Volume 45, Issue 1, Pages 144-159 (July 2006) Present status and perspectives of cell-based therapies for liver diseases  Andreas Nussler, Sarah Konig, Michael Ott, Etienne Sokal, Bruno Christ, Wolfgang Thasler, Marc Brulport, Geredn Gabelein, Wiebke Schormann, Maren Schulze, Ewa Ellis, Matthias Kraemer, Frank Nocken, Wolfgang Fleig, Michael Manns, Steven C. Strom, Jan G. Hengstler  Journal of Hepatology  Volume 45, Issue 1, Pages 144-159 (July 2006) DOI: 10.1016/j.jhep.2006.04.002 Copyright © 2006 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Transplantation of adult hepatocytes and liver repopulation. Primary hepatocytes were injected into the portal vein of dipeptidylpeptidase IV (DPPIV) deficient rats pre-treated with retrorsine and subjected to 30% partial hepatectomy to ensure selective donor growth. Donor cell integration and repopulation was studied by co-localising transplanted cells (DPPIV-positive=red) with hepatic gap junctions (Connexin-32=green) to visualize the intact hepatic architecture, cell nuclei (DAPI=blue). Multilayer immunofluorescence imaging, magnification as indicated). (A) Shortly after transplantation (4h), donor cells form microemboli in the distal branches of the portal vein and translocate further to the sinusoids. (B) From day 3 onwards, donor cells are fully integrated and display re-established gap junctions with neighbouring cells. (C) Donor cells and descendents form clusters and grow out from the periportal areas (21 days). (D) Two months following transplantation, donor cell-derived cell clusters are nearly confluent and range up to 200 cells in number. The integration process is remarkably homogeneous and the architecture of the parenchyma is perfectly maintained without any sign of displacement. Journal of Hepatology 2006 45, 144-159DOI: (10.1016/j.jhep.2006.04.002) Copyright © 2006 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Morphology of extrahepatic stem cells after transplantation into livers of immunodeficient mice from published studies. (A) Danet et al. [13]: transplantation of purified human Lin−CD38−CD34−or+C1qRp+ cells isolated from human umbilical cord blood. Livers from transplanted mice were recovered 8–10 weeks posttransplant and immunostained for human albumin. Bar: 10μm. (B) Newsome et al. [56]: transplantation of unsorted mononuclear cell preparations of human cord blood and immunostaining for HepPar1. Bar: 20μm. (C) Wang et al. [76]: transplantation of CD34+ cells from human umbilical cord blood and immunostaining for human albumin. Magnification: 100×. (D) Kollet et al. [39]: transplantation of CD34+ cells isolated from human cord blood. Immunostaining for human albumin. Magnification: 100×. (E) Kakinuma et al. [33]: transplantation of adherently proliferating cells isolated from human cord blood. Immunostaining for HepPar1. Bar: 10μm. (F) Von Mach et al. [74]: transplantation of nestin-positive islet-derived adherently proliferating cells. Immunostaining for human albumin. Magnification: 400×. (G) Von Mach et al. [74]: transplantation of nestin-positive islet-derived adherently proliferating cells. Immunostaining for human (red) and mouse (green) albumin. Magnification: 630-fold. (H) Ruhnke et al. [63]: transplantation of primary human hepatocytes (left side) and human blood monocyte derived hepatocyte-like cells (right side). Immunostaining for albumin. Bar: 50μm. (I) Sharma et al. [64]: transplantation of unsorted human cord blood cells. Immunostaining for albumin. Magnification: 40×. Journal of Hepatology 2006 45, 144-159DOI: (10.1016/j.jhep.2006.04.002) Copyright © 2006 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Heterogeneity of human albumin positive cell types. Adherently proliferating cord blood cells derived from a single colony were directly injected into the left liver lobe of SCID/NOD mice followed by immunohistochemistry visualizing human albumin 3 weeks after transplantation. (A) Human albumin positive cells with hepatocyte-like morphology. Magnification: 200×. (B) Human albumin positive cells that do not show a hepatocyte like morphology, but rather resemble monocytes Magnification: 400×. (Hengstler and Brulport, unpublished data). Journal of Hepatology 2006 45, 144-159DOI: (10.1016/j.jhep.2006.04.002) Copyright © 2006 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Milestones in defining hepatocyte-like cells: (A) Marking of cells before transplantation. In this example human fetal hepatocytes have been marked by red fluorescent nanoparticles. (B–D) Identification of cells of human origin in mouse livers by in situ hybridization. (B) SCID/NOD mouse liver tissue after in situ hybridization with mouse major satellite probes (visualized by pink fluorescence). (C) Human liver tissue after in situ hybridization with alu-probes (visualized by green fluorescence). (D) Visualization of human hepatocytes after transplantation into the liver of a SCID/NOD mouse by combined in situ hybridization with alu- and mouse major satellite probes [80]. (E) Double immunohistochemistry for human (red) and mouse (green) albumin, demonstrating that the transplanted cell expresses human but not mouse albumin. (F) Confirmation of the human origin of a human albumin expressing cell. Human albumin is immunohistochemically detected (visualized by red fluorescence), whereas human DNA is identified by in situ hybridization with alu-probes (visualized by green fluorescence). Blue fluorescence: nuclear staining with DAPI. Magnification: A,E,F: 630×; D: 200×. Journal of Hepatology 2006 45, 144-159DOI: (10.1016/j.jhep.2006.04.002) Copyright © 2006 European Association for the Study of the Liver Terms and Conditions