Article first published online: 7 JAN 2013
Copyright © 2012 American Association for the Study of Liver Diseases
Volume 57, Issue 1, pages 311–319, January 2013
How to Cite
Herrera, M. B., Fonsato, V., Bruno, S., Grange, C., Gilbo, N., Romagnoli, R., Tetta, C. and Camussi, G. (2013), Human liver stem cells improve liver injury in a model of fulminant liver failure. Hepatology, 57: 311–319. doi: 10.1002/hep.25986
Potential conflicts of interest: Drs. Fonsato and Herrera Sanchez received grants from Sis-Ter S.p.a. Drs. Camussi and Tetta is an employee of and received grants from Fresenius Medical Care.
Funded by Regione Piemonte, Piattaforme Biotecnologiche, Pi-Stem project, Converging Technologies NanoIGT.
- Issue published online: 7 JAN 2013
- Article first published online: 7 JAN 2013
- Accepted manuscript online: 24 JUL 2012 10:04AM EST
- Manuscript Accepted: 16 JUL 2012
- Manuscript Received: 15 DEC 2011
Additional Supporting Information may be found in the online version of this article.
|HEP_25986_sm_SuppFig1.tif||1138K||Supporting Information Figure 1. Liver functions and HLSCs localization in L.P mice with FLF. Alanine aminotransferase and aspartate aminotransferase were measured in serum of healthy SCID mice (Ctrl) and in FLF mice L.P. injected with 0.5 x 106 HLSCs and sacrificed 7, 14 or 21 days after injection. Data are expressed as mean ±SD U/L of five different SCID per group. Analysis of variance with Newman-Keuls multicomparison test was performed; *p<0.05 FLF mice L.P. injected with 0.5 x 106 HLSCs and sacrificed at 7 days vs Ctrl; #p<0.05 FLF mice L.P. injected with 0.5 x 106 HLSCs and sacrificed at 14 or 21 days vs FLF mice L.P. injected with 0.5 x 106 HLSCs and sacrificed at 7 days. (B) Quantification of CFSE+ cells per hpf (40x) in livers of healthy mice (white columns) and in liver of FLF mice L.P. injected with 0.5 x 106 HLSCs (black columns) and sacrificed 7 or 21 days after injection. (C) Quantification of fluorescence intensity, calculated in ROI, expressed as the mean of average radiance ±SD of mice L.P. injected with DiD labeled-HLSCs after induction of FLF (black bar). White bars are not treated control mice. Vehicle represent the intensity of liver of mice treated with PBS instead of HLSCs. Data are expressed as mean ±SD of 3 different SCID mice injected with HLSCs without FLF. (D) Representative fluorescence images of mice with or without FLF injury injected with DiD labeled-HLSCs and sacrificed after 24 hours 3 or 7 days. Abbreviations hours=h and days=d.|
|HEP_25986_sm_SuppFig2.tif||8413K||Supporting Information Figure 2. Detection of HLSCs in spleens and lungs after in vivo injection. Representative micrographs of spleen and lung paraffin section of SCID mice injected with 2x106 of CFSE labeled HLSCs and stained with anti-CFSE antibody (brown staining). A small number of HLSCs were detectable within spleen and lung after 24 hours and 7 days, and disappeared after 21 days. Original Magnification x250. Abbreviations hours=h and days=d.|
|HEP_25986_sm_SuppFig3.tif||5653K||Supporting Information Figure 3. In vitro effect of HLSCs-CM on murine hepatocytes. (A) Apoptosis was evaluated by Tunel assay on murine hepatocytes incubated with vehicle alone (white column) in the presence of GalN 5mM (black column) and in the presence of 29 μg/ml of concentrated HLSC-CM (grey column). (B) Proliferation evaluated by BrdU incorporation on murine hepatocytes incubated with vehicle alone (black column) or with different concentrations of concentrated HLSC-CM (29 μg/ml; 0.12-0.46-0.93 mg/ml) (grey columns). (C) Apoptosis performed on murine hepatocytes isolated from mice after 30 minutes from induction of FLF (white column) and cultured in the presence or absence 29 μg/ml of CM (black column). Results are expressed as mean ±SD of three different experiments performed in triplicate. Analyses of variance with Newmann-Keuls multicomparison test was performed; *p<0.05 hepatocytes incubated with GalN 5mM versus vehicle alone; #p<0.05 hepatocytes incubated with 29 μg/ml of concentrated HLSC-CM vs hepatocytes incubated with GalN 5mM. (A and C). (D) Proliferation evaluated by BrdU incorporation on murine hepatocytes isolated from mice after 30 minutes from induction of FLF cultured in the presence of vehicle alone (black columns) or with different concentration of concentrated HLSC-CM (grey columns). Results are expressed as mean ±SD of three different experiments performed in triplicate. Analyses of variance with Newmann-Keuls multicomparison test was performed; *p<0.05 hepatocytes incubated with 0.46; 0.93 mg/ml of CM vs hepatocytes incubated with vehicle alone (B and D).|
|HEP_25986_sm_SuppFig4.tif||5055K||Supporting Information Figure 4. Bioclarma assay. A multiplex biometric immunoassay, Bioclarma was used to quantify the amount of cytokines contained in HLSC-CM and in MSC-CM. Data are expressed as ng/ml and as mean ±SD of three different experiments performed in duplicate. A) High expressed cytokines and growth factors; B) low expressed cytokines and growth factors. Analysis of variance with Newman-Keuls multicomparison test was performed; *p<0.05 HLSC-CM vs MSC-CM; #p<0.05 MSC-CM vs HLSC-CM.|
|HEP_25986_sm_SuppFig5.tif||11997K||Supporting Information Figure 5. Characterization and potency of HLSC. (A) Representative confocal micrographs showing expression of several embryonic, mesenchymal stem cells and hepatic markers on HLSCs. (B) FACS analysis of HLSC showing the expression of CD105, CD73, CD29 and albumin. (C) Urea production by HLSCs in RCCS at different time-points was evaluated and compared with that of HLSCs cultured in adhesion with growth factors. Data are expressed as mg/dL and as mean ±SD of three different experiments. ANOVA was performed; *p<0.05 HLSCs cultured in RCCS vs HLSCs cultured in adhesion.|
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.