Volume 126, Issue 4, Pages 955-963 (April 2004) A significant proportion of myofibroblasts are of bone marrow origin in human liver fibrosis  Stuart J. Forbes, Francesco P. Russo, Virginia Rey, Patrizia Burra, Massimo Rugge, Nicholas A. Wright, Malcolm R. Alison  Gastroenterology  Volume 126, Issue 4, Pages 955-963 (April 2004) DOI: 10.1053/j.gastro.2004.02.025

Figure 1 (A) Female cirrhotic control liver immunostained with α-SMA and direct in situ hybridization for the Y chromosome. Myofibroblast cells are positive for α-SMA (red); no Y chromosomes are visualized. (B) Male cirrhotic control liver immunostained with α-SMA and direct in situ hybridization for the Y chromosome. Myofibroblast cells are positive for α-SMA (red); Y-probe staining is positive in many cells both in fibrotic and nonfibrotic areas (green, arrow). (C) Cirrhotic liver from a male patient with hepatitis C, B, and D who received a liver transplant from a female donor and subsequently developed recurrent hepatitis and cirrhosis. Indirect in situ hybridization for the Y chromosome (brown dot at the edge of the nucleus, arrow) is combined with immunohistochemistry for α-SMA (red); Y chromosome—positive myofibroblasts are seen within a band of cirrhosis (arrow). (D) Liver biopsy specimen from a male patient with hepatitis B who received a liver transplant from a female donor and subsequently developed recurrent hepatitis B and fibrosis. Direct in situ hybridization for the Y chromosome (green fluorescent spot at the edge of the nucleus, arrow) is combined with immunostaining for α-SMA (red). (E) Cirrhotic band within a liver from the female patient who received a bone marrow transplant from a male donor 10 years earlier and subsequently developed hepatitis C-induced cirrhosis. The section is immunostained with α-SMA and direct in situ hybridization for the Y chromosome. Cells of a myofibroblast phenotype are positive for α-SMA; Y-probe staining is positive in some of the α-SMA—positive cells (arrows). (F) Cirrhotic liver from the female patient who received a bone marrow transplant from a male donor immunostained for vimentin and direct in situ hybridization for the Y chromosome. Fibrotic cells are positive for vimentin; Y-probe staining is positive in both vimentin-positive (arrows) and vimentin-negative (asterisk) cells. (G) Cirrhotic liver from the female patient who received a bone marrow transplant from a male donor immunostained for fibulin-2 and direct in situ hybridization for the Y chromosome. Y-probe staining is positive in a proportion of the fibulin-2—positive cells within the collagen bands (arrows). (H) Cirrhotic liver from a male patient with hepatitis B who received a liver transplant from a female donor and developed recurrent hepatitis. The section is immunostained for CD45 (red) and direct in situ hybridization for the Y chromosome (green). Inflammatory cells are positive for CD45. CD45 staining is positive in some Y-probe—positive cells (asterisk) but negative in a proportion of the Y-probe—positive cells (arrows). (Original magnification: A and B, 40×; C—H, 60×.) Gastroenterology 2004 126, 955-963DOI: (10.1053/j.gastro.2004.02.025)

Figure 2 Validation of combined immunostaining for CD45 and Y chromosome detection byin situ hybridization. (A and B) CD45 immunoreactivity was present in splenocytes in both female and male spleens, respectively, but only Y-probe—positive cells (green dots) were seen in the male. (C and D) Intestinal mucosal cells bordering the crypt lumina (L) were CD45 negative in both female and male specimens, respectively; however, as expected, there were occasional CD45-positive cells in the adjacent lamina propria (red); only Y-probe positive cells (green dots) were seen only in the male. Gastroenterology 2004 126, 955-963DOI: (10.1053/j.gastro.2004.02.025)

Figure 3 (A) Representative image series from confocal microscopy of the liver from the female patient who received a bone marrow transplant from a male donor and then developedhepatitis C—induced cirrhosis. The Y chromosome (green, asterisk) is appropriately located at the edge of the nucleus in the cell that is immunoreactive for fibulin-2 (red). This shows that the Y chromosome “belongs” to the fibulin-2—positive cell and not related to an adjacent inflammatory cell. (B) A confocal image of the liver from the female patient who received a bone marrow transplant from a male donor and then developed hepatitis C. Double immunostaining for CD45 (red) and fibulin-2 (blue) has been performed followed by in situ hybridization for the Y chromosome (green). The nuclei are not colored in this section by 4′,6-diamidino-2-phenylindole staining, because this would obscure the fibulin immunopositivity. A fibulin-2—positive cell can be seen that is also positive for the Y chromosome at the edge of the nuclear space (arrow). A CD45-positive cell (asterisk) is seen in the same section. Gastroenterology 2004 126, 955-963DOI: (10.1053/j.gastro.2004.02.025)