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

Platelet-rich plasma (PRP) as a source of growth factors may induce tissue repairing and improve fibrosis. This study aimed to assess the effects of PRP on kidney regeneration and fibrosis in gentamicin (GM)-induced nephrotoxicity rat model by stereological study. Thirty-two male rats were selected. Nephrotoxicity was induced in animals by administration of GM (80 mg/kg/daily, intraperitoneally [IP], 8 day) and animals were treated by PRP (100 µL, intra-cortical injection using surgical microscopy, single dose). Blood samples were collected for determine blood urea nitrogen (BUN) and creatinine (Cr) before and after PRP therapy. At the end of experiment, right kidneys were sectioned by Isotropic Uniform Random (IUR) method and stained with H & E and Masson's Trichrome. The stereological methods were used for estimating the changes in different structures of kidney. PRP increased the number of epithelial cells in convoluted tubules, and decreased the volume of connective tissue, renal corpuscles and glomeruli in GM-treated animals (P < 0.05). Our findings indicate that PRP had beneficial effects on proliferation of epithelial cells in convoluted tubules and ameliorated GM-induced fibrosis.
Animals ; Blood Urea Nitrogen ; Connective Tissue ; Creatinine ; Epithelial Cells ; Fibrosis ; Gentamicins ; Humans ; Intercellular Signaling Peptides and Proteins ; Kidney* ; Male ; Methods ; Microscopy ; Models, Animal ; Platelet-Rich Plasma* ; Rats ; Regeneration*

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.
Antibodies ; Collagen ; Collagen Type I ; Constitution and Bylaws ; Extracellular Matrix ; Fetal Proteins ; Gels ; Glucose-6-Phosphatase ; Glycogen ; Heparin ; Hepatocytes* ; Humans ; Keratin-18 ; Keratin-19 ; Mesenchymal Stromal Cells* ; Phenotype ; Wharton Jelly

Human breast milk stem cells (hBSCs) contain a population of cells with the ability to differentiate into various cell lineages for cell therapy applications. The current study examined the differentiation potential of hBSCs into hepatocytes- like cells. The cells were isolated from the breast milk and were treated with hepatogenic medium containing hepatocyte growth factor, insulin-like growth factor and dexamethasone for 7 days subsequently; Oncostatin M was added to the culture media. RT-PCR and immunocytochemistry were performed to detect the hepatogenic markers. The glycogen storage and the ability of the cells to absorb and release indocynanin green were also tested. The data showed that most of the differentiated cells formed cell aggregates after the 30th day, with more cells accumulated to form spheroids. RT-PCR revealed the expression of the hepatic nuclear factor, albumin, cytokeratin 18 and 19, cytochrome P2B6, glucose-6-phospahtase and claudin. The functional assays also showed glycogen storage and omission of indicynine green. Our study demonstrated hBSCs are novel population that can differentiate into hepatocyte-like cells.
Breast* ; Cell Culture Techniques ; Cell Lineage ; Cell- and Tissue-Based Therapy ; Culture Media ; Cytochromes ; Dexamethasone ; Glycogen ; Hepatocyte Growth Factor ; Hepatocytes ; Humans ; Immunohistochemistry ; Keratin-18 ; Mesenchymal Stromal Cells* ; Milk, Human ; Oncostatin M ; Stem Cells