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Fibrogenic potential of human multipotent mesenchymal stromal cells in injured liver.

Baertschiger RM, Serre-Beinier V, Morel P, Bosco D, Peyrou M, Clément S, Sgroi A, Kaelin A, Buhler LH, Gonelle-Gispert C - PLoS ONE (2009)

Bottom Line: Expansion capacity of pediatric MSC was significantly higher when compared to adult MSC.In conclusion, when transplanted into an injured or regenerating liver, MSC differentiated into myofibroblasts with development of fibrous tissue, regardless of donor age.These results indicate that MSC in certain circumstances might be harmful due to their fibrogenic potential and this should be considered before potential use of MSC for cell therapy.

View Article: PubMed Central - PubMed

Affiliation: Surgical Research Unit, Department of Surgery, University Hospital Geneva, Geneva, Switzerland.

ABSTRACT
Multipotent mesenchymal stromal cells (MSC) are currently investigated clinically as cellular therapy for a variety of diseases. Differentiation of MSC toward endodermal lineages, including hepatocytes and their therapeutic effect on fibrosis has been described but remains controversial. Recent evidence attributed a fibrotic potential to MSC. As differentiation potential might be dependent of donor age, we studied MSC derived from adult and pediatric human bone marrow and their potential to differentiate into hepatocytes or myofibroblasts in vitro and in vivo. Following characterization, expanded adult and pediatric MSC were co-cultured with a human hepatoma cell line, Huh-7, in a hepatogenic differentiation medium containing Hepatocyte growth factor, Fibroblast growth factor 4 and oncostatin M. In vivo, MSC were transplanted into spleen or liver of NOD/SCID mice undergoing partial hepatectomy and retrorsine treatment. Expression of mesenchymal and hepatic markers was analyzed by RT-PCR, Western blot and immunohistochemistry. In vitro, adult and pediatric MSC expressed characteristic surface antigens of MSC. Expansion capacity of pediatric MSC was significantly higher when compared to adult MSC. In co-culture with Huh-7 cells in hepatogenic differentiation medium, albumin expression was more frequently detected in pediatric MSC (5/8 experiments) when compared to adult MSC (2/10 experiments). However, in such condition pediatric MSC expressed alpha smooth muscle more strongly than adult MSC. Stable engraftment in the liver was not achieved after intrasplenic injection of pediatric or adult MSC. After intrahepatic injection, MSC permanently remained in liver tissue, kept a mesenchymal morphology and expressed vimentin and alpha smooth muscle actin, but no hepatic markers. Further, MSC localization merges with collagen deposition in transplanted liver and no difference was observed using adult or pediatric MSC. In conclusion, when transplanted into an injured or regenerating liver, MSC differentiated into myofibroblasts with development of fibrous tissue, regardless of donor age. These results indicate that MSC in certain circumstances might be harmful due to their fibrogenic potential and this should be considered before potential use of MSC for cell therapy.

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Human albumin is not expressed in mouse liver engrafted with pediatric MSC and adult MSC.A) Staining with anti-human albumin Ab did not detect any albumin positive cells within mouse liver parenchyma (a,b,c). Scale bars indicate magnification. B) Total RNA was extracted from sham injected or transplanted mouse liver. Expression of human vimentin, human αFP and human albumin was analyzed by RT-PCR in several liver tissues after intra-hepatic transplantation with human MSC. Sham: sham injected mouse liver. C+: positive control for human vimentin was RT-PCR on MSC extract. Positive control for αFP and albumin was RT-PCR on human hepatocytes extract. C-: negative control without polymerase. Vimentin was expressed in liver tissue demonstrating engraftment of MSC. Human αFP and albumin were never detected. Each figure shows one representative result of several independent experiments (see table 1).
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pone-0006657-g006: Human albumin is not expressed in mouse liver engrafted with pediatric MSC and adult MSC.A) Staining with anti-human albumin Ab did not detect any albumin positive cells within mouse liver parenchyma (a,b,c). Scale bars indicate magnification. B) Total RNA was extracted from sham injected or transplanted mouse liver. Expression of human vimentin, human αFP and human albumin was analyzed by RT-PCR in several liver tissues after intra-hepatic transplantation with human MSC. Sham: sham injected mouse liver. C+: positive control for human vimentin was RT-PCR on MSC extract. Positive control for αFP and albumin was RT-PCR on human hepatocytes extract. C-: negative control without polymerase. Vimentin was expressed in liver tissue demonstrating engraftment of MSC. Human αFP and albumin were never detected. Each figure shows one representative result of several independent experiments (see table 1).

Mentions: In order to analyze effects of liver parenchyma on MSC, we injected aMSC and pMSC directly into liver parenchyma. Immunohistochemistry studies showed that pMSC were still present after 8 weeks (Fig. 5B). Extend of engraftment varied from a restricted localization near the border of the liver capsule to a diffuse engraftment in the whole liver lobe where cell injection had been performed (see Table 1: Summary of in vivo experiments). Using an anti-human albumin Ab, we analyzed liver sections at 3 levels. We did not detect human albumin within the liver at any stage after transplantation (Fig. 6A). In accordance to this, RT-PCR on liver samples showed that neither CK18, cytochrome P450 (CYP3A4) (data not shown), αFP, nor albumin but vimentin was expressed, demonstrating that MSC are present but differentiation into hepatocytes did not occur (Fig. 6B). The outcome was identical when hepatectomy was preceded by retrorsine treatment, a treatment blocking endogenous hepatocyte proliferation [24].


Fibrogenic potential of human multipotent mesenchymal stromal cells in injured liver.

Baertschiger RM, Serre-Beinier V, Morel P, Bosco D, Peyrou M, Clément S, Sgroi A, Kaelin A, Buhler LH, Gonelle-Gispert C - PLoS ONE (2009)

Human albumin is not expressed in mouse liver engrafted with pediatric MSC and adult MSC.A) Staining with anti-human albumin Ab did not detect any albumin positive cells within mouse liver parenchyma (a,b,c). Scale bars indicate magnification. B) Total RNA was extracted from sham injected or transplanted mouse liver. Expression of human vimentin, human αFP and human albumin was analyzed by RT-PCR in several liver tissues after intra-hepatic transplantation with human MSC. Sham: sham injected mouse liver. C+: positive control for human vimentin was RT-PCR on MSC extract. Positive control for αFP and albumin was RT-PCR on human hepatocytes extract. C-: negative control without polymerase. Vimentin was expressed in liver tissue demonstrating engraftment of MSC. Human αFP and albumin were never detected. Each figure shows one representative result of several independent experiments (see table 1).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2722022&req=5

pone-0006657-g006: Human albumin is not expressed in mouse liver engrafted with pediatric MSC and adult MSC.A) Staining with anti-human albumin Ab did not detect any albumin positive cells within mouse liver parenchyma (a,b,c). Scale bars indicate magnification. B) Total RNA was extracted from sham injected or transplanted mouse liver. Expression of human vimentin, human αFP and human albumin was analyzed by RT-PCR in several liver tissues after intra-hepatic transplantation with human MSC. Sham: sham injected mouse liver. C+: positive control for human vimentin was RT-PCR on MSC extract. Positive control for αFP and albumin was RT-PCR on human hepatocytes extract. C-: negative control without polymerase. Vimentin was expressed in liver tissue demonstrating engraftment of MSC. Human αFP and albumin were never detected. Each figure shows one representative result of several independent experiments (see table 1).
Mentions: In order to analyze effects of liver parenchyma on MSC, we injected aMSC and pMSC directly into liver parenchyma. Immunohistochemistry studies showed that pMSC were still present after 8 weeks (Fig. 5B). Extend of engraftment varied from a restricted localization near the border of the liver capsule to a diffuse engraftment in the whole liver lobe where cell injection had been performed (see Table 1: Summary of in vivo experiments). Using an anti-human albumin Ab, we analyzed liver sections at 3 levels. We did not detect human albumin within the liver at any stage after transplantation (Fig. 6A). In accordance to this, RT-PCR on liver samples showed that neither CK18, cytochrome P450 (CYP3A4) (data not shown), αFP, nor albumin but vimentin was expressed, demonstrating that MSC are present but differentiation into hepatocytes did not occur (Fig. 6B). The outcome was identical when hepatectomy was preceded by retrorsine treatment, a treatment blocking endogenous hepatocyte proliferation [24].

Bottom Line: Expansion capacity of pediatric MSC was significantly higher when compared to adult MSC.In conclusion, when transplanted into an injured or regenerating liver, MSC differentiated into myofibroblasts with development of fibrous tissue, regardless of donor age.These results indicate that MSC in certain circumstances might be harmful due to their fibrogenic potential and this should be considered before potential use of MSC for cell therapy.

View Article: PubMed Central - PubMed

Affiliation: Surgical Research Unit, Department of Surgery, University Hospital Geneva, Geneva, Switzerland.

ABSTRACT
Multipotent mesenchymal stromal cells (MSC) are currently investigated clinically as cellular therapy for a variety of diseases. Differentiation of MSC toward endodermal lineages, including hepatocytes and their therapeutic effect on fibrosis has been described but remains controversial. Recent evidence attributed a fibrotic potential to MSC. As differentiation potential might be dependent of donor age, we studied MSC derived from adult and pediatric human bone marrow and their potential to differentiate into hepatocytes or myofibroblasts in vitro and in vivo. Following characterization, expanded adult and pediatric MSC were co-cultured with a human hepatoma cell line, Huh-7, in a hepatogenic differentiation medium containing Hepatocyte growth factor, Fibroblast growth factor 4 and oncostatin M. In vivo, MSC were transplanted into spleen or liver of NOD/SCID mice undergoing partial hepatectomy and retrorsine treatment. Expression of mesenchymal and hepatic markers was analyzed by RT-PCR, Western blot and immunohistochemistry. In vitro, adult and pediatric MSC expressed characteristic surface antigens of MSC. Expansion capacity of pediatric MSC was significantly higher when compared to adult MSC. In co-culture with Huh-7 cells in hepatogenic differentiation medium, albumin expression was more frequently detected in pediatric MSC (5/8 experiments) when compared to adult MSC (2/10 experiments). However, in such condition pediatric MSC expressed alpha smooth muscle more strongly than adult MSC. Stable engraftment in the liver was not achieved after intrasplenic injection of pediatric or adult MSC. After intrahepatic injection, MSC permanently remained in liver tissue, kept a mesenchymal morphology and expressed vimentin and alpha smooth muscle actin, but no hepatic markers. Further, MSC localization merges with collagen deposition in transplanted liver and no difference was observed using adult or pediatric MSC. In conclusion, when transplanted into an injured or regenerating liver, MSC differentiated into myofibroblasts with development of fibrous tissue, regardless of donor age. These results indicate that MSC in certain circumstances might be harmful due to their fibrogenic potential and this should be considered before potential use of MSC for cell therapy.

Show MeSH
Related in: MedlinePlus