Biological Characterization Of Cardiac Progenitor Cells Derived From Heart Tubes And Their Differentiation Into Pacemaking Cells

1.Objective and backgroundIt is critical to seek ideal seeding cells for the development of cardiovascular tissue engineering(CvTE).Currently autologous vascular wall cells(AVWCs) and marrow stromal cells(MSCs) represent established cell sources for CvTE.However,the invasive harvesting of vessel segments or bone marrow,a wound brought to body,are required duing cells isolation.Furthermore,these autologous cells were greatly limited in clinical applications,because the fussy experiment in vitro culture can be performed only with necessitating the appearance of a case.Additionally,it is controversial about the differentiatal potential and proliferative ability of MSCs.So both AVWCs and MSCs are not preferable cell sources for tissue engineer,whereas stem cells derived from embryos are better than the two types of cells.In early embryos,there are two cardiac progenitor cell lineages,cardiomyogenic cells and endocardial endothelial cells.The cardiomyogenic cells occupy 95 percent of cardiac progenitor cells.The cells with potential of differentiate into cardiomyocytes,could express cardiac transcription factors gata-4,nkx2.5 and so on, which were considered as molecular markers of cardiac progenitor cells.It has been reported that nkx2.5 positive cells were isolated from embryoid bodies and proved to be capable of differentiating into various cardiac cell types.This suggests the possibility that cardiac stem cells can be cultured from embryos.In this study,we isolated embryonic cardiac progenitor cells as an alternative cell source for cardiovascular tissue engineering.2.Materials and Methods2.1 Biological characteristics of human embryonic cardiac progenitor cells2.1.1 Embryonic cardiac progenitor cells culture and identification2.1.1.1 Human embryonic cardiac progenitor cells culture Heart tubes were isolated under a dissecting microscope by surgical nippers,and then incubated in 0.25%trypsin at 37℃.Cell suspensions obtained in this way were pelleted by centrifugation at 800×g for 5 min.The cells were then resuspended in normal growth medium at 10⁵/ml.The cell suspensions was seeded into 12-well cluster dishes.The cultures were maintained at 37℃,95%humidity,and 5%CO₂. The normal growth medium was DMEM supplemented with 15%fetal calf serum, 10ug/L VEGF,10⁶U/L LIF,0.375%NaHCO₃,100 U/mL penicillin G,100μg/mL streptomycin,2 mmol/L L-glutamine.After 5 or 8-day primary culture,with routine passage methods cell suspension was obtained and the cells were passaged.2.1.1.2 Identification of Human embryonic cardiac progenitor cellsHuman embryonic cardiac progenitor cells at passage 5 were detected for the expression of Nkx2.5,Isl-1,α-smooth muscle actin,cytokeratin,factorâ…§,C-kit by immunocytochemistry,and gata-4 mRNA by reverse transcriptase polymerase chain reaction(RT-PCR).2.1.2 Biological characteristics of embryonic cardiac progenitor cells2.1.2.1 Hasic biological characteristics embryonic cardiac progenitor cellsMorphological characters of cultured cells were investigated by transmission electron microscope(TEM) and inverted phasecontrast microscope.To check growth characteristics,the growth curves were plotted and the mitotic index was tested.2.1.2.2 Embryonic cardiac progenitor cells differentiation characteristics2.1.2.2.1 Differentiation of embryonic cardiac progenitor cells into cardiomyocytesTo investigate the differentiation of embryonic cardiac progenitor cells into cardiomyocytes,the cells at passage 15 were treated with 5-azacytidine.The expression of cardiogenic and myogenic specific genes(gata-4,α-MHC) were detected by RT-PCR at day 14,and the expression ofα-sarcomeric actin was analyzed by the immune fluorescence assay and Western Blot.2.1.2.2.2 Differentiation of embryonic cardiac progenitor cells into smooth muscle cellsEmbryonic cardiac progenitor cells were cocultured with canine vascular endothelial cells(CVECs) in direct cell-cell contact to find out the possibility of their differentiation into vascular smooth muscle cells(VSMCs).α-smooth muscle actin and smooth muscle-myosin heavy chain were detected in induced Embryonic cardiac progenitor cells.2.1.3 Differentiation of embryonic cardiac progenitor cells into pacemaking cells2.1.3.1 Rat embryonic cardiac progenitor cells cultureMale and female rats were mated.After 10.5 days heart tubes were isolated under a dissecting microscope.In succession,the other performation refered to 2.1.1.12.1.3.2 Rat embryonic cardiac progenitor cells identificationRat embryonic cardiac progenitor cells at primary passage,5 passageand 20 passage,were detected for the expression of Nkx2.5,OCT-4 and SSEA-4 by immunocytochemistry.2.1.3.3 Differentiation into pacemaking cellsTo induce differentiation of cardiac progenitor cells towards cardiac pacemaking cells,endothelin-1(10^-7M) was added to the cells in the growth medium without LIF. After inducement culture,the cells was detected for morphological characters, electrophysiology and expression of molecule marker.3.Results3.1 Biological characteristics of human embryonic cardiac progenitor cell3.1.1 Culture and identification of human embryonic cardiac progenitor cellsThe primary cultured embryonic cardiac progenitor cells are mainly round and long spindle-shaped,however,the passaged cells were long spindle and triangon-shaped. Embryonic cardiac progenitor cells stained positive for Nkx2.5,Isl-1 and negative forα-smooth muscle actin,cytokeratin,factorâ…§,C-kit.GATA-4 expression of the cells was higher than that of the control group embryonic limb bud mesenchymal cells (p＜0.05).3.1.2 Biological characteristics of human embryonic cardiac progenitor cells3.1.2.1 Basic biological characteristicsTEM showed that the cells had elements of typical fetal-type cells.The cell population doubling time was 56 hours.The cells had been passaged continuously for more than 2 months(20 passages),and could proliferate actively in vitro.Growth curves and mitotic index curves were plotted.3.1.2.2 Differentiation of embryonic cardiac progenitor cells3.1.2.2.1 Differentiation into cardiomyocytesThe expressions ofα-MHC and GATA-4 were increased in embryonic cardiac progenitor cells treated with 5-azacytidine in contrast to the untreated.The fluorescence intensity ofα-sarcomeric actin of cells induced by 5-azacytidine was stronger than that of the control group.Similarly the protein level ofα-sarcomeric actin was higher in cardiomyogenic differentiation cells than the control group (p＜0.05).The isolated cells exhibited sarcomeres and intercalated discs after being treated with 5-azacytidine.So it was confirmed that the isolated Nkx2.5 positive cells could differentiate into cardiomyocytes by 5-azacytidine.3.1.2.2.2 Differentiation into smooth muscle cellsEmbryonic cardiac progenitor cells exhibited positive staining ofα-smooth muscle actin when cocultured with CVECs,however,smooth muscle-myosin heavy chain was not detected in the induced cells.Similarly the protein level ofα-smooth muscle actin was higher in the induced cells than the control group(p＜0.05).3.2 Differentiation into cardiac pacemaking cells3.2.1 Rat embryonic cardiac progenitor cells culture and identificationEmbryonic cardiac progenitor cells derived from rat stained positive for Nkx2.5, OCT-4 and negative for SSEA-4 at primary,5 and 20 passage.The shape of the cells were the same as human embryonic cardiac progenitor cells.3.2.2 Differentiation into cardiac pacemaking cellsEndothelin-1 was added to induce differentiation of the cells towards cardiac pacemaking cells.After the inducement the cells exhibited more spontaneous beating. There was Connexin-45 and Connexin-43 staining at interfaces between the cells treated with endothelin-1.In contrast,no staining for the both protein was seen between undifferentiated embryonic cardiac progenitor cells.The induced cells exhibited positive staining of HCN2,HCN4 andα-sarcomeric actin.After being treated with endothelin-1,the cells all displayed spontaneously electrical activity(a slow diastolic depolarization phenomenon).Ultrastructural observation showed that the induced cells exhibited some new elements.4.Discussion4.1 Biological characteristics of human embryonic cardiac progenitor cell4.1.1 Identification of embryonic cardiac progenitor cellsIt was reported that murine cardiac cell progenitors can express cardiac transcription factors GATA-4 and Nkx2-5 which were considered as molecular markers of cardiac progenitor cells.In our study cardiac progenitor cells derived from embryonic hearts tubes expressed a high level of Nkx2-5,Isl-1,OCT-4 and GATA-4. This characterization of the cells is similar to that of Isl1⁺ cardiac progenitors from postnatal hearts and Nkx2-5⁺ cardiac precursor cells from murine embryonic stem cells which have been reported.It is demonstrated that the cells mainly consisted of cardiogenic progenitors.These results suggested that the cells might differentiate into cardiomyocytes more easily than ESCs and MSCs do.C-Kit is expressed in adult cardiac progenitor cells and some cardiac stem cells are isolated using c-kit as a marker(Antonio et al.,2003).However,C-Kit was stained negative in hCPCs.Immunocytochemistry of the cells revealed negative staining for several differentiated cell markers such as factorâ…§,α-SMA,CK andα-sarcomeric actin.These results confirmed that there are no endothelial cells,mature myocytes and epithelia in the cells.4.1.2 Biological characteristics of human embryonic cardiac progenitor cells4.1.2.1 Basic biological characteristicsBeing isolated from early embryos,cardiac cell progenitors had elements of typical fetal-type cells which ultrastructural observation have showed.These primitive and fetal cells were capable of self-renewal and continuous passage.From the growth curve and mitotic index curve,it can be seen that cardiac cell progenitors grew vigorously and proliferate actively.The growth properties of cardiac cell progenitors were consistent from 8 passage to 20 passage.These results imply that cardiac cell progenitors could be expanded to a large quantity which is required for CTE.4.1.2.2 Differentiation of embryonic cardiac progenitor cells After cardiac cell progenitors were induced toward myocytes,some myocytes markers(α-MHC,α-smooth muscle actin andα-sarcomeric actin) were expressed. These results indicated that hCPCs possess potential to differentiate into cardiac muscle-like cells and smooth muscle-like cells.It is suggested that cardiac cell progenitors may be a new source of cardiac muscle cells for CTE.But whether hCPCs differentiate into functional mature cardiomyocytes with a high efficiency or not and how many subpopulations of cardiac progenitor cells there are in this cell line await further study.4.2 Differentiation into cardiac pacemaking cellsAfter cardiac progenitor cells were treated with endothelin-1,the cells all displayed spontaneously electrical activityand and spontaneous beating.These cells with spontaneous depolarization generated sinus nodal and atrial-like pacemaking action potentials.The markers of sinus node cells,including Connexin-45,Connexin-43, HCN2,HCN4,were stained positive in the induced cells.It is confirmed that cardiac progenitor cells treated with endothelin-1 can differentiate into cardiac pacemaking cells.So cardiac progenitor cells can be used as a cell source of creating biological cardiac pacemakers and tissue engineered sinus node.5.ConclusionIn this study,embryonic cardiac progenitor cells have been isolated,cultured, identificatified.Our results suggest that these cells be able to differentiate into cardiomyocytes,smooth muscle cells and pacemaking cells.The cells,which have the ability to undergo self-renew,belong to cardiac progenitor cells and are multipotent. These cells maybe have enormous potential in the application to cardiovascular tissue engineering.