Hepatogenic differentiation of mesenchymal stem cells induced by insulin like growth factor-I

Abstract

Aim: To improve hepatic differentiation of human mesenchymal stem cell (MSC) using insulin growth factor 1 (IGF-I), which has important role in liver development, hepatocyte differentiation and function.

Methods: Bone marrow of healthy donors was aspirated from the iliac crest. The adherent cells expanded rapidly and were maintained with periodic passages until a relatively homogeneous population was established. The identification of these cells was carried out by immunophenotype analysis and differentiation potential into osteocytes and adipocytes. To effectively induce hepatic differentiation, we designed a protocol based on a combination of IGF-I and liver specific factors (hepatocyte growth factor, oncostatin M and dexamethasone). Morphological features, hepatic functions and cytological staining were assessed to evaluate transdifferentiation of human marrow-derived MSCs.

Results: Flow cytometric analysis and the differentiation potential into osteoblasts and adipocytes showed that more than 90% of human MSCs which were isolated and expanded were positive by specific markers and functional tests. Morphological assessment and evaluation of glycogen storage, albumin and α-feto protein expression, as well as albumin and urea secretion revealed a statistically significant difference between the experimental groups and control.

Conclusion: In vitro differentiated MSCs using IGF-I were able to display advanced liver metabolic functions, supporting the possibility of developing them as potential alternatives to primary hepatocytes.

Keywords: Differentiation; Hepatocyte; Human; Insulin-like growth factor 1; Mesenchymal stem cell.

Figure 1

Osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells in the 4th passage. A: Osteogenic differentiation was positive for alizarin red staining; B: The adipose droplet in differentiated cells after staining with oil red.

Figure 2

Immunophenotyping of bone marrow derived mesenchymal stem cells in the 3rd passage. A: The black histogram shows the profile of the isotype control; B-E: The cells were negative for CD40, CD80, CD45 and HLA-DR respectively; F: The cells were positive for CD90.

Figure 3

Morphological features of the hepatogenic differentiated cells. A: The mesenchymal stem cells obtained from human culture before differentiation; B: The differentiated cells under hepatogenic conditions on day 7; C: The binucleated cells on day 19; D: The differentiated cells with cuboidal morphology on day 21; E: These cells exhibited a polygonal shape with cytoplasmic granules.

Figure 4

The immunocytochemistry analysis of the hepatogenic differentiated cells at day 21. A: PE-fluorescent staining for albumin; B: FITC-fluorescent staining for α-feto protein; C-D: Nuclear staining using DAPI for albumin and α-feto protein respectively.

Figure 5

Albumin levels in hepatogenic differentiated cells (test) in comparison to undifferentiated cells (control) by enzyme linked immunosorbent assay. Albumin levels are expressed as μg/mL (Y-axis) on days 0, 7, 14 and 18 of differentiation (X-axis). The treated cells produced significantly higher levels of albumin compared to undifferentiated cells (P < 0.001).

Figure 6

Periodic Acid-Schiff staining assay in hepatogenic differentiated cells. A: Glycogen uptake by the cells was first seen after 21 d upon treatment; B: Nuclei were stained with haematoxylin.

Figure 7

Production of urea in hepatogenic differentiated cells (group-1) compared to undifferentiated cells (group-2). The urea production as mg/dL (Y-axis) was significantly increased over culture time (P < 0.001).