1D-G). The ALDH− population

contained nonparenchymal (NP) cells (liver sinusoidal endothelial cells [LSECs], HSCs, and Kupffer cells) and some hepatocytes, which were removed after a brief wash, leaving only HSCs and fibroblast-like cells after 1 day in culture. In contrast, at day 1 (after washing) the ALDH+ population remained phase-bright and seemed to consist of two types of cells, small round cells and fibroblast-like cells. During culture, only the small round cells kept their ALDH activity and grew on top of the fibroblast-like cells that had lost their ALDH activity earlier on in culture (Fig. 1I and Supporting Fig. 3). The ALDH+ population was analyzed immediately after sorting by immunohistochemistry of cytospins, thereby avoiding cell culture-induced artifacts. The “surface marker footprints” of the two freshly isolated populations JQ1 nmr is displayed in Fig. 2 (for antibodies, see Supporting Table 1). These show that the two populations are clearly distinct, as demonstrated by the very low percentage of cells that expressed endothelial (CD31, CD146), HIF inhibitor Kupffer (CD11b, F4/80), LSEC (MRC1, CD32b), and HSC markers (Bodipy, GFAP) and the complete absence of hepatocyte markers (PMP70, Connexin-32, CYP1A1) in the ALDH+ population

(Fig. 2A). In contrast, the ALDH+ population was enriched in cells expressing markers find more that have been reported to be suitable for the isolation and/or identification of LPCs like CK19, EpCAM, CD133, SOX9, ABCG2, MRP2, CD117, and CD49f (Fig. 2B-D). The complete absence of Sca-1-, CD34-, and CD45-positive cells in the ALDH+ population indicated the absence of any contaminating hematopoietic (stem) cells (data not shown). The ALDH+ population was also enriched in cells

expressing ALB, AFP, and CK-7, -8, -14, -18, and -19, suggesting their bipotentiality (Fig. 2D). In addition, markers found to be expressed by a variety of LPCs isolated from different liver injury mouse models were also readily detected in the ALDH+ population (Connexin43, CD24, CD26, CD29, Claudin-3, and Integrin β4) (Fig. 2E). To test the overall efficiency of isolating LPCs using the ALDH strategy, we investigated whether this method could be improved by combining it with an established method like Percoll gradient enrichment of LPCs.20 Cells were isolated either by their ALDH activity, through a Percoll gradient, or by both methods combined (Fig. 3A,B). ALDH activity sorting resulted in a higher enrichment of CK19+, EpCAM+, CD133+, and E-cadherin+ cells compared with the Percoll gradient method. Moreover, ALDH activity could enrich for this population when applied subsequently to the Percoll gradient but did not result in a higher proportion of desired cells compared with the ALDH activity sorting method alone (Fig. 3C).