Volume 119, Issue 6, Pages 1720-1730 (December 2000) Correction of liver disease by hepatocyte transplantation in a mouse model of progressive familial intrahepatic cholestasis  J.Marleen L. De Vree, *, Roel Ottenhoff, *, Piter J. Bosma, *, Alexander J. Smith, *,§, Jan Aten, ‡, Ronald P.J. Oude Elferink, *  Gastroenterology  Volume 119, Issue 6, Pages 1720-1730 (December 2000) DOI: 10.1053/gast.2000.20222 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) Patient with PFIC type 3 (cryosection stained for Ki67); (B) mdr2(−/−) mouse on a cholate-supplemented diet (paraffin-embedded section, stained for PCNA). The arrows indicate proliferating hepatocytes. (C–H) Immunohistochemical detection of MDR3-expressing hepatocytes in mdr2(−/−) mice using P3II26, a monoclonal antibody against MDR3 P-gp. (C) One week after transplantation (arrows indicate MDR3-expressing hepatocytes); (D) 8 weeks after transplantation on a cholate-supplemented diet; (E) 16 weeks after transplantation on a control diet; (F) 16 weeks after transplantation on a cholate-supplemented diet; (G) 50 weeks after transplantation on control diet; (H) 50 weeks after transplantation on a cholate-supplemented diet. (Original magnifications: 50× [C]; 25× [A, B, D, F]; 16× [E, G, H].) Gastroenterology 2000 119, 1720-1730DOI: (10.1053/gast.2000.20222) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Lipid secretion after hepatocyte transplantation and repopulation in mdr2(−/−) mice. Mice were transplanted with MDR3-expressing hepatocytes and fed (A–C) a control diet or (D–F) a cholate-supplemented diet. ●, Data from transplanted mdr2(−/−) mice; ○, the same parameter in mdr2(+/+) fed the same diet for 6 weeks. (A and D) Phospholipid secretion (absolute values); (B and E) cholesterol secretion (absolute values); (C and F) phospholipid/bile salt ratio. The data represent means ± SD and are derived from 5, 3, 4, 4, and 6 animals in the control diet group after 1, 4, 8, 16, and 52 weeks, respectively, and from 4 animals at each time point in the cholate-fed group. Gastroenterology 2000 119, 1720-1730DOI: (10.1053/gast.2000.20222) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Number of MDR3-expressing cells in mdr2(−/−) livers at various times after transplantation. Frozen sections were prepared from 4 different lobes of each mouse and stained with P3II26. The number of MDR3-expressing cells in each positive field was counted. ▿, Animals on a control diet; ▾, animals on a cholate-supplemented diet. The data represent means ± SD are derived from the same numbers of animals as in Figure 2. Gastroenterology 2000 119, 1720-1730DOI: (10.1053/gast.2000.20222) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 The relationship between the number of MDR3-expressing hepatocytes per positive field (the mean of all counted fields in 4 lobes per mouse; see Figure 3) and the biliary phospholipid secretion (see Figure 2) for each mouse. Data are from individual mice on a control diet (▿) or a cholate-supplemented diet (▾). The correlation coefficient (r2) for the combined data was 0.832 (n = 39), which is highly significant (P < 0.001). Gastroenterology 2000 119, 1720-1730DOI: (10.1053/gast.2000.20222) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Parameters of liver pathology at various time points after transplantation of MDR3-expressing hepatocytes in mdr2(−/−) mice. (A and B) Histopathologic score in mdr2(−/−) mice at different time points after hepatocyte transplantation on control and cholate-supplemented diets. The scoring was performed as described in Materials and Methods and a previous report.16 A maximum score of 15 can be reached, indicating strong pathology. (A) Mice on a control diet; (B) mice on a cholate-supplemented diet. (C and D) Serum alkaline phosphatase activity in transplanted mdr2(−/−) mice. (C) Mice on a control diet; (D) mice on a cholate-supplemented diet. Note that the ordinate has a logarithmic scale. Closed symbols represent transplanted mdr2(−/−) mice; open symbols represent untransplanted mdr2(−/−) mice kept under identical conditions. Gastroenterology 2000 119, 1720-1730DOI: (10.1053/gast.2000.20222) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Liver histology 1 year after hepatocyte transplantation in (A and C) a transplanted mdr2(−/−) mouse on a control diet and (B and D) a transplanted mouse on a cholate-supplemented diet. (A and B) Immunohistochemical detection of proliferating hepatocytes by PCNA staining (original magnification 25×). (C and D) Liver histology 1 year after hepatocyte transplantation (H&E staining; original magnification 25×). Reversal of liver pathology was observed in transplanted mice on a control diet (C), in contrast to the sustained liver pathology in mice on a cholate-supplemented diet (D). Gastroenterology 2000 119, 1720-1730DOI: (10.1053/gast.2000.20222) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Immunohistochemical detection of transplanted mdr2(+/+) hepatocytes. Mdr2(−/−) mice were transplanted with normal mdr2(+/+) hepatocytes and fed a control diet for 6 months. Liver tissue was stained with REG3, a polyclonal antibody against mdr2 P-gp (original magnification 33×). Gastroenterology 2000 119, 1720-1730DOI: (10.1053/gast.2000.20222) Copyright © 2000 American Gastroenterological Association Terms and Conditions