Foxl1-Cre-marked adult hepatic progenitors have clonogenic and bilineage differentiation potential

Abstract

Isolation of hepatic progenitor cells is a promising approach for cell replacement therapy of chronic liver disease. The winged helix transcription factor Foxl1 is a marker for progenitor cells and their descendants in the mouse liver in vivo. Here, we purify progenitor cells from Foxl1-Cre; RosaYFP mice and evaluate their proliferative and differentiation potential in vitro. Treatment of Foxl1-Cre; RosaYFP mice with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet led to an increase of the percentage of YFP-labeled Foxl1(+) cells. Clonogenic assays demonstrated that up to 3.6% of Foxl1(+) cells had proliferative potential. Foxl1(+) cells differentiated into cholangiocytes and hepatocytes in vitro, depending on the culture condition employed. Microarray analyses indicated that Foxl1(+) cells express stem cell markers such as Prom1 as well as differentiation markers such as Ck19 and Hnf4a. Thus, the Foxl1-Cre; RosaYFP model allows for easy isolation of adult hepatic progenitor cells that can be expanded and differentiated in culture.

Figure 1.

Labeling of the Foxl1-Cre lineage in RosaYFP mice. YFP⁺ cells are present within the periportal region in an injured mouse liver.

Figure 2.

YFP⁺ cell number increases after treatment with a DDC-supplemented diet. (A) FACS data of nonparenchymal cells from the liver of Foxl1-Cre; RosaYFP mice fed chow or DDC diets. CD45 is a marker for hematopoietic cells. (Rectangle) CD45⁻/YFP⁺ fraction. (B) A time-dependent increase of CD45⁻/YFP⁺ cells is shown. Data are represented as mean ± SEM.

Figure 3.

Foxl1⁺ cells are capable of self-renewal. (A) YFP⁺ cells are enriched for Foxl1. mRNA levels of Foxl1 are shown; (*) P < 0.05, liver versis YFP⁺; (^) P < 0.05, YFP⁻ versus YFP⁺ (n = 4). (B) Percent of colonies over total number of cells. (*) P < 0.05 (n = 6). Data are represented as mean ± SEM. (C) YFP⁺ and YFP⁻ cells from Foxl1-Cre; RosaYFP mice were cultured for 7 d. A representative colony is shown.

Figure 4.

Foxl1⁺ cells are capable of differentiating into cholangiocytes and hepatocytes. (A) Foxl1⁺ cells differentiate into duct-like cells when stimulated by collagen type 1A and TNFα. (B) Immunofluorescence staining of duct-like cells for CK19 (red) and nuclei (blue). (C,D) Periodic acid–Schiff staining of untreated cells (C) and cells differentiated into hepatocytes for 21 d (D). (E) mRNA levels of hepatocyte markers. Foxl1⁺ cells were treated with Matrigel and oncostatin M for 7 d (n = 4). (L) Liver; (U) undifferentiated; (T) differentiated. Data are represented as mean ± SEM. (*) P < 0.05, untreated versus treated. (F) Number of clonal cell lines that have bilineage potential.

Figure 5.

Microarray analyses of Foxl1⁺ and Foxl1⁻ cells. Foxl1-Cre; RosaYFP mice were fed a chow diet (day 0) or a DDC diet for 3, 7, and 14 d (n = 4). Expression levels are shown relative to day 0 nonparenchymal cells. (Closed triangle, solid line) YFP⁺; (open circle, dotted line) YFP⁻; (*) false discovery rate (FDR) < 20, versus day 0.

Figure 6.

Foxl1-Cre; YFP⁺ cells are a subset of Sox9⁺ cells. Representative images from the liver of a Foxl1-Cre; RosaYFP mouse fed a DDC diet for 7 d are shown.