of hHpSCs but showed distinctions in expressing ICAM-1 (not NCAM) and AFP and P450-A7 (data not shown). The activation of angioblasts, which gave rise to hHpSTCs and endothelial cell precursors, was associated with dramatically elevated levels of CD146 (Fig. 3) and with elevated levels of ASMA and desmin (data not shown); this all correlated with the formation of cords of hHBs and committed progenitors from the colonies of hHpSCs. Later lineage stages of parenchymal cells were partnered with either endothelia (hepatocytes) or hepatic stellate cells, pericytes, and myofibroblasts (cholangiocytes). The data from cultures of these epithelial-mesenchymal partnerships are not shown except in summary form in Supporting Information Fig. 7, although we provide data on the identified paracrine signals from those stages of mesenchymal cells.