Study On The Differentiation Of Mesenchymal Stem Cells Into Cardiomyocytes In A Simulated Cardiac Microenvironment

A large number of animal experiments have verified that mesenchymal stem cells(MSCs) can spontaneously differentiate into cardiomyocytes(CM)after being transplanted into heart tissue.Cardiac microenvironment is thought to be the key inducer in directing the site-specific differentiation of MSCs.Possible factors might include direct cell-cell contact, electric stimulus, mechanical forces, cytokines secreted by CM and other elements.However for the in vivo nature,the ingredients of microenviroment and relating converting mechanisms remain unknown.It is extremely difficult to investigate whether one or more elements play critical roles in the progress of trans-differentiation.Recent evidence demonstrates that co-culturing stem cells with certain type of cells in vitro can be useful for simulating them in vivo phenomena and representing microenvironment to some extent.In such a simulated system stem cells can successfully differentiate towards certain type of cells by being co-cultured.Such method is more visualized than that in vivo and all the process can be observed consistently and timely.So co-culture techniques provide a good model to examine the converting process of MSCs which will help us to elucidate what composes microenvironment and how they trigger such differentiation.Therefore we designed this study as follows:Objective:We simulated the cardiac environment with co-culture composed of MSCs and CM to evaluate the cardiomyogenic differentiation in mesenchymal stem cells.Methods:Preparation of MSCs:The full marrow pellet was collected by centrifugation in vitro from SD rat.After having isolated with a density gradient (Percoll), MSCs were cultured and then stained by DAPI before being co-cultured.Preparation of CM:Hearts from 1 to 2-day-old newborn rats werescissored to small blocks and then digested by enzymatical solution repeatedly. Cardiomyocytes released from the minced tissue were collected and then cultured for 2 days.The two types of cells were cultured and co-cultured in different conditions as follows:In group l,MSCs were cultured in the normal medium as control specimens; In group2 DAPI-MSCs were added to rhythmically beating CM at three ratios of 1:1 -. \:2> 2:1 seperately to make up co-culture; In group 3,MSCS were cultured in the presence of media conditioned by separate cultures of cardiomyocytes; In group 4,the beating of CM were completely abolished in the presence of isoptin followed by additional plating of DAPI-MSCs in a ratio of 1:1.During the 5 days, the morphology and growth properties of MSCs were observed respectively with microscope system.And the coverslips in each group carrying cells were taken out to receive immunofluorescence examination for cardiac troponin T since the fourth day of culture.Results: 1.In normal medium group,MSCs proliferated rapidly and remained the same in shape of spindle.During the 5 days none of them showed any contraction. And all the cells stained negative for cardiac Troponin T.2.In group2, MSCs replicated more slowly than other groups.One part of the MSCs attached to CM become larger and the spindle-shape gradually transformed into broad flattened or triangle-like shape.On the fourth day, these cells started to contract synchronously with cardiomyocytes and were identified by the positive staining for Troponin T which indicated that these cells have converted to cardiomyocytes. And the converting rate was increased among days (P<0.05) .3.1n group3, none of the cells showed any contraction during the 5 days.Most of them remained spindle-like.We can not find any cardiac Troponin T-positive cell in this group.This indicated that MSCs could not converted into cardiomyocytes when fed with conditioned mediumAIn group3, when contracting of CM were abolished by isoptin, the differentiation in MSCs was suppressed.None of the MSCs showed any contraction during the 5 days and those cells were all stained negatively for Troponin T.Conclusions:l.MSCs can differentiate into cardiomyocytes in a simulated cardiac environment in vitro.As a control test,MSCs cannot convert into cardiomyocytes when cultured in normal medium.2.Direct cell-cell contact between MSCs and CM was the prerequisite for the trans-differentiation of MSCs into CM.In contrast,MSCs treated by cardiomyocytes conditioned medium that separated the effects of cell-cell contact did not express cardiomyocytes proteins.lt suggested that the cytokines and other soluble elements are not sufficient to stimulate the stem cells to differentiate into CM.3.Electro-mechanical stimulus of beating CM was also necessary for the trans-differentiation of MSCs into CM.If that element was abolished, the differentiation in MSCs was suppressed either.