Heparin/Collagen 3D Scaffold Accelerates Hepatocyte Differentiation of Wharton's Jelly-Derived Mesenchymal Stem Cells

Abstract

Both mature and stem cell-derived hepatocytes lost their phenotype and functionality under conventional culture conditions. However, the 3D scaffolds containing the main extracellular matrix constitutions, such as heparin, may provide appropriate microenvironment for hepatocytes to be functional. The current study aimed to investigate the efficacy of the differentiation capability of hepatocytes derived from human Wharton's jelly mesenchymal stem cells (WJ-MSCs) in 3D heparinized scaffold. In this case, the human WJ-MSCs were cultured on the heparinized and non-heparinized 2D collagen gels or within 3D scaffolds in the presence of hepatogenic medium. Immunostaining was performed for anti-alpha fetoprotein, cytokeratin-18 and -19 antibodies. RT-PCR was performed for detection of hepatic nuclear factor-4 (HNF-4), albumin, cytokeratin-18 and -19, glucose-6-phosphatase (G6P), c-met and Cyp2B. The results indicated that hepatogenic media induced the cells to express early liver-specific markers including HNF4, albumin, cytokeratin-18 and 19 in all conditions. The cells cultured on both heparinized culture conditions expressed late liver-specific markers such as G6P and Cyp2B as well. Besides, the hepatocytes differentiated in 3D heparinized scaffolds stored more glycogen that indicated they were more functional. Non-heparinized 2D gel was the superior condition for cholangiocyte differentiation as indicated by higher levels of cytokeratin 19 expression. In conclusion, the heparinized 3D scaffolds provided a microenvironment to mimic Disse space. Therefore, 3D heparinized collagen scaffold can be suggested as a good vehicle for hepatocyte differentiation.

Keywords: Collagen type I; Gel; Heparin; Hepatocytes; Scaffold.

Fig. 1

The gross structure of the gel and scaffold. The heparin immobilization was shown by toluidine blue staining. The heparinized gel A and scaffold B were stained purple (metachromatic) while the non-heparinized one stained blue (orthochromatic). The gross structure of the lyophilized scaffolds was also shown C

Fig. 2

The supernatant solution was assessed by safranin O after crosslinking processes. A safranin O is red (left) and after adding heparin, it turns to yellow (right) due to metachromatic property of the dye in the presence of heparin. B The optical density of the control (non-crosslinked scaffold with the same heparin concentration) had the significant lower optical density than the experiment samples. This indicated the presence of a less amount of heparin in the supernatant solution and as a result, crosslinking of heparin with the scaffold. * Significant difference with control (p < 0.05). (Color figure online)

Fig. 3

MTT test showed the cells cultured on heparinized, non-heparinized gels and culture dishes were viable and the various culture conditions had no significant effect on the number of viable cells. The cells also could proliferate in all conditions. However, in long term, the proliferation rate in conventional culture condition was higher than on the gels. * Significant difference with control cultured for 14 days (p < 0.05); ¥ Significant difference with control cultured for 7 days (p < 0.05); δ Significant difference with control cultured for 14 days (p < 0.05); £ Significant difference with cells cultured on heparinized gels for 14 days (p < 0.05)

Fig. 4

The flow cytometry showed that the frequencies of the Wharton’s jelly mesenchymal stem cells that expressed CD90, CD73 and CD44 were high, the frequencies of the cells expressed CD106 and CD109 were moderate and those expressing CD34 and CD144 was very low A. Wharton’s jelly mesenchymal stem cells also showed the differentiation potential toward adipocytes B and osteocytes C

Fig. 5

Immunohistochemistry and cytochemisrty of the cells cultured in 3D scaffolds and 2D gels in heparinized and non-heparinized conditions for alpha-fetoprotein (AFP, at the top), cytokeratin 18 (CL18, at the middle) and cytokeratin 19 (CK19, at the bottom). The cytoplasm of the differentiated cells was stained brown and the nuclei of undifferentiated cells stained with hematoxylin but the cytoplasm remained unstained. The arrows point collagen fibers in the scaffolds

Fig. 6

RT-PCR shows the liver–specific marker expression by the cells in various conditions. The best microenvironment for cells to express liver specific markers was 3D heparinized condition. Semi-quantifications of the RT-PCR products of each liver-specific marker were depicted in the graphs. HepG2 cell line was used as positive control and undifferentiated Wharton’s jelly mesenchymal stem cells were used as negative control. * Significant difference with non-heparinized scaffold, gel, heparinized gel and negative control (undifferentiated WJ-MSCs) (p < 0.05); β Significant difference with both negative (undifferentiated WJ-MSCs) and positive controls (HepG2 cells) (p < 0.05); δ Significant difference with non-heparinized gel (p < 0.05); γ Significant difference with non-heparinized scaffold, gel, negative (undifferentiated WJ-MSCs) and positive controls (HepG2 cells) (p < 0.05); € Significant difference with non-heparinized scaffold, gel and negative control (undifferentiated WJ-MSCs) (p < 0.05); ¥ Significant difference with non-heparinized gel, negative (undifferentiated WJ-MSCs) and positive controls (HepG2 cells) (p < 0.05); £ Significant difference with non-heparinized gel and negative control (undifferentiated WJ-MSCs) (p < 0.05)

Fig. 7

PAS staining showed all cells exposed to hepatogenic media were stored glycogen. The best condition for glycogen storage was heparinized 3D condition