Fibrogenic potential of human multipotent mesenchymal stromal cells in injured liver

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.

Figure 1. Characterization of adult and pediatric multipotent mesenchymal stromal cells.

A) Fibroblast-like morphology of adult (a) and pediatric (b) MSC. Both cell types showed similar morphology. B) Flow cytometry of cells stained for CD34, CD36, CD44, CD45, CD54, CD90, CD105, CD106, HLA ABC and isotype controls showing similar expression of surface antigens in adult (a) and pediatric (b) MSC. A representative example of adult and pediatric MSC is shown. Red histograms: specific marker; unfilled black histograms: isotype controls. C) Adipogenic differentiation was induced with adipocyte differentiation medium. After 3 weeks aMSC (a) and pMSC (b) were fixed and stained with Oil-red-O. In control medium (Insets), very few cells contained lipid droplets. In adipogenic differentiation medium a majority of cells contained lipid droplets stained in red. D) Chondrogenic differentiation was induced in MSC pellet culture in chondrogenic differentiation medium. After 4 weeks, pellets were fixed and sections were analyzed using Masson trichrome staining (a,b blue) and anti-collagen type II immunohistochemistry (c,d red). The images represent zoomed portion (delimitated by white frames) of the picture shown in the insets. The pellet structure appeared compact and contained abundant collagen fibrils. E) Osteogenic differentiation was induced by culturing aMSC (a) and pMSC (b) in osteogenic differentiation medium. After 4 weeks, cells were washed and incubated with Fast red and Sodium alpha-naphtyl phosphate solutions for 30 min at 4°C. Nuclei were stained with hemalun. MSC expressing alkaline phosphatase showed brown colored cytoplasm and displayed typical star-shaped cell morphology. In control medium (Insets), only few cells showed alkaline phosphatase expression.

Figure 2. Telomerase activity in adult and pediatric MSC.

Telomerase activity of adult and pediatric MSC from 4 different donors and at different passages (1 to 4) was measured and normalized to a provided positive control set as 100 percent. Immortalized human hepatocytes (IHH) were used as quality controls of cell extracts containing telomerase; IHH are immortalized cells transduced with lentivectors coding for telomerase . As compared to control, telomerase activity was low and not significantly different between aMSC and pMSC (p>0.4).

Figure 3. Induction of hepatocyte specific genes in adult and pediatric MSC after co-culture with Huh-7 cells.

A) aMSC were cultured for 4 weeks in hepatogenic differentiation medium containing HGF, FGF4 and Oncostatin M. RT-PCR analysis of αFP and albumin expression shows no induction of hepatocyte specific gene expression. C-, negative control, PCR without polymerase. Huh-7, positive control for αFP and albumin expression. Lane 3, 4, 5 and 6: aMSC from different donors. B) Adult MSC were co-cultured with Huh-7 cells in a transwell system, with or without hepatogenic differentiation medium (Diff. Media+/−). C-, negative control. RT-PCR analysis was done on total RNA extracts from aMSC co-cultured with Huh-7 cells for αFP, albumin and API. Huh-7 cells from transwell of same experience were used as a positive control for αFP and albumin. In 2 of 10 independent experiments, we observed in MSC co-cultured with Huh-7 cells in hepatogenic differentiation medium. The result represents one of the two positives results obtained. C) PMSC were co-cultured under same conditions as aMSC. C-, negative control. In 5 of 8 independent experiments, we observed αFP, albumin and API expression in MSC, independently of the presence of hepatogenic differentiation medium. The result represents one of the 5 positives results obtained. API: alpha1 anti-trypsin; αFP: alpha fetoprotein; Huh-7: human hepatoma cell line.

Figure 4. Alpha smooth muscle actin expression in adult and pediatric MSC cultured in various conditions.

A) Cell extracts from aMSC and pMSC cultured with or without Huh-7 cells in hepatogenic differentiation medium during 4 weeks were analyzed for their expression of αSMA and compared to aMSC cultured in control condition with low FCS concentration (IMDM 2% FCS) by Western blotting. B) Adult MSC and pMSC and EDX cells where incubated with medium (DMEM 5% FCS) conditioned by Huh-7 cells during 7 days. A and B show representative results of multiple blots. αSMA: alpha smooth muscle actin; aMSC: adult multipotent mesenchymal stromal cells; pMSC: pediatric multipotent mesenchymal stromal cells; EDX: human foreskin fibroblasts.

Figure 5. Liver engraftment of MSC in a mouse model of liver injury, after intrasplenic or intra-hepatic transplantation.

A) When injected into the spleen of NOD/SCID mice, human adult and pediatric MSC engrafted and survived for 8 weeks in the spleen (a). Only few cells migrated to the liver with maximum 3 cells per high-power field observed (arrow, b). Anti-human vimentin Ab (panel a,b), Hoechst (panel c,d), overlay (lower panel e,f). B) When injected into liver parenchyma, MSC engrafted and high numbers of cells were detected (a,b). However, they retained their spindle shape morphology (b). Anti-human vimentin Ab (panel a,b), Hoechst (panel c,d), overlay (panel e,f). Higher magnification (b,d,f).

Figure 6. 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).

Figure 7. Engrafted MSC express alpha smooth muscle actin and merge with collagen deposition in mouse liver.

Eight weeks after injection, sections from MSC injected (A) or sham injected (B) livers were stained with an Ab specific against human vimentin (panels a) or an Ab against αSMA (panels b) or with Hoechst (panels e). MSC retained their spindle like morphology and formed band-like structures similar to fibrotic livers. Overlay images of Hoechst and vimentin (panels c) or Hoechst and αSMA (panels d) are shown. Serial sections were stained with Masson to visualize collagen deposition (blue staining). Comparing collagen stained by Masson (blue) with staining of vimentin and αSMA demonstrate similar localization (Aa, Ab, Af).