Optimisation of the differentiation of cord originated mesenchymal stem cells into hepatocyte cells

Liver diseases are amongst the crucial health problems the world and Turkey face. Liver diseases such as cirrhosis and liver cancer have become common in the recent years and they cause a loss in hepatocyte functions. Despite the number of methods used to treat liver diseases, an effective treatment hasn't yet been found. Therefore, alternative methods based on stem cells are currently being investigated. Some of the commonly used stem cell components are bone marrow, adipose tissue, cord blood and cord matrix (Wharton gel). The isolated mesenchymal stem cells obtained by using numerous methods present give promising results to cure liver diseases with their high reproduction potential, low immünogenecity and capacity to differentiate. In order to treat patients with liver diseases; different protocols containing cytokines, growth hormones and chemical agents were used to differentiate mesenchymal stem cells into hepatocyte cells. Furthermore, mesencyhmal stem cells isolated by using different origins don't have the same potential to differentiate into hepatocyte cells. The potential for mesencyhmal stem cells to differentiate into hepatocyte cells is a result of not only the type of the source being used to isolate, but also the combination of cytokines, growth hormones and chemical agents involved in the process. As a result of this, until now an effective protocol hasn't been developed to differentiate mesencyhmal stem cells into hepatocyte cells despite the ongoing investigations. The purpose of this study is to investigate a relatively effective new protocol to differentiate mesencyhmal stem cells into hepatocyte cells using human cord blood, cord vein and cord matrix (Wharton gel), and continuously culture and cryopreservation which will be the first time for this kind of study to be conducted in Turkey. The additional aim was to investigate the effect of the nanoparticles such as titanium dioxide, titanium silver, silver and zinc on mesencyhmal stem cells obtained from cord matrix in order to be used in tissue engineering. The methods used in the experiments were; different isolation (Ficoll, enzymatic, explant), microscopic, culture, cell count and viability analysis 3 - (4,5-dimethyltriazole-2-yl) -2,5-diphenyltetrazolium bromide (MTT), histologically (Periodic Acid Shift Dye), molecular (Real Time Polymerase Chain Reaction) and Flow cytometric. The data was evaluated using SPSS version 15.0 for Windows and p < 0,05 was taken as the statistically significant. As a result of the microscopic and flow sytometric analysis, surplus number of healthy mesencyhmal stem cells were isolated from cord matrix using explant method in a short period of time without using enzymes. According to the histological and molecular results, with this new protocol; using 20 ng/ml bFGF, 50 ng/ml HGF, 10 ng/ml FGF-4, 20 ng/ml EGF, 1 µM/L Dexamethasone, 5 mM Nicotinamide, 50 mg/ml ITS, 20 mM Napyruvate containing 10% FBS added 1:1 ratio DMEM-F12 (Non phenol red)-IMDM as the differentiating culture medium, and 10 ng/ml FGF-4, 20 ng/ml HGF, 50 ng/ml OSM, 5 mM Nicotinamide, 1% DMSO, 50 mg/ml ITS, 1 µM Dexamethasone containing 10% FBS added 1:1 ratio DMEM (Non low glucose phenol red)- William's E as the maturing culture medium, the most effective hepatogenic differentiation was conducted. Comparing with the other protocols, the hepatogenic cells obtained by using this new protocol in comparison to the control (mesenchymal stem cell) and hepatocyte (Hep 3B) cell groups; Real Time Polymerase Chain Reaction made as a result of albumin (ALB) and alpha fetoprotein (AFP) gene expression levels in a statistically significant way to express that most were determined (PANAVO *** <0.001). Cytokeratin 18 (CK-18) gene expression has increased four times more relative to the hepatocyte (Hep 3B) cell group and twice more than the control (mesencyhmal stem cells). Consequently, dissimilar to the previous protocols, this new one enabled the differentiation of a more hepatocyte characterized hepatogenic cells from cord matrix originated mesencyhmal stem cells. Additionally, MMT results revealed that titanium, titanium silver, silver and zinc nanoparticles' overdose has a toxic effect on cord originated mesencyhmal stem cells as well as decreasing the proliferation. In cord originated mesencyhmal stem cells titanium EC50 is 20,58, titanium silver EC50 is 33,3, silver EC50 is 12,11 and zinc EC50 is 3,1. Due to the lowest nanoparticle toxic effect, titanium silver with the highest EC50 was determined as the most suitable application for the cord matrix (Wharton gel) originated mesencyhmal stem cells. In consequence of the experiments conducted, the most effective protocol so far to differentiate the cord matrix originated mesencyhmal stem cells into hepatogenic (hepatocyte like) cells was developed. In conclusion, this new protocol differentiating cord matrix originated mesencyhmal stem cells into hepatocyte cells can be applied to the mesencyhmal stem cells obtained from other origins. The acquired hepatogenic cells by using this method can be used in cell based treatment of the liver diseases as well as in tissue engineering to develop new medicine, toxicity research, and also enabling the non-toxic concentrations of titanium, titanium silver, silver and zinc nanoparticles to be used as medicine, vaccine and in different fields of tissue engineering.