Volume 134, Issue 3, Pages 823-832 (March 2008) Purification of Fetal Liver Stem/Progenitor Cells Containing all the Repopulation Potential for Normal Adult Rat Liver  Michael Oertel, Anuradha Menthena, Yuan–Qing Chen, Børge Teisner, Charlotte Harken Jensen, David A. Shafritz  Gastroenterology  Volume 134, Issue 3, Pages 823-832 (March 2008) DOI: 10.1053/j.gastro.2008.01.007 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Enrichment of rat ED14 fetal liver stem/progenitor cells. Magnetic bead sorting (MACS) was used to select Dlk-1+ cells. Unfractionated fetal liver cells contained 5.7% Dlk-1+ cells (A); after enrichment with magnetic microbeads, this increased to 94.9% (B). Panel C shows Dlk-1+ cells at higher magnification. Cells were immunostained for Dlk-1 (red). DAPI was used for staining of nuclei (blue). Data are shown as merged images. Original magnification, 200× (A and B), 900× (C). (D) RT-PCR analysis for Dlk-1 in unfractionated (lane 1), isolated Dlk-1+ (lane 2) and Dlk-1− (lane 3) fetal liver cells, and adult liver (lane 4). GAPDH was used as internal standard. FACS analysis for Dlk-1 in the Dlk-1+ (E) and Dlk-1− (F) cell fractions. Elimination of 99.5% autofluorescence of the Dlk-1+ and Dlk-1− fractions was used to set gates for determining positively stained cells in each fraction. Similar results were obtained using unfractionated fetal liver cells incubated with isotype-matched irrelevant antibody as a control. Gastroenterology 2008 134, 823-832DOI: (10.1053/j.gastro.2008.01.007) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Expression of epithelial, stem/progenitor, and hematopoietic cell markers in the Dlk-1+ and Dlk-1− fractions after cell sorting. ED14 fetal liver cells were isolated and enriched using anti-Dlk-1 magnetic microbeads. (A–C) Dlk-1+ (A and B) and Dlk-1− (C) liver cell fractions were characterized by immunocytochemical analysis for Dlk-1 (red fluorescence) and AFP expression (green fluorescence). Only single Dlk-1+ cells within the Dlk-1+ cell fraction were negative for AFP (inset, B), and none of the Dlk-1− cells were positive for AFP (C). (D–F) Double immunocytochemistry for Dlk-1 (green) and CK-19 (red) within the Dlk-1+ (D and E) and Dlk-1− (F) fetal liver cells. (G–J) Simultaneous detection for Dlk-1 (G), CK-19 (H), and AFP (I) in the Dlk-1+ cell fraction shows that all Dlk-1-expressing CK-19+ cells coexpressed AFP (J, merged). (K–M) Simultaneous detection for Dlk-1 (green) and E-Cadherin (red) in the Dlk-1+ (K and L) and Dlk-1− (M) fractions. (N–W) Dlk-1+ and Dlk-1− cells were costained for Dlk-1/EpCAM (N and O), Dlk-1/Vimentin (P and Q), Dlk-1/Nestin (R and S), Dlk-1/Thy-1 (T and U), and Dlk-1/CD45 (V and W). DAPI was used for nuclear staining (blue). Data are shown as merged images. Original magnification, 200× (A, D, K), 900× (B, C, E, F, G–J, L, M, N–W). Gastroenterology 2008 134, 823-832DOI: (10.1053/j.gastro.2008.01.007) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Gene expression profile of Dlk-1+ and Dlk-1− fetal liver cells. Using RT-PCR analysis, mRNA from unfractionated ED14 fetal liver cells (lane 1) was compared with mRNA from Dlk-1+ cells (lane 2), from Dlk-1− cells (lane 3), and from adult liver (lane 4). Gastroenterology 2008 134, 823-832DOI: (10.1053/j.gastro.2008.01.007) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Comparison between Dlk-1+ and Dlk-1− fetal liver cells in culture. After enrichment with anti-Dlk-1 magnetic microbeads, 0.1 × 106 Dlk-1+ and 1.7 × 106 Dlk-1− cells were plated on gelatin-coated, 2-well chamber slides and cultured for up to 5 days. (A–F) Dlk-1+ fetal liver cells were cultured for 2 days (A–C) and 5 days (D–F). (G–I) Dlk-1− fetal liver cells were cultured for 2 days. A small epithelial cell colony is highlighted by an arrow (H). (J–L) Dlk-1+ (J and K) and Dlk-1− (L) cells were cultured for up to 2 days, followed by immunocytochemistry for E-Cadherin. (C, F, I, J, K, L) Nuclei were identified with DAPI (blue fluorescence). Data are shown as merged images. Original magnification ×40 (A, D, G), ×100 (B, E, H), 200× (J, L), 400× (C, F, I, K). Gastroenterology 2008 134, 823-832DOI: (10.1053/j.gastro.2008.01.007) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 Proliferation and phenotypic characterization of Dlk-1+ cells in vitro. After enrichment by magnetic microbeads, Dlk-1+ cells were cultured for up to 5 days after cell plating; cells were released from chamber slides, using Trypsin/EDTA digestion, and fixed to cytospin slides. (A–D) Dlk-1+ cells were stained for Ki-67 on days 2 (A and C) and 5 (B and D) in culture. (E–L) Immunocytochemistry for AFP (E–H) and CK-19 (I–L) on days 2 (E, G, I, K) and 5 (F, H, J, L) after cell plating. (M–R) Immunocytochemistry for EpCAM (M and N), Vimentin (O and P), and Nestin (Q and R) on day 5 in cell culture. Nuclei were identified with DAPI (blue). Data are shown as merged images. Original magnification 200× (E, F, I, J, M, O, Q), 400× (A and B), 600× (G, H, K, L, N, P, R), and 900× (C and D). Gastroenterology 2008 134, 823-832DOI: (10.1053/j.gastro.2008.01.007) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Repopulation of normal rat liver by anti-Dlk-1 magnetic microbead selected ED14 fetal liver cells. Equal numbers of Dlk-1+ (A and C) and Dlk-1− ED14 (B and D) fetal liver cells from DPPIV+ F344 rats were transplanted into DPPIV− F344 rats in conjunction with two-thirds PH (0.9 × 106 cells, A and B; 1.9 × 106 cells, C and D). (A–D) Whole liver sections from animals killed at 6 months after cell transplantation. (E and F) Selected areas from recipient livers at 6 months after cell transplantation at higher magnifications show transplanted cells differentiating into hepatocytes and mature bile duct structures. Original magnification, 100× (E) and 400× (F). Gastroenterology 2008 134, 823-832DOI: (10.1053/j.gastro.2008.01.007) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Functional incorporation of transplanted Dlk-1+ ED14 fetal liver stem/progenitor cells into the hepatic parenchyma. After purification by MACS, 0.9–1.9 × 106 Dlk-1+ fetal liver cells were transplanted into DPPIV− rats in conjunction with two-thirds PH; animals were killed at 6 months. Specific areas on serial tissue sections A and C, where DDPIV+ cells have proliferated and replaced host hepatocytes (B), are outlined by dashed lines. In these serial sections, DPPIV+ cell clusters (B) showed normal glycogen storage (A) and normal expression of glucose-6-phosphatase (G6P) (C) at all locations within the hepatic lobule. Original magnification, 100×. (D–F) Double-label immunohistochemistry for DPPIV and albumin (Alb) (D), DPPIV/asialoglycoprotein receptor (ASGPR) (E), and DPPIV/UDP-glucuronosyl transferase (UGT1A1) (F) shows that transplanted Dlk-1+ fetal liver cells differentiate into mature hepatocytes and express unique hepatocyte-specific proteins. Panels G–L show differentiation of transplanted DPPIV+ cells into mature bile duct structures expressing DPPIV/CK-19 (G), DPPIV/OV-6 (H), DPPIV/EpCAM (I), DPPIV/CD44 (J), DPPIV/Connexin 43 (K), and DPPIV/Claudin-7 (L). Nuclei were identified by DAPI (blue fluorescence). Data shown as merged images. Original magnification, 400×. Gastroenterology 2008 134, 823-832DOI: (10.1053/j.gastro.2008.01.007) Copyright © 2008 AGA Institute Terms and Conditions

Figure 8 Distribution and phenotypic characterization of DPPIV+ cells in different organs, derived from ED14 fetal liver cells transplanted into normal adult rat liver. (A–F) Six × 107 unfractionated fetal liver cells were transplanted by infusion through the portal vein in conjunction with PH. Sections from the lung were taken at 6 months after cell transplantation and analyzed using combined enzyme histochemistry for detection of DPPIV (A, C, E; red) and immunohistochemistry for detection of CD45, Thy-1, or CK-19 (B, D, F; red fluorescence), all shown as merged images with nuclear staining with DAPI (blue). Black and white arrows point to cells positive for DPPIV and show coexpression of DPPIV+ cells with CD45+ and rarely with Thy-1+ but not with CK-19+ cells. (G and H) At 6 months after transplantation of 32 × 106 Dlk-1− cells, no liver repopulation was observed in recipients; however, substantial numbers of hematopoietic DPPIV+ cells were detected in bone marrow (G) and spleen (H). Nuclei were counterstained with hematoxylin. Original magnification, 400×. Gastroenterology 2008 134, 823-832DOI: (10.1053/j.gastro.2008.01.007) Copyright © 2008 AGA Institute Terms and Conditions