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The idea of regenerating lost myocardium via cell-based therapies remains as highly considerable. C-kit? stem/progenitor cells are represented to be suitable candidates for cardiac regeneration compared to other stem cells.A multitude of cytokines from these cells are known to give such multifunctional properties; however, theassociated mechanisms of these factors are yet to be totally understood. The aim of the present study was toinvestigate the in vitro effect of L-carnitine (LC) on cardiac differentiation of c-kit? cells using a cytokinessecretion assay. For this purpose, bone-marrow-resident-c-kit? cells were enriched by MACS method, andwere differentiated to cardiac cells using cardiomyocyte differentiation medium in the absence (control group)and presence of LC (experimental group). Also, characterization of enriched c-kit? cells was performed usingflow cytometry and immunocytochemistry. In the following, the cells were subjected to real-time PCR andWestern blotting assay for gene and protein assessment, respectively. Afterward, culture medium was collectedfrom both control (-LC) and experimental groups (? LC) for cytokine measurement. It was found that 0.2mM LC significantly increased the mRNA and protein expression of cardiac markers of Ang-1, Ang-2, C-TnI,VEGF, vWF, and SMA in c-kit?-cardiomyogenic-differentiated cells. Also, the significant presence of IL-6,IGF-1, TGF-b, and VEGF were obvious in the cultured media from the experimental group compared with thecontrol group. It can be concluded that the mentioned in vitro effects of LC on cardiac differentiation of c-kit?cells could have resulted from the secreted cytokines IL-6, IGF-1, TGF-b, and VEGF.
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Incomplete development and severe malformation of the heart result in miscarriage of embryos because of its malfunction as a pump for circulation. During cardiogenesis, development of the heart is precisely coordinated by the genetically-primed program that is revealed by the sequential expression of transcription factors. It is important to investigate how spatial allocation of the heart containing cardiomyocytes and other mesoderm-derived cells is determined. In addition, the molecular mechanism underlying cardiomyocyte differentiation still remains elusive. The location of ectoderm-, mesoderm-, and endoderm-derived organs is determined by their initial allocation and subsequent mutual cell-cell interactions or paracrine-based regulation. In the present work, we provide an overview of cardiac development controlled by the germ layers and discuss the points that should be uncovered in future for understanding cardiogenesis.
Subject(s)
Female , Humans , Pregnancy , Abortion, Spontaneous , Cilia , Embryonic Development , Embryonic Structures , Germ Layers , Heart , Myocytes, Cardiac , Transcription Factors , ZebrafishABSTRACT
Objective:To verify DNA methyltransferase 1(DNMT1) is the direct target of miR-148a and explore the internal mechanism by which miR-148a regulates the cardiac differentiation of mesenchymal stem cells(MSCs)by directly targeting DNMT1.Methods:miRNA microarray was used to screen out the abnormally expressed miRNAs in MSCs before and after 5′-azacytidine(5′-aza) treatment.Target scan was uesd to predict the target of miR-148a.miR-148a mimics and DNMT1-wt,scramble and DNMT1-wt,miR-148a and DNMT1-mut,scramble and DNMT1-mut were co-transfected in MSCs cells and luciferase activity were detected by single photon.MSCs cells were transfected with miR-148a lentivirus plasmid.Respectively extract DNA,RNA and protein 1,7,14 and 28 days after transfection.The CpG methylation level on DNA regulatory sequences of Gata-4 upstream gene was detected by methylation detection,the mRNA and protein expressions of myosin heavy chain(MHC),cardiac troponin T(cTnT),CD90,CD29,Nkx2.5 and Gata-4 were detected by qRT-PCR and Western blot.Results:miRNA Microarray screened out the abnormally expressed miRNAs in MSCs before and after 5′-aza-induced,including miR-146a-5p,miR-148a,miR-539,etc.Among which miR-148a was remarkably upregulated.Targetscan,Luciferase Reporter Gene and qRT-PCR verified that DNMT1 was the direct target of miR-148a.By infected MSCs cells with miR-148a lentivirus plasmid,we confirmed that the methylation level of Gata-4 gene upstream was changed,and with prolong of transfection,the methylation level of Gata-4 was decreased,the expression of MHC and cTnT were increased,while CD90 and CD2 were continually decreased,the mRNA expression of Nkx2.5 and Gata-4 were increased MSCs cells started myocardial differentiation.Conclusion:miR-148a can regulate the cardiac differentiation of MSCs by directly targeting DNMT1.
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Objective To investigate whether hepatocyte growth factor ( HGF ) and insulin-like growth factor (IGF1) induce cardiac stem cells (CSCs) to proliferate and directly differentiate into cardiomyocytes in vitro.Methods The myocardial tissues were dissected for primary culture of CSCs with the method of explants .The expressions of c-kit and CD34 were examined with immunofluorescence .Primary cells were purified with c-kit by flow cytometry.CFDA SE fluorescent probe was used to detect the proliferation of c-kit+CSCs.C-kit +CSCs were divided into two groups , and cardiac stem cells group and co-cultured with cardiomyocytes group , both group were cultured with HGF and IGF 1.An inverted microscope was used to observe changes in cell number and morphology in different periods .Living cells workstation was used to observe CFDA SE fluorescence intensity , to acquire images and do statistical analysis .Immunofluorescence technique was used to detect the expression of Nkx 2.5 and cardiac troponin T .Results In cardiac stem cells group ,CSCs had no obvious changes in cell number .In co-cultured with cardiomyocytes group , CSCs proliferated and had changes in morphology .Nkx2.5 and cTnT were positively expressed . Several CSCs differentiated into beating cardiomyocytes . Conclusion In co-cultured with cardiomyocytes condition , HGF and IGF1 may promote CSCs to proliferate and differentiate into beating cardiomyocytes .
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Objective To observe the expression changes in microRNA (miR)-379 in the developmental process of the mouse heart and during the differentiation of P19 cells into cardiac myocytes,and to explore the possible relationship between miR-379 and the differentiation of cardiacmyocytes.Methods Heart tissues were collected from fetal mice in pregnant ones at their gestational age (8.5,11.5,14.5 and 18.5 days) respectively.Heart tissue sections of the fatal mice were obtained to observe the heart development process.Then total RNA was isolated from heart tissues by using the TRIzol method.Complementary DNA was synthesized from 1 μg total RNA by using a Reverse Transcriptase Kit.Finally,real-time PCR (RT-PCR) was employed to detect the expression of miR-379.At the same time,P19 cells were cultured with 10 mL/L Dimethyl sulfoxide in suspension for 4 days to form cell aggregation,and these aggregations were transferred into 6-wells plate for culturing by adherence.Beating cells were detected with microscopy on the 10th day after induction.Afterwards,total RNA was extracted from cultured P19 cells at different time points.Reverse transcription was executed to get DNA.At last,RT-PCR was used to explore the expression of miR-379 on 0,4,6,10 days after aggregation.Results The expression level of miR-379 was down-regulated gradually in the developing heart (at gestational age of 8.5,11.5,14.5,16.5 days,respectively),and there were significant differences on the different days (F =21.13,P ＜ 0.05).On the other hand,myocardial markers of troponin T represented an increasing trend during the process of P19 cells induction,which demonstrated that P19 cells were successfully induced into cardiomyocyte-like cells.Meanwhile,miR-379 showed a low expression on day 0 of P19 cells aggregation.On day 4,miR-379 demonstrated a higher level.Afterwards,miR-379 proved to be down-regulated gradually.Conclusions miR-379 plays a role in the process of the heart development,but the specific mechanisms need further research.
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Objective: To explore the mechanism about the cardiac differentiation of human mesenchymal stem cells(MSCs) induced by rat cardiomyocytes.Methods:To induce the cardiac differentiation of human MSCs by using rat cardiomyocytes in different ways(three dishes for each way):(1) Cocultured with rat cardiomyocytes at the ratio of 1:1;(2)Cultured with the conditional medium,which got from the culture of rat cardiomyocytes and mixed with fresh medium by 1:1;(3) Cultured with the supernatant,which got from the minced rat cardiomyocytes and mixed with fresh medium by 1:10.The human MSCs were harvested at the day of 2,5 and 7,respectively.Then the cells were stained with desmin and cardiac troponin I monoclonal-antibodies,and fluorescent in situ hybridization to human DNA was used to mark the cells from human.Results: No desmin or cTnI was detected in the human MSCs cultured with conditional medium or supernatant.While for the human MSCs cocultured with rat cardiomyctes,desmin was detectable at the day of 2 and cTnI at the day of 5.Until the day of 7,about 35% of cocultured-human MSCs expressed those cardiac specific antigens.Conclusion:Human MSCs could be induced to differentiate into cardiomyocyte-like cells by coculture with rat cardiomyocytes,but not by the conditional medium or the supernatant of rat cardiomyocytes.
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Objective To explore the cardiac differentiation of human embryonic stem cells(ESCs)in coculturing with human fetal cardiomyocytes.Methods Fetal cardiomyocytes and fibroblasts were obtained from the aborted fetus,and human ESCs were from the inner cell mass(ICM)of the surplus embryos for tube babies,with the treated fibroblast as feeder layer.Human ESCs of passage 2 to 5 were cocultured with fetal cardiomyocytes at the ratio of 1∶2,and the XY chromosome types of these two kinds of human cells were different to each other in order to be marked.At the same time,human ESCs without coculture were used as negative control.The cocultured cells were harvested 5 days later and were double-stained with human XY chromosome probe and cardiac specific antibody(desmin or cardiac troponin I,cTnI).Results On coculture day 5,40%-50% human ESCs expressed cardiac specific antigens,and human ESCs without coculture did not express those antigens.Conclusion Human ESCs could be induced into cardiac differentiation by the coculture with fetal cardiomyocytes,and hopefully they will be the candidate for cell transplantation of heart.
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Objective:To study the survival and the cardiac differentiation of mesenchymal stem cells from human fetal livers(FMSCs) after being transplanted into infarted rat myocardium. Methods:FMSCs were cultured from human fetal livers, the average age of which was 9 days. The infarction model was built by the ligation of left anterior descending artery and 3.5?106cells were transplanted into infarction area by direct injection one week after ligation. Rats were killed at 1 and 2 weeks after transplantation, and hearts were collected for the examination of the cells' survival and differentiation toward cardiac tissue by fluorescent in situ hybridization(FISH)and immunohistochemistry staining. Results:The FACS results showed that FMSCs expressed antigen CD29,CD44,CD166, SH2(CD105),SH3 and SH4, but did not express CD14,CD34 and CD45.The transplanted cells survived at 7th day after transplanted,but dissappeared at 14th day. No cardiac differentiation was observed during this period. Conclusion:The cells we cultured are testified as FMSCs by their phenotypic expression,and FMSCs could survive for a short period after transplanted into rat heart infarction model,but no cardiac differentiation is induced.