Integrin and Extracellular Matrix Interactions Regulate Engraftment of Transplanted Hepatocytes in the Rat Liver  Vinay Kumaran, Brigid Joseph, Daniel Benten, Sanjeev Gupta  Gastroenterology  Volume 129, Issue 5, Pages 1643-1653 (November 2005) DOI: 10.1053/j.gastro.2005.08.006 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Attachment of primary hepatocytes was ECM and integrin dependent. Attachment of hepatocytes to collagen or fibronectin-like polymer (FLP)-coated dishes was 3.8- ± 1.3-fold and 4.3- ± 0.3-fold greater, respectively, than on plastic. Moreover, prior incubation of hepatocytes with RGD peptide abolished cell attachment as shown in dishes coated with FLP, indicating capacity for ECM interactions. Asterisks, P < .001, t tests. The experiment was reproduced twice. Gastroenterology 2005 129, 1643-1653DOI: (10.1053/j.gastro.2005.08.006) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Engraftment of ECM-bound transplanted cells in DPPIV− rat liver. Shown is engraftment of cells 4 days following cell transplantation. (A) Saline-treated control hepatocytes (arrows) adjacent to a periportal area. (B) A recipient of cells incubated with 8 μg/mL laminin with transplanted cells in a periportal area, similar to the previous panel. (C) Cells incubated with 45 μg/mL collagen showing a large transplanted cell cluster (arrow). (D) Transplanted cells incubated with 6.6 μg/mL FLP showing more cells in a periportal area (arrows), although cells were additionally in clusters. Transplanted cells were identified by DPPIV histochemistry. Original magnification, ×200; hematoxylin counterstain. Gastroenterology 2005 129, 1643-1653DOI: (10.1053/j.gastro.2005.08.006) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Effect of ECM infusion on cell engraftment in the liver of DPPIV− rats. (A) Shows transplanted cells (arrows) adjacent to a portal area (Pa) in a control rat with only saline treatment. (B) A cell recipient given 9 μg collagen with transplanted cells predominantly in portal spaces (arrowheads), with some cells in the parenchyma (arrow). (C and D) Cell recipients given 1.3 μg FLP showing multiple transplanted cells in parenchyma (C, arrows) and vascular spaces (D, arrowhead). (E) Morphometric analysis showing transplanted cell numbers in various engraftment conditions. Asterisks indicate P < .05 vs controls. Cv, central vein. Original magnification, ×100; hematoxylin counterstain. Gastroenterology 2005 129, 1643-1653DOI: (10.1053/j.gastro.2005.08.006) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Identification of FAC in hepatocytes. (A and B) FAC in hepatocytes cultured for 2 hours on cell culture plastic (A) or FLP-coated dishes. Note dot-like structures, crescents, and annular shapes of FAC (dark color, arrowheads), which were more frequent in FLP-coated dishes (B). (C and D) Localization of transplanted cells (DPPIV+, red, arrows and asterisks) and FAC (arrowheads) in saline-treated control and FLP-treated rats 2 hours after cell transplantation. Transplanted cells showed FAC, which were more prevalent in FLP-treated livers. Inset shows a magnified view of transplanted cells with linear and dot-like FAC. Original magnification, A and B ×200; C and D, ×400; inset, ×1000. Gastroenterology 2005 129, 1643-1653DOI: (10.1053/j.gastro.2005.08.006) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Morphometric analysis showing differences in hepatocyte engraftment after integrin-receptor binding with RGD peptide. Data are normalized against transplantation of control hepatocytes. Transplantation of RGD-treated cells in unmanipulated animals showed a trend for lower cell engraftment. In rats given FLP followed by transplantation of RGD-treated cells, engraftment of cells was approximately 4-fold lower. Asterisks, P < .05. Gastroenterology 2005 129, 1643-1653DOI: (10.1053/j.gastro.2005.08.006) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Liver repopulation in animals conditioned with retrorsine and partial hepatectomy. (A) Shows a control animal without ECM treatment with scattered foci of transplanted hepatocytes. (B) Recipient of 9 μg collagen showing similar findings with no change in liver repopulation. (C and D) Recipient of 1.3 μg FLP with far more transplanted cell foci (C), along with larger transplanted cell foci. Original magnification, A, B, and D, ×100; C, ×40; hematoxylin counterstain. Gastroenterology 2005 129, 1643-1653DOI: (10.1053/j.gastro.2005.08.006) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 Molecular analysis of liver repopulation in DPPIV− rats. (A) Shows Nde1 restriction enzyme digests in mixtures of DPPIV cDNA with 100% DPPIV− rat liver in lane 2 and 100% DPPIV+ F344 rat liver in lane 7, as indicated. Nde1 enzyme treatment cleaved the 568-bp DPPIV cDNA fragment into 379- and 190-bp fragments, although the samples were not digested completely as seen in lanes 3–7. MW, molecular weight marker. (B) Shows densitometric analysis of the 3 bands in (A), indicating that the assay was capable of detecting greater than 10% liver repopulation with the combined density of bands 379 and 190. (C) Analysis of liver samples from retrorsine-PH conditioned rats following cell transplantation with Nde1 digests. Lanes 2–5 show control rats treated with only saline infusion followed by cells. In these animals, Nde1 digestion did not produce cDNA fragments, indicating that liver repopulation was <10%. Lanes 6–9 show animals treated with FLP infusion prior to cell transplantation, which resulted in the appearance of Nde1 digests in 2 of 4 rats (lanes 8 and 9), and densitometric scanning indicated >10% liver repopulation. Gastroenterology 2005 129, 1643-1653DOI: (10.1053/j.gastro.2005.08.006) Copyright © 2005 American Gastroenterological Association Terms and Conditions