Isolation, expansion and characterization of bone marrow derived mesenchymal stem cells and their differentiation into hepatocytes in canines
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Date
2018-07
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G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand)
Abstract
Mesenchymal stem cells (MSCs), as adult stem cells (ASCs) able to divide into a variety of different cells, are of utmost importance for stem cell research. Mesenchymal stem cells (MSC) isolated from bone marrow and differentiated into hepatocyte-like cells have increasingly gained attention for clinical cell therapy of liver diseases because of their high regenerative capacity. The main focus of current study was on canine bone marrow derived mesenchymal stem cells and their differentiation towards hepatic lineage in vitro. The bone marrow was aspirated from iliac crest of young non-descript male dog under xylazine and ketamine anesthesia. The mononuclear cells along with mesenchymal stem cells were harvested by density gradient centrifugation. The mesenchymal stem cells were isolated on the basis of their plastic adherence property. The cells were seeded in tissue culture flasks and allowed to grow in monolayer till the attainment of 80-90% of confluency. At this confluency passaging was done using trypsin-EDTA and cultured with two different densities of 16 cells/cm2 and 4000 cell/cm2 in T-25 flasks. The MSCs cultured with initial seeding density of 4000 cell/cm2 showed higher yield up to 10 passages with faster expansion and followed standard growth kinetic curve. Thus, the fourth passage of these MSCs were made to differentiate into hepatocytes in stepwise manner including induction, differentiation and maturation steps. The differentiation was done by using growth factors HGF, bFGF and nicotinamide for induction, HGF, ITS and Dexamethasone for differentiation and OSM, ITS and dexamethasone for maturation. The cells were cultured for 30 days in an incubator maintained at 5% CO2 and 37oC.The morphology of cells was observed at regular intervals and cells were characterized by PAS staining, ICG uptake, gene expression analysis and SEM. The cells demonstrated positive PAS staining by giving pink to purple red colour. The cells also affirmed the ICG uptake by manifestation of dark green colour nuclei. The RT-PCR analysis revealed the expressions of hepatocyte specific markers alfa fetoprotein (AFP), albumin (ALB), tyrosine aminotransferase (TAT) and alpha-1 antitrypsin (_1-AT) in differentiated cells. The cells when subjected to scanning electron microscopy also revealed hepatocyte like morphology. On the basis of this study it was concluded that canine bone marrow derived mesenchymal stem cells were expanded successfully up to 10 passages with optimum initial seeding density and possessed potential for differentiating into hepatocyte-like cells in vitro. The exploitation of this in vitro harvested hepatocyte like cells may prove an effective therapy for liver diseases.
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