Trans-differentiation of human mesenchymal stem cells generates functional hepatospheres on poly(l-lactic acid)-co-poly(ε-caprolactone)/collagen nanofibrous scaffolds

Journal of Materials Chemistry B1323972-398

In vitro hepatic trans-differentiation of human mesenchymal stem cells using sera from congestive/ischemic liver during cardiac failure.

Cellular therapy for end-stage liver failures using human mesenchymal stem cells (hMSCs)-derived hepatocytes is a potential alternative to liver transplantation. Hepatic trans-differentiation of hMSCs is routinely accomplished by induction with commercially available recombinant growth factors, which is of limited clinical applications. In the present study, we have evaluated the potential of sera from cardiac-failure-associated congestive/ischemic liver patients for hepatic trans-differentiation of hMSCs. Results from such experiments were confirmed through morphological changes and expression of hepatocyte-specific markers at molecular and cellular level. Furthermore, the process of mesenchymal-to-epithelial transition during hepatic trans-differentiation of hMSCs was confirmed by elevated expression of E-Cadherin and down-regulation of Snail. The functionality of hMSCs-derived hepatocytes was validated by various liver function tests such as albumin synthesis, urea release, glycogen accumulation and presence of a drug inducible cytochrome P450 system. Based on these findings, we conclude that sera from congestive/ischemic liver during cardiac failure support a liver specific microenvironment for effective hepatic trans-differentiation of hMSCs in vitro

Cell proliferation assay for hMSCs in various hepatogenic culture conditions.

<p>(A) <i>In vitro</i> cytotoxicity assay showing MTT activity across 9 days of induction in different culture conditions. Error bars represents mean ± S.D. The differences in cell proliferation between the groups were considered statistically significant at <i>p</i><0.05 and <i>p</i>-values are indicated on the graph. (*) indicates significant increase in cell proliferation at day 9 compared to day 3 and day 6 in 5% patient sera group. (#) indicates no significant difference in hMSC proliferation at different time points across 9 days of culture. (B) Cell proliferation assay by direct cell counting reveals higher proliferation rate in 10% FBS, which was comparable to that of 10% normal sera (NS) induction group. 10% patient sera (PS) induction group did not show any increase in cell proliferation. However, 5% PS group showed increase in proliferation, which was significantly more compared to 10% PS group (*<i>p</i><0.05). (C) Cell death assay by annexinV-FITC/PI flow cytometric quantification revealed that 10% patient sera caused mostly necrotic cell death with increase in cell death across 9 days, whereas in 5% PS group cell death was comparably minimal. However, there was negligible cell death in control groups.</p

Assessment of hepatic trans-differentiation efficiency of hMSCs.

<p>Representative flow cytometry data showing average number of albumin positive cells (A) and endoglin (CD105) positive cells (B) after hepatic trans-differentiation of hMSCs in hepatogenic cocktail induction group (i), patient sera induction group (ii) and normal sera induction group (iii). Untreated hMSCs grown in FBS (iv) and HepG2 cells (v) were used as negative and positive control respectively. Histograms showing isotype control (blue area, quantified by M1) and the positive expression of either albumin or endoglin (green area, quantified by M2). Results are representative of three different experiments. Comparison of the total number of albumin positive cells (C) and endoglin positive cells (D) in different hepatogenic culture conditions as well as controls represents the trans-differentiation efficiency. Differences in cell number between the groups were considered statistically significant at <i>p</i>< 0.05 and <i>p</i>-values are indicated on the graph.</p

Functional characterization of hMSCs-derived hepatocytes.

<p>(Ai) Albumin ELISA of the culture media collected from various hepatic induction groups and controls. Differences in albumin concentration between the groups were considered statistically significant at <i>p</i><0.05 and <i>p</i>-values are indicated on the graph. (*) indicates significant increase in albumin release by hepatocytes in patient sera induction group as well as cocktail induction group at day 28 compared to their day 21 counterparts. (#) represents no significant difference in albumin concentration in undifferentiated hMSCs of normal sera induction group and untreated hMSCs at 28 compared to earlier days. (Aii) Urea assay showed higher concentration in hMSCs derived hepatocytes compared to controls on day 28, but was less than that of HepG2 cells. (B) Glycogen accumulation was represented by PAS staining, which showed positive expression (magenta color) in hMSCs-derived hepatocyte colonies of patient sera induction group (i) and defined growth factor induction group (ii). Normal sera treated hMSCs were PAS negative (iii). (Original magnifications, ×100) (Scale bars  = 100 μM). (C) PROD Assay for phenobarbital inducible cytochrome P450 was confirmed by positive expression (red fluorescence) in hMSC-derived hepatocytes in patient sera induction group (i) and growth factor cocktail induction group (ii). HepG2 cells were used as positive control (iii). (Original magnifications × 100) (Scale bars  = 100 μM). (D) Spectrofluorimetric quantification of PROD activity revealed a significant increase in fluorescence intensity after phenobarbital induction in hMSC-derived hepatocytes in patient sera induction group and defined growth factor induction group (* <i>p</i><0.05). However, negligible PROD activity was observed in untreated hMSCs and normal sera treated hMSCs even after phenobarbital induction. HepG2 cells with higher level of fluorescence activity compared to hMSC-derived hepatocytes did not show significant elevation in PROD activity after phenobarbital induction.</p