Original Article
Copyright ©2012 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Hepatol. Oct 27, 2012; 4(10): 274-283
Published online Oct 27, 2012. doi: 10.4254/wjh.v4.i10.274
Bone marrow progenitor cells do not contribute to liver fibrogenic cells
Bruno Diaz Paredes, Lanuza Alaby Pinheiro Faccioli, Luiz Fernando Quintanilha, Karina Dutra Asensi, Camila Zaverucha do Valle, Paulo César Canary, Christina Maeda Takiya, Antonio Carlos Campos de Carvalho, Regina Coeli dos Santos Goldenberg
Bruno Diaz Paredes, Lanuza Alaby Pinheiro Faccioli, Luiz Fernando Quintanilha, Karina Dutra Asensi, Camila Zaverucha do Valle, Christina Maeda Takiya, Antonio Carlos Campos de Carvalho, Regina Coeli dos Santos Goldenberg, Carlos Chagas Filho Biophysics Institute, Rio de Janeiro 21941-902, Brazil
Paulo César Canary, Radiology Department, Clementino Fraga Filho University Hospital , Rio de Janeiro 22909-538, Brazil
Author contributions: Paredes BD and Faccioli LAP contributed equally to this work and participated in the experimental design, performed flow cytometry, fluorescence assays, and analyzed the data; Quintanilha LF performed mouse liver injury and data analysis; Asensi KD performed bone marrow transplantation and data analysis; Zaverucha-do-Valle C performed confocal microscopy and data analysis; Canary PC performed mouse whole-body irradiation and data analysis; Takiya CM performed histology and data analysis; Campos-de-Carvalho AC participated in the experimental design and statistical analysis; Goldenberg RCS designed and supervised the study, execution, analysis and approved the final version; and all authors participated in manuscript preparation.
Supported by Brazilian Council for Scientific and Technological Development; Coordination for the Improvement of Higher Education Personnel; Rio de Janeiro State Research Supporting Foundation and Ministry of Health
Correspondence to: Regina Coeli dos Santos Goldenberg, PhD, Carlos Chagas Filho Biophysics Institute, Carlos Chagas Filho Avenue, 373, Health Sciences Center, Corridor G, 2nd floor/room 53-University City, Rio de Janeiro 21941-902, Brazil. rcoeli@biof.ufrj.br
Telephone: +55-21-25626559 Fax: +55-21-25626559
Received: December 6, 2011
Revised: October 24, 2012
Accepted: October 26, 2012
Published online: October 27, 2012

AIM: To investigate the contribution of bone marrow (BM) cells to hepatic fibrosis.

METHODS: To establish a model of chimerism, C57Bl/6 female mice were subjected to full-body irradiation (7 Gy) resulting in BM myeloablation. BM mononuclear cells obtained from male transgenic mice expressing enhanced green fluorescent protein (GFP) were used for reconstitution. Engraftment was confirmed by flow cytometry. To induce liver injury, chimeric animals received carbon tetrachloride (CCl4) 0.5 mL/kg intraperitoneally twice a week for 30 d (CCl4 30 d) and age-matched controls received saline (Saline 30 d). At the end of this period, animals were sacrificed for post mortem analysis. Liver samples were stained with hematoxylin and eosin to observe liver architectural changes and with Sirius red for collagen quantification by morphometric analysis. α-smooth muscle actin (α-SMA) was analyzed by confocal microscopy to identify GFP+ cells with myofibroblast (MF) characteristics. Liver tissue, BM and peripheral blood were collected and prepared for flow cytometric analysis using specific markers for detection of hepatic stellate cells (HSCs) and precursors from the BM.

RESULTS: Injury to the liver induced changes in the hepatic parenchymal architecture, as reflected by the presence of inflammatory infiltrate and an increase in collagen deposition (Saline 30 d = 11.10% ± 1.12% vs CCl4 30 d = 12.60% ± 0.73%, P = 0.0329). Confocal microscopy revealed increased reactivity against α-SMA in CCl4 30 d compared to Saline 30 d, but there was no co-localization with GFP+ cells, suggesting that cells from BM do not differentiate to MFs. Liver flow cytometric analysis showed a significant increase of CD45+/GFP+ cells in liver tissue (Saline 30 d = 3.2% ± 2.2% vs CCl4 30 d = 5.8% ± 1.3%, P = 0.0458), suggesting that this increase was due to inflammatory cell infiltration (neutrophils and monocytes). There was also a significant increase of common myeloid progenitor cells (CD117+/CD45+) in the livers of CCl4-treated animals (Saline 30 d = 2.16% ± 1.80% vs CCl4 30 d = 5.60% ± 1.30%, P = 0.0142). In addition the GFP-/CD38+/CD45- subpopulation was significantly increased in the CCl4 30 d group compared to the Saline 30 d group (17.5% ± 3.9% vs 9.3% ± 2.4%, P = 0.004), indicating that the increase in the activated HSC subpopulation was not of BM origin.

CONCLUSION: BM progenitor cells do not contribute to fibrosis, but there is a high recruitment of inflammatory cells that stimulates HSCs and MFs of liver origin.

Keywords: Bone marrow, Liver, Fibrosis, Progenitor cells, Chimeric mice, Green fluorescent protein+ cells