Int J Stem Cells.  2017 Nov;10(2):218-226. 10.15283/ijsc17003.

Hepatogenic Differentiation Capacity of Human Wharton’s Jelly Mesenchymal Stem Cell in a Co-culturing System with Endothelial Cells in Matrigel/collagen Scaffold in the Presence of Fetal Liver Extract

• Khodabandeh Z
• Vojdani Z
• Talaei-Khozani T
• Bahmanpour S

• KMID: 2400875
• DOI: http://doi.org/10.15283/ijsc17003

Abstract

BACKGROUND
Human Wharton's jelly mesenchymal stem cells (HWJMSCs) isolated from medical waste product can be considered as an accessible source of cells in regenerative medicine. Stem cell-derived hepatocytes have poor function and need appropriate niche to reconstruct the liver structure. Therefore, we attempted to find a novel approach in differentiating HWJMSCs into functional hepatic cells using 3D culture conditions and liver extract that recapitulates vital stage in liver development.
MATERIALS AND METHODS
HWJMSCs were extracted from human Wharton's jelly, characterized by flow cytometry, and differentiated towards osteogenic and adipogenic lineages. HWJMSCs were co-cultured with HUVECs in 3D matrigel/collagen scaffolds in the presence of fetal liver extract for 14 days. The expression of specific liver genes were evaluated by lectins, PAS and immunocytochemistry.
RESULTS
According to flow cytometry data, isolated cells from HWJMSCs were shown to express MSC markers. HWJMSCs co-cultured with HUVECs in matrigel/collagen scaffold with extract expressed albumin, lectins UEA and PNA. Immunohistochemistry of the cells in matrigel/collagen scaffold with or without extract exhibited a positive reaction for CK19.
CONCLUSIONS
Co-culturing of the HWJMSC/HUVEC in 3D matrigel/collagen scaffold is bimimicary of in vivo cell condition. The results showed that administration of the liver extract in 3D matrigel/collagen culture of HWJMSC/HUVEC can induce hepatocyte marker expression.

Keyword

Collagen; Matrigel; 3D culture; Hepatocyte; Liver extract

MeSH Terms

Collagen
Endothelial Cells*
Flow Cytometry
Hepatocytes
Humans*
Immunohistochemistry
Lectins
Liver*
Medical Waste
Mesenchymal Stromal Cells*
Regenerative Medicine
Collagen
Lectins
Medical Waste

Figure

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  Fig. 1 The flow cytometry showed the frequency of omitted HWJMSCs which reacted to CD44, CD90, CD105, CD106 and CD73 was high; however, the frequency of the cells which reacted to CD34 and CD 144 was negligible (A). The blue line is positive cells, Oil red O staining showed that the cells stored lipid droplets in the presence of adipogenic medium (B), alizarin red S showed that the cells deposited Ca+ in the presence of osteogenic medium (C).

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  Fig. 2 The Figure shows the morphology of HWJMSCs after supplementation with or without extract for 7 days in 2D culture (A, B). The effects of matrigel/collagen scaffold and fetal liver extract on the cell morphology (C, D). Some cells are arranged into the structure with tubular shape in matrigel/collagen scaffold with or without extract.

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  Fig. 3 Immunofluorescence micrographs show the HWJMSCs co-cultured with HUVECs expressed albumin in matrigel/collagen scaffold with or without extract, FITC-conjugated albumin (up) and DAPI (down).

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  Fig. 4 Immunohistochemical staining of co-culturing HWJMSCs with HUVECs within the matrigel/collagen scaffold with or without extract administration for CK 19.

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  Fig. 5 The micrographs of the co-cultured cells in the matrigel/collagen scaffold with or without extract stained with lectins UEA. FITC- conjugated lectin (up) and DAPI (down). The unstained cells are non-endothelial cells.

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  Fig. 6 The micrographs of the bile duct formation in the matrigel/collagen scaffold with or without extract stained with lectins PNA. FITC-conjugated lectin (green) and DAPI (blue).

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  Fig. 7 The light micrograph of 3D culture in matrigel/collagen scaffolds stained with PAS. The luminal space that surrounded by the cells is also depicted; (A&C) without extract; (B&D) with extract.

Reference

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