Generation Of Induced Cardiac Progenitor Cells And Construction Of Their 3D Hydrogel Culture System

Heart disease is one of the main reasons for causing human death.Its morbidity is second only to cancer.Especially,myocardial infarction(MI),which is resulted from coronary artery occlusion,often causes irreversible damage or death of cardiomyocytes(CMs).Due to the poor regenerative ability of CMs after birth,there is still no effective radical cure for MI.A large number of exogenous CMs are required for transplantation in vivo to treat MI,which are prone to leading immune rejection,and the source of these cells is limited.Embryonic stem cells(ESCs)and induced pluripotent stem cells(i PSCs)can differentiate into heart cells.However,due to the difficulty in controlling their differentiation directions in vivo,non-cardiac cells are easy to be generated,and even they have the potential capacity to induce tumorigenesis.The differentiated somatic cells can be reprogrammed into induced cardiac progenitor cells(iCPCs)via indirect lineage transformation technology.Since their‘self-renewal’and differentiation potential of cardiac lineage cells,iCPCs can provide a new cell resource,and effectively reduce their oncogenicity and heterogeneity of the differentiated cells,so they have a broad application prospect.Since the traditional two-dimensional(2D)cell culture system cannot accurately simulate cell state in vivo,a large number of research results on heart diseases will not be replicated in laboratory animals and clinical trials.Three-dimension(3D)culture system(such as hydrogel)constructed by using extracellular matrix(ECM)can simulate cell microenvironment in vivo,which is more conducive to studying heart diseases.Chitosan,a kind of natural polysaccharide,is composed of glucosamine and acetylglucosamine,which has the properties of non-toxicity,degradability,antibacterial activity,antifungal activity and so on.It is expected to reduce pollution caused by ECM and improve the stability of hydrogel.Therefore,human embryonic kidney 293T cells(HEK-293T cells)were reprogrammed here into iCPCs by the overexpressions of five kinds of cardiac transcription factors including Gata4,Nkx2.5,Tbx5,Baf60c,and Mesp1.Collagen,chitosan,and ECM were utilized to construct the3D hydrogel cell culture system,and cell behavior and induced differentiation characteristics of iCPCs in the system were explored,which laid a foundation for the mechanism research,drug screening,model building and cell therapy of heart disease.Part 1:Formation and identification of iCPCsiCPCs were generated from HEK-293T cells via Lipofectamine LTX-mediated overexpressions of cardiac transcription factors including Gata4,Nkx2.5,Tbx5,Baf60c,and Mesp1.They were identified by the morphological observation,immunocytochemical staining,real-time quantitative PCR(RT-qPCR),and induced differentiation.The results showed that:(1)After being reprogrammed,the generated cells from HEK-293T cells had cardiac progenitor cell(CPC)-like morphological characteristics.These cells were round or elliptical in shape,and they had no processes.Their immunocytochemical staining for Gata4,Nkx2.5,and Irx4 were positive,and the corresponding positive cell rates were 61.36±1.08%,60.97±1.28%,and 61.73±1.18%,respectively.Compared with HEK-293T cells,the relative expression levels of IRX4,Tbx20,Mesp1,and Nkx2.5 genes in iCPCs were significantly up-regulated(P<0.01),and the relative expression levels of FSP1 and COL5A1 genes were significantly down-regulated(P<0.01).(2)iCPCs could differentiate into CMs with short cylindrical shape and nucleus in the center of the cell,endotheliocytes(ECs)with polygonal shape and irregular edge,and smooth muscle cells(SMCs)in long spindle shape without striation after being treated with cardiac lineage induction solution.After being immunocytochemically stained,the differentiated cells showed positive for CM specific marker protein-cardiac actin,EC specific marker protein-CD31,or SMC specific marker protein-SM-MHC,the corresponding positive cell rate were 49.37±84%,48.61±0.74%,and 47.57±0.77%,respectively.The relative expression levels of c Tn T and Actinin-α1(CM specific marker genes),PEACAM1(EC specific marker gene),and MYL2(SMC specific marker gene)in differentiated cells were significantly higher than that in i CPC control group,respectively(P<0.01).Therefore,overexpression of Gata4,Nkx2.5,Tbx5,Baf60c,and Mesp1 could reprogram HEK-293T cells into CPC-like cells,the generated iCPCs had the capacity of differentiating into CMs,ECs,and SMCs.Part 2:Construction of 3D hydrogel scaffold system for iCPCsCollagen,chitosan,and ECM at the different volume ratios including 4:0:3(control group),4:1:3(chitosan treatment group 1),4:2:3(chitosan treatment group 2),or 4:3:3(chitosan treatment group 3)were utilized to prepare 3D hydrogels.In order to construct 3D hydrogel scaffold system for iCPCs,the morphological structure,porosity rate,swelling rate,and degradation rate of 3D hydrogels,as well as cell recovery method were detected,effects of 3D hydrogel on i CPC behaviors and differentiation were also explored.The results showed that:(1)3D hydrogels with different components showed stereoscopic network structure under the scanning electron microscope.In the control group,the hydrogel presented large pore size and loose distribution of collagen.The hydrogel in chitosan treatment group 1showed a relatively dense structure,uniform pore size,rough surface,and through-pore each other.The hydrogels in chitosan treated group 2 and 3 had smaller pore size,disorderly distribution of collagen fibers,and different size of bulges.(2)The porosity rate(99±0.30%),swelling rate(97±0.87%),and degradation rate(44±0.03%)of3D hydrogels,as well as proliferation times(58±0.24),survival rate(73±0.60%)of iCPCs in chitosan treated group 1 were significantly higher than these in control group and chitosan treated groups 2 and 3,respectively(P<0.01).(3)No significant difference in the number of cells recovered from the same 3D hydrogel digested by0.30%trypsin or 0.25%collagenase was found(P>0.05),but which was significantly higher than that in 0.45%pepsin group,respectively(P<0.01).After digestion with different concentrations of trypsin,collagenase,or pepsin,the number of recovered cells in the chitosan treatment group 1 was significantly higher than that in the other 3D hydrogel treatment groups(P<0.01).(4)In 3D hydrogel,the morphological characteristics of iCPCs and their differentiated cells were not obviously different from these of 2D cultured cells except for the slightly smaller size.Compared with the 2D culture system,the number of differentiated cells including cardiac actin~+CMs,CD31~+ECs,and SM-MHC~+SMCs from iCPCs in 3D hydrogel was significantly higher than that in the 2D culture system,respectively(P<0.01),the relative expression levels of c Tn T,actinin-α1,PEACAM1,and SMCs genes in differentiated cells were also significantly up-regulated(P<0.01).Therefore,it was suitable to use the protocol of chitosan treatment group 1 to prepare hydrogel for 3D culture of iCPCs,0.25%collagenase or 0.30%trypsin was suggested to digest hydrogel for recovering cells.Compared with 2D culture system,3D hydrogel was more conducive to the differentiation of iCPCs into CMs,ECs,and SMCs in vitro.Conclusions:Lipofectamine LTX mediated overexpression of five kinds of cardiac transcription factors including Gata4,Nkx2.5,Tbx5,Baf60c,and Mesp1 could reprogram HEK-293T cells into iCPCs,the generated iCPCs had the ability to differentiate into CMs,ECs and SMCs.Collagen,chitosan,and ECM at the ratio of4:1:3 were suggested to construct 3D hydrogels with strong mechanical properties and high stability.This cell culture system supported the survival and proliferation behaviors of iCPCs.0.25%collagenase or 0.30%trypsin was suitable for digesting hydrogel to recycle cells.Compared with 2D culture system,3D hydrogel was more favorable for the differentiation of iCPCs into cardiac lineage cells including CMs,ECs and SMCs.The generation of iCPCs and the establishment of 3D hydrogel culture system provided a research platform for analyzing the mechanism of heart development,injury,and repair,building heart disease model,screening and developing drugs,and exploring new strategies for cell therapy.