Effects of 5-azacytidine on DNA methylation and index components in patchouliol-type Pogostemon cablin / 中国中药杂志

This study aims to explore the relationship of DNA methylation with the contents of the index components as well as the growth and development of Pogostemon cablin. The demethylation reagent 5-azacytidine(5-azaC) was used to treat the tissue culture seedlings of patchouliol-type P. cablin. High performance liquid chromatography was employed to evaluate the changes of DNA methy-lation in P. cablin, and GC-MS to detect the contents of index components in P.cablin. The agronomic characters of P.cablin were measured using the common methods. The results showcased that DNA methylation of P.cablin was significantly reduced by 5-azaC in a concentration-dependent manner. Thirty days after treatment with 5-azaC at different concentrations, the content of patchouli alcohol changed slightly; compared with that in the control group, the content of pogostone in 50 μmol·L~(-1) and 100 μmol·L~(-1) 5-azaC groups was significantly up-regulated. The 100 μmol·L~(-1) 5-azaC group had the largest differences in contents of pogostone and patchouli alcohol compared with the control group, followed by the 50 μmol·L~(-1) 5-azaC group. Ninety days after disinhibition, the content of pogostone in the treatment group was significantly increased and the content of patchouli alcohol was significantly decreased. In addition, 5-azaC significantly inhibited the growth and development of P.cablin in a dose-dependent manner. These results indicate that DNA methylation regulates the biosynthesis of the index components in patchouliol-type P.cablin and proper demethylation can directly promote the synthesis of pogostone and indirectly affect the accumulation of patchouli alcohol.

Transcriptional Abundance of Myosin Light Chain 2 Gene in Cardiac Differentiated Canine Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) are promising cell source for cardiac tissue engineering and cell based therapies for heart repair as they can be expanded in vitro and differentiated into most cardiovascular cell types, including cardiomyocytes. During embryonic heart development, this differentiation occurs under the influence of internal and external stimuli that guide cells to go down the cardiac lineage. The aim of this study was to characterize the cardiac differentiation potential of a canine iPS cell. With the use of a standard embryoid body–based differentiation protocol for iPS cells were differentiated for 24 days. In vitro differentiations of canine iPSCs via embryoid bodies (EBs) were produced by ‘Hanging Drop’ method. EB’s were differentiated using 5-azacytidine (5-Aza). During differentiation, EBs were collected on day 4, 6, 8, 12, 16, 20 and 24 to evaluate the expression of cardiomyocyte specific marker. Analyses on molecular, structural, and functional levels demonstrated that iPS cell– derived cardiomyocytes show typical features of ES cell– derived cardiomyocytes. Reverse transcription polymerase chain reaction analyses demonstrated expression of marker genes. The differentiated cells expressed cardiac-specific gene myosin light chain 2 (MYL2) which started from day 8 of differentiation and highest expression was observed on day 16. Immunocytochemistry and relative expression of cardiac specific genes revealed that iPS cells differentiate into functional cardiomyocytes and allow to derivation of autologous functional cardiomyocytes for cellular cardiomyoplasty and myocardial tissue engineering

Comparison of differentiation effects of BMSCs into cardiomyocytes induced by three culture methods in vitro / 吉林大学学报(医学版)

Objective: To induce the differentiation of bone marrow mesenchymal stem cells (BMSCs)by three culture methods in vitro, and to explore the best method of inducing the BMSCs to differentiate into the cardiomyocytes in vitro. Methods: The rat BMSCs were separated and cultured with density gradient centrifugation combined with adherence culture method. The rat BMSCs were cultured with 5-azacytidine (5-aza), the myocardial cell cleavage of the dilated cardiomyopathy (DCM) model rats and the serum of DCM model rats + 5-aza in vitro, respectively; control group was set up at the same time (The BMSCs were cultured in the L-DMEM medium containing 10% FBS under the same condition). The morphology of BMSCs were observed and the expression of troponin T (cTnT) was detected by RT-PCR, Western blotting method and immunohistochemical staining. Results: The rat BMSCs were obtained by d ensity gradient centrifugation combined with adherence culture method. The BMSCs highly expressed the characteristic surface marker CD44, CD29 and CD105 of mesenchymal stem cells (MSCs), and did not express the characteristic surface marker of hematopoietic stem cells CD34. Under the specific induction conditions, the BMSCs were differentiated into the osteoblasts and the aliphatic cells. Ire vitro, three culture methods could induce the BMSCs to express cTnT and differentiate into the cardiomyoid cells. In 5-aza group, the cell growth status was poor, and the cells in the other two groups had better growth status. The positive expression rate of cTnT was the highest in DCM model rat serum + 5-aza group. Conclusion: The effect of DCM model rat serum combined with 5-aza to induce the BMSCs to differentiation into the myocardial cells is the best.

Elabela promotes differentiation of wharton’s jelly-derived mesenchymal stem cells into cardiomyocyte-like cells / 中国组织工程研究

BACKGROUND: Elabela is a new type of endogenous receptor of APJ discovered in recent years. It is widely distributed in the adult cardiovascular system and has a certain influence on cardiovascular diseases. However, the effect of Elabela on the differentiation of stem cells into cardiomyocytes and the expression of APJ in cardiomyocyte differentiation has not been studied yet. OBJECTIVE: To investigate the effect of Elabela on the differentiation of Wharton’s jelly-derived mesenchymal stem cells into cardiomyocytes. METHODS: The frozen mesenchymal stem cells were resuscitated. 5-Azacytidine was used to induce Wharton’s jelly-derived mesenchymal stem cells to differentiate into cardiomyocytes when the cell confluence reached 80%-90%. After 24 hours, the medium was replaced by low-glucose medium containing Elabela and 10% fetal bovine serum in the experimental group, and by low-glucose medium containing 10% fetal bovine serum in the control group. At 7, 14, 21, and 28 days after induction, cell morphology was observed. The total RNA and total protein of each group were collected. The myocardial specific markers Nkx2.5, cTnT and Connexin 43 mRNA and protein expression levels were detected by real-time fluorescent quantitative PCR and western blot assay. The expression of APJ in the induced cardiomyocytes was detected by real-time fluorescent quantitative PCR and flow cytometry. RESULTS AND CONCLUSION: (1) The expression levels of myocardial specific markers Nkx2.5, cTnT and Connexin 43 mRNA and protein were higher in the experimental group than in the control group in all stages of differentiation, and the expression of APJ was also higher in the experimental group than in the control group. (2) In summary, Elabela plays a certain promoting role in the differentiation of Wharton’s jelly-derived mesenchymal stem cells into oriented cardiomyocytes. Elabela, as another agonist of APJ, can promote the expression of APJ during the induced cell differentiation.

Enhancement of Osteogenic Differentiation by Combination Treatment with 5-azacytidine and Thyroid-Stimulating Hormone in Human Osteoblast Cells

BACKGROUND AND OBJECTIVES: The role of thyroid-stimulating hormone (TSH) signaling on osteoblastic differentiation is still undetermined. The aim of this study was to investigate the effects of 5-aza-2′-deoxycytidine (5-azacytidine) on TSH-mediated regulations of osteoblasts. MATERIALS AND METHODS: MG63, a human osteoblastic cell-line, was treated with 5-azacytidine before inducing osteogenic differentiation using osteogenic medium (OM) containing L-ascorbic acid and β-glyceophosphate. Bovine TSH or monoclonal TSH receptor stimulating antibody (TSAb) was treated. Quantitative real-time PCR analyses or measurement of alkaline phosphatase activities were performed for evaluating osteoblastic differentiation. RESULTS: Studies for osteogenic-related genes or alkaline phosphatase activity demonstrated that treatment of TSH or TSAb alone had no effects on osteoblastic differentiation in MG63 cells. However, treatment of 5-azacytidine, per se, significantly increased osteoblastic differentiation and combination treatment of 5-azacytidine and TSH or TSAb in the condition of OM showed further significant increase of osteoblastic differentiation. CONCLUSION: Stimulating TSH signaling has little effects on osteoblastic differentiation in vitro. However, in the condition of epigenetic modification using inhibitor of DNA methylation, TSH signaling positively affects osteoblastic differentiation in human osteoblasts.

Enhanced differentiation of mesenchymal stromal cells by three-dimensional culture and azacitidine

BACKGROUND: Mesenchymal stromal cells (MSCs) are useful for cell therapy because of their potential for multilineage differentiation. However, MSCs that are expanded in traditional two-dimensional (2D) culture systems eventually lose their differentiation abilities. Therefore, we investigated whether azacitidine (AZA) supplementation and three-dimensional culture (3D) could improve the differentiation properties of MSCs. METHODS: 2D- or 3D-cultured MSCs which were prepared according to the conventional or hanging-drop culture method respectively, were treated with or without AZA (1 µM for 72 h), and their osteogenic and adipogenic differentiation potential were determined and compared. RESULTS: AZA treatment did not affect the cell apoptosis or viability in both 2D- and 3D-cultured MSCs. However, compared to conventionally cultured 2D-MSCs, AZA-treated 2D-MSCs showed marginally increased differentiation abilities. In contrast, 3D-MSCs showed significantly increased osteogenic and adipogenic differentiation ability. When 3D culture was performed in the presence of AZA, the osteogenic differentiation ability was further increased, whereas adipogenic differentiation was not affected. CONCLUSION: 3D culture efficiently promoted the multilineage differentiation of MSCs, and in combination with AZA, it could help MSCs to acquire greater osteogenic differentiation ability. This optimized culture method can enhance the therapeutic potential of MSCs.

Analysis of DNA methylation with 5-Azac induced immune hyporesponsiveness following acute graft-versus-host disease / 天津医药

Objective To analyse the change of DNA methylation with 5-Azac injection in acute graft-versus-host dis- ease (aGVHD) mouse model, which received allogeneic bone marrow transplantation, and explore the immunomodulatory ef-fects of 5-Azac. Methods Male C57BL/6 (H-2b)and female BALB/c (H-2d) mice were selected as donor and recipient of complete allotransplantation. BABL/c mice were divided into two groups, transplantation control group and 5-Azac experi-mental group. At 1-7, 14, 21 and 28-day after transplantion, 5-Azac 0.25 mg/kg (0.3 mL/time) was injected by tail vein in experimental group, while the control group were injected with sterile water 0.3 mL/time. Peripheral blood DNA samples were collected from three control mice and three experimental mice, then mixed with equal amount respectively. The MeDIP-seq method was selected to detect methylation changes in mice, and the differential DNA methylation in the biological path-ways was analyzed. Results The survival time was prolonged, and the rejection reaction was decreased in 5-Azac experi-mental group, which suggested immune hyporesponsiveness post aGVHD. The MeDIP-seq result showed that 369 different DNA methylation located in the promoter regions, including 239 up-regulated genes and 130 down-regulated genes. There were 184 differential DNA methylation genes located in the exon regions, including 113 up-regulated genes and 71 down-regulated genes. Differential DNA methylation genes involved in 10 immunological signaling pathways, respectively. Among them, TGF-β, GSK-3β, SYK, PI3K, NFAT, CD28 andα4β7 were closely related to the development of aGVHD. Conclu-sion 5-Azac can effectively induce immune hyporesponsiveness post aGVHD by changing the gene methylation status.

Effects of 5-azacytidine on radiation-induced epithelial-mesenchymal transition in rat alveolar type Ⅱ epithelial cell line / 中华放射肿瘤学杂志

Objective To investigate the effects of 5-azacytidine on radiation-induced epithelialmesenchymal transition in rat alveolar type Ⅱ epithelial cell line (RLE-6TN) and explore their working mechanisms,and to provide experimental evidence for the potential drug-based treatment of radiationinduced pulmonary fibrosis.Methods RLE-6TN cells were cultured in vitro and divided into four groups according to the experimental purposes:control group (C),radiation group (R),5-azacytidine group (A),and radiation followed by 5-azacytidine group (R + A).The microstructural changes in cells were determined by transmission electron microscopy.Inverted phase-contrast microscopy revealed the morphological changes in cells.The mRNA expression levels of E-cadherin and α-SMA were measured by quantitative real-time polymerase chain reaction (qRT-PCR).The protein expression levels of E-cadherin,GSK3 β,and p-GSK3 β (Ser9) were measured by Western blot.The one-way analysis of variance was used for pairwise comparison.Results The cells in group R became spindle-like.Similar morphological changes were not observed in cells in group R + A.Osmiophilic lamellar bodies disappeared at last in cells in group R.RT-PCR results showed that compared with group C,group R had a significantly lower mRNA expression level of E-cadherin ((0.23 ± 0.06) vs.(1.00 ± 0.00),P =0.002)) and a significantly higher mRNA expression level of α-SMA ((2.91 ± 0.01) vs.(1.00 ± 0.00),P =0.000)).However,compared with group R,group R + A had a significantly higher mRNA expression level of E-cadherin ((0.47 ± 0.05) vs.(1.00 ± 0.00),P =0.024)) but a significantly lower mRNA expression level of α-SMA ((2.50 ± 0.02) vs.(1.00 ±0.00),P =0.037)).The results of Western blot showed that the protein expression level of Ecadherin was significantly reduced ((0.07 ± 0.01) vs.(0.48 ± 0.02),P =0.028)),while the protein expression level of p-GSK3β was significantly increased in Group R than in Group C ((0.85 ± 0.04) vs.(0.23 ± 0.03),P =0.031)).However,compared with group R,group R + A had a significantly lower protein expression level of E-cadherin ((0.25 ± 0.00) vs.(0.07 ± 0.01),P =0.024)) and significantly less up-regulation of the protein expression level of p-GSK3β ((0.39 ± 0.03) vs.(0.85 ± 0.04),P =0.014)).Conclusions X-ray radiation can induce the epithelial-mesenchymal transition in epithelial cells.5-azacytidine suppresses radiation-induced epithelial-mesenchymal transition by inhibition of the activity of p-GSK3β in RLE-6TN cells.

Preliminary study of NDRG1 gene promoter methylation status in prostate cancer / 中华泌尿外科杂志

Objective To evaluate the methylation status of prostate cancer NDRG1 gene promoter region,and to explore the influence of methylation inhibitor 5-azacytidine on NDRG1 gene's mRNA expression in prostate cancer cells and its effects on cell proliferation.Methods Bisulfite-sequencing PCR (BSP) were used to detect the NDRG1 gene promoter methylation status in prostate cancer and BPH tissue,prostate cancer cell lines (PC3,22RV1,LNCaP and DU145) and human normal prostate cell line's RWPE-1.After 10 μmol/L 5-azacytidine were used on LNCaP and DU145 cells for 72 h,5-azacytidine's influence on cell proliferation was analyzed with MTT,two prostate cancer cell lines NDRG1 mRNA expressions were detected with RT-PCR.Results The methylation rates of NDRG1 gene in prostate cancer cell lines PC-3,22RV1,LNCaP and DU145 were (24.8 ± 3.3) ％,(36.2 ± 2.5) ％,(48.6 ± 2.8) ％ and (69.5 ± 1.7) ％,respectively.Methylation rate of Human normal prostate cell lines RWPE-1 was (4.8 ± 4.5) ％;prostate carcinoma was (48.6 ± 5.3) ％,BPH tissue was (4.3 ± 2.1) ％.The differences between groups were statistically significant.After 10 μmol/L 5-azacytidine added on LNCaP and DU145 cells for 72 h,NDRG1 gene demethylation occurred in both cells,its mRNA expression enhanced 8-9 times compared with previous and its cell growth was inhibited (P ＜ 0.05).Conclusions NDRG1 gene promoter region's hypermethylation is one of the reasons of its aberrant expression in prostate cancer.5-azacytidine can reverse NDRG1 gene promoter methylation status,regulate the expression of the gene and can inhibit prostate cancer cell proliferation.

Effects of 5-azacytidine’s demethylation for P16 gene on the proliferation and apoptosis of hemangioma cell / 中国生化药物杂志

Objective To study the effects of 5-azacytidine’s demethylation for P16 gene on hemangioma cell’s proliferation and apoptosis.Methods Bisulfite sequencing PCR was applied to detect P16 gene′s promoter methylation status in 5-azacytidine treated and untreated EOMA cell line.RT-PCR and western blot were used to detect the P16 gene mRNA and protein expressions.Flow cytometry was used to detect cell proliferation, cell cycle and cell apoptosis.The differences of P16 gene′s promoter methylation status,mRNA and protein expressions,cell proliferation,cell cycle and apoptosis in two groups were compared. Results The methylation rates in 1st and 13th CGs were 0%after 5-azacytidine treatment in EOMA hemangioma cell line,which were lower than in untreated cells.The mRNA and protein expressions increased after 5-azacytidine treatment,which were significantly higher than in untreated cells.The absorbance,S phase and G2/M phase and PI after 5-azacytidine treatment were lower than untreated cells,while the G0/G1 phase and apoptosis rates were higher.Conclusion The P16 gene promoter is hypermethylated in hemangioma cells with silent gene expressions.5-azacytidine could reverse P16 gene′s promoter methylation and silent gene expressions,which inhibit hemangioma cell’s proliferation and promote apoptosis.

5-azacytidine induces apoptosis of bone marrow mesenchymal stem cells in mouse / 中华实用儿科临床杂志

Objective To investigate the effect of 5-azacytidine(5-AZA) on apoptosis of bone marrow mesenchymal stem cells(BMSCs).Methods BMSCs were isolated from bone marrow of mouse tibia and femur; the expression of MSC specific markers CD44 and CD90 in BMSCs was measured by immunofluorescence staining;BMSCs were cultured in vitro in the medium supplemented with 0,10 and 20 μmol/L 5-AZA for 48 hours.Cell apoptosis was measured with fluorescent labeled inhibitor of caspases (FLICA) apoptosis kit and 4',6-diamidino-2-phenylindole (DAPI) staining ;the expression of apoptosis-related proteins Annexin V and Caspase-3 in the treated BMSCs was detected by Western blot.Results In this study,BMSCs positively expressed MSC specific markers CD44 and CD90.DAPI staining and Caspase-3 staining both showed that 10 and 20 μmol/L 5-AZA markedly increased apoptotic rate of BMSCs;the apoptosis-positive rate in DAPI staining was (21.086 ± 2.601) ％,(34.467 ± 3.724) ％ and (46.512 ± 3.864) ％,the apoptosis-positive rate in Caspase-3 staining was (5.354 ± 0.735)％,(15.462 ± 2.385)％ and (28.190 ± 4.190)％ in the controls,10 and 20 μmol/L 5-AZA groups,and there were significant differences among the control group and 5-AZA treated groups(all P ＜0.01).Western blot assay showed that Annexin V and Caspase-3 were both markedly upregulated in 5-AZA treated cells;the relative level of Annexin V expression was(26.612 ±2.184)％,(42.873 ±4.313)％ and (50.056 ± 4.457) ％,the relative level of Caspase-3 expression was (19.231 ± 2.683) ％,(38.618 ± 5.385) ％ and(91.235 ± 7.116)％ in the controls,10 and 20 μmol/L 5-AZA groups,and there were significant differences among the control group and 5-AZA treated groups (all P ＜ 0.01).Conclusion The commonly used doses of 5-AZA can induce apoptosis of BMSCs.

Uso tópico de 5-azacitidina melhora a cicatrização de feridas cutâneas de roedores por meio do sistema ativina/folistatina / Topical 5-azacytidine accelerates skin wound healing in rats

O desenvolvimento de métodos que tem por objetivo acelerar e melhorar a qualidade do processo de cicatrização de feridas tem impacto positivo na condução de distúrbios de cicatrização associados a inúmeras condições médicas. Neste estudo, avaliamos os efeitos moleculares, celulares e clínicos da aplicação tópica de 5-azacitidina na cicatrização de feridas em ratos. De acordo com estudos pregressos, a 5-azacitidina reduz a expressão de folistatina, que é um regulador negativo das ativinas. Estas, por sua vez, promovem o crescimento de células em diferentes tecidos, incluindo a pele. Ratos Wistar machos com oito semanas de vida foram submetidos a um ferimento cutâneo com punch de oito milímetros na região dorsal. A seguir os ratos foram aleatoriamente separados em grupo controle (veículo) ou submetidos à aplicação tópica de 5-azacitidina (10 mM), uma vez por dia por até 12 dias, iniciando-se no terceiro dia após a lesão. A documentação fotográfica e coleta de amostras ocorreram nos dias 5, 9 e 15. O emprego desta droga resultou em aceleração da cicatrização da ferida, (99,7±7,0% versus 71,2±2,8% no dia 15, p <0,01). Este resultado clínico foi acompanhado pela redução de aproximadamente três vezes na expressão protéica de folistatina. O exame histológico da pele revelou re-epitelização eficiente com aumento da expressão de queratinócitos e aumento significativo na expressão do gene de TGF-β além da diminuição significativa de citocinas, tais como TNF-α e IL-10. Analisamos também a proliferação celular na lesão de pele através do método de incorporação de BrdU. O número de células positivas para BrdU aumentou significativamente quando comparado ao controle. No entanto, quando folistatina exógena foi aplicada na pele em paralelo ao tratamento tópico de 5-azacitidina a maioria dos benefícios do medicamento foi perdida.

The development of new methods aimed at improving wound healing may have an impact on the outcomes of a number of medical conditions. Here we evaluate the molecular and clinical effects of topical 5-azacytidine, a compound used in myelodysplasia, on the wound healing in rats. According to previous studies, 5-Azacytidine decreases the expression of follistatin 1, which is a negative regulator of activins. Activins, in turn, promote cell growth in different tissues, including the skin. Eight-week old male Wistar rats were submitted to an 8 mm punchwound in the dorsal region. After three days, rats were randomly assigned to either control or topical application of a solution containing 5-azacytidine (10mM), once a day. Photo documentation and collection of samples occurred at days 5, 9 and 15. Overall, 5-azacytidine resulted on a significant acceleration of complete wound healing (99.7% ±0.7.0 vs. 71.2%±2.8 on days 15; n=10; p<0,01). This was accompanied by an up to 3-fold reduction in follistatin expression. Histological examination of the skin revealed efficient reepithelization with increase in gene expression of TGF-β and keratinocytes markers, involucrin and citokeratin, besides the significant decrease of cytokines such as TNF-α and IL-10. In addition, we analyzed cell proliferation in injured skin employing the BrdU incorporation method. The treatment with 5-azacytidine led to a progressive increase of BrdU positive cells. Finally when recombinant follistatin was employed in the skin in parallel to topical 5-azacytidine most of the benefits of the drug were lost. Thus, 5-azacytidine acts, at least in part, through the follistatin/activin pathway to improve wound healing in rats. This study belongs to online research process Caring in Nursing and Health.

Diferenciação de células-tronco mesenquimais derivadas do tecido adiposo em cardiomiócitos / Differentiation of adipose tissue-derived mesenchymal stem cells into cardiomyocytes

FUNDAMENTO: O potencial de renovação e proliferação dos cardiomiócitos, in vivo, é pequeno, e por isso, o músculo cardíaco apresenta limitada capacidade de repor células perdidas. Na tentativa de minimizar os danos oriundos de lesões hipóxico-isquêmicas e daquelas que acometem o sistema de condução do coração, a terapia celular com células-tronco mesenquimais (MSC) vem sendo utilizada, inclusive com cardiomiócitos diferenciados a partir de MSC. OBJETIVO: O presente trabalho comparou três protocolos distintos de indução de diferenciação objetivando a sugestão de um método viável para a diferenciação de maior número de células funcionais que expressem fenótipo cardiomiogênico. MÉTODOS: Culturas de MSC obtidas de tecido adiposo de ratos jovens da linhagem Lewis transgênicos para proteína verde fluorescente (GFP) foram submetidos a três diferentes meios de diferenciação cardiogênica: Planat-Bérnard, 5-azacitidina e meio Planat-Bérnard + 5-azacitidina e observadas quanto a expressão de marcadores celulares cardíacos. RESULTADOS: Nos três protolocos utilizados observou-se formação da proteína alfa-actinina sarcomérica no citoesqueleto das células submetidas à diferenciação, expressão de conexina 43 na membrana nuclear e citoplasmática e formação de gap junctions, necessárias para a propagação do impulso elétrico no miocárdio, contudo, em nenhum protocolo foi observada contração espontânea das células submetidas à diferenciação cardiogênica. CONCLUSÃO: A indução com 5-azacitidina proporcionou diferenciação celular cadiomiogênica efetiva e similar à encontrada com o meio Planat-Bénard e, por ser um protocolo mais simples, rápido e com menor custo torna-se o método de eleição.

BACKGROUND: Cardiomyocytes have small potential for renovation and proliferation in vivo. Consequently, the heart muscle has limited capacity of self-renewal. Mesenchymal stem cells (MSC) therapy, as well as MSC differentiated into cardiomyocytes, has been used in the attempt to minimize the effects of ischemic-hypoxic lesions and those affecting the electrical conduction system of the heart. OBJECTIVE: The present study compared three distinct protocols for induced differentiation of MSC into cardiomyocytes aimed at finding a viable method for producing a large number of functional cells expressing cardiomyogenic phenotype. METHODS: Mesenchymal stem cells were obtained from the adipose tissue of young transgenic Lewis rats expressing green fluorescent protein (GFP), and submitted to three distinct differentiation-inducing media: 1) Planat-Bérnard, 2) 5-azacytidine, and 3) Planat-Bérnard + 5-azacytidine; further, these cells were identified based on the expression of cardiac cell markers. RESULTS: All three protocols detected the expression of sarcomeric-alpha-actinin protein in the exoskeleton of cells, expression of connexin-43 in the nuclear and cytoplasmic membrane, and formation of gap junctions, which are necessary for electrical impulse propagation in the myocardium. However, no spontaneous cell contraction was observed with any of the tested protocols. CONCLUSION: Induction with 5-azacytidine provided an effective cadiomyogenic cellular differentiation similar to that obtained with Planat-Bénard media. Therefore, 5-azacytidine was the method of choice for being the simplest, fastest and lowest-cost protocol for cell differentiation.

Expression of specific genes of cardiomyocytes differentiated from human umbilical cord mesenchymal stem cells / 中国组织工程研究

BACKGROUND: Intramyocardial transplantation of autologous umbilical cord-derived mesenchymal stem cells for repair of myocardial tissue damage is paid increasing attention in the cardiovascular field. OBJECTIVE: Human umbilical cord mesenchymal stem cells were isolated and cultured. Passage 2 human umbilical cord mesenchymal stem cells were treated with various concentratins of 5-azacytidine (2.5, 5, 10, 20, 40, 80 μmol/L) for 24 hours . After removal of 5-azacytidine, cells were cultured for another 4 weeks. RESULTS AND CONCLUSION: Before 5-azacytidine treatment, filament-like structures or particles were not observed in the cells, but the amount of cytoplasm was less and uniform, nuclear/cytoplasm ratio was high, cells exhibited typical fusiform appearance and grew in a swirl-like manner, and nucleolus was obvious. After treatment with 5-azacytidine for 24 hours, some cells died in each group, and typical fusiform appearance turned into stick-like or column-like appearance, especial y in the 40 and 80 μmol/L 5-azacytidine groups. Reverse transcription-PCR results showed that atrial natriuretic peptide and α-skeletal actin gene expression levels were detected on human umbilical cord mesenchymal stem cells after treatment with 2.5 or 40 μmol/L 5-azacytidine for 4 weeks or with 5, 10, 20 μmol/L 5-azacytidine for 1, 2, 3 and 4 weeks. These findings suggest that 5-azacytidine-induced human umbilical cord mesenchymal stem cells express the specific gene of myocardiocytes.

Cardiac troponin I expression in human umbilical cord mesenchymal stem cells after 5-azacytidine induction in vitro / 中国组织工程研究

10.3969/j.issn.2095-4344.2013.23.005

Mouse embryonic hepatic stem cells differentiate into cardiomyocyte-like cells / 中国组织工程研究

BACKGROUND:In recent years, embryonic hepatic stem cel s have attracted more attention, but there are few reports on the potential of embryonic hepatic stem cel s to differentiate into cardiomyocyte-like cel s as wel as the related differentiation conditions. OBJECTIVE:To investigate the moderate condition to induce mice embryonic hepatic stem cel s to differentiate into cardiomyocyte-like cel s in vitro with chemical reagents. METHODS:Dimethylsulfoxide in combination with 5-azacytidine with different concentrations and time were used to induce the embryonic hepatic stem cel s of 13.5 days mice and to observe the differentiation effect. RESULTS AND CONCLUSION:Under in vitro conditions, 0.8%dimethylsulfoxide+5μmol/L 5-azacytidine could induce the mouse embryonic hepatic stem cel s to express the specific markers of myocardial cel s, while increasing the concentration of the inducer and extending the induction time could not improve the induction efficacy.

Effects of mechanical stimulus on mesenchymal stem cells differentiation toward cardiomyocytes.

Background: Mesenchymal stem cells (MSCs) known to be sensitive to mechanical stimulus. This type of stimulus plays a role in cellular differentiation, so that it might affect MSCs differentiation toward cardiomyocytes. Objectives: Investigate the effect of mechanical stimulus on MSCs differentiation toward cardiomyocytes. Methods: The adipose tissue-derived MSCs were induced to differentiate with 5-azacytidine, and stimulated by one Hz mechanical stretching up to 8%. After 10 days, the cell’s cardiac markers and cardiogenesis-related genes were detected by immumohistochemistrical staining and reverse transcriptase-polymerase chain reaction, and the cell’s ATPase activity was detected. Results: The cyclic mechanical stretching enhanced the expression of cardiogenesis-related genes and cardiac markers, and stimulated the activity of Na+-K+-ATPase and Ca2+-ATPase in the MSCs treated with 5-azacytidine. Without 5-azacytidine pre-treatment, cyclic mechanical stretch alone has little effect. Conclusion: Mechanical stretch combined with 5-azacytidine treatment could accelerate MSCs differentiation toward cardiomyocytes.

5-azacytidine induces PD-1 gene promoter demethylation and PD-1 protein expression in human lymphoid cell series Molt-4 cells / 中华微生物学和免疫学杂志

Objective To investigate the demethylation and changes in gene expression of programmed death receptor-1 ( PD-1) caused by methylation inhibitor 5- azacytidine (5-Zac) in lymphocyte series Molt-4 cells and its mechanism. Methods Molt-4 cells were cultured in different concentrations of 5-Zac(0, 5, 10 Umol/L)for 72 h, ratio of cell expressing PD-1 and apoptosis rate were detected by FCM, transcription of PD-1 gene mRNA was detected by RT-PCR. Molt-4 cell DNA of all groups were disposed by sodium bisulfite, PD-1 gene promoter fragment binded with transcription factor Brn-2 was amplified by PCR,these amplification fragments were transformed into E. coli. Positive clones were selected by sequencing,methylation status of the fragments binded with transcription factor Brn-2 was examined. Results S-Zac could increase the PD-1 expression of Molt-4 cells. PD-1 expression rate in 0 μmol/L 5-Zac( 1. 13%±0.01% ) treated cells was found more lower than that in both 5 μmol/L and 10 μmol/L 5-Zac treated cells (18. 96% ±1. 87% , 63. 09% ± 6. 25% , P < 0. 05 ) , and they showed concentration-dependent (P <0.01). Cells apoptosis rate and PD-1 mRNA expression were also observed increased significantly with 5-Zac treating. Demethylation probability of CG points showed significant difference between transcription factor Brn-2 binding site and other four locations (P < 0.05 ). Conclusion 5 -Zac inhibits cell grouth in human lymphoid cell series Molt-4 by inducing PD-1 gene expression and promoter demethylation. PD-1 gene promoter binding transcription factor Brn-2 fragment CG point demethylation may be one of the important mechanisms in 5-Zac treated Molt-4 cells.

The mechanism underlying the differentiation of human umbilical cord-derived mesenchymal stem cells into myocardial cells induced by 5-azacytidine.

Objective: To investigate the molecular mechanism underlying the differentiation of human umbilical cord-derived mesenchymal stem cells (hUCMSCs) into myocardial cells induced by 5-azacytidine (5-aza), and to explore the expression and significance of DLL4-Notch signaling in this process. Materials and Methods: hUCMSCs were isolated and purified from the umbilical cords of normal or cesarean term deliveries under sterile conditions. After treatment with 5-aza for 24 h, hUCMSCs was continued to culture, the expression of GATA4 and NKx2.5 at 4 weeks after induction, DLL4 and Notch1 mRNA at 1d, 3d, 5d, 7d after induction were detected. The expression of cardiac troponin I (cTnI) after 4 weeks was determined by immunocytochemistry. Results: hUCMSCs treated with 5-aza were stained positively for cTnI 4 weeks after induction. The expression of Notch1 and DLL4 mRNA in the 5-aza-induced group was stable and significantly higher than that in the control group (mean Ct value for the Notch1 gene: 0.51 ± 0.21 in the 5-aza-induced group vs. 7.85 ± 0.35 in the control group; mean Ct value for the DLL4 gene: 1.60 ± 0.49 in the 5-aza-induced group vs. 12.42 ± 0.73 in the control group). Similar results were observed for Nkx2.5 and GATA4 genes. The expressions of Nkx2.5 and GATA4 mRNA in the 5-aza group were 4.72 ± 0.58 and 3.76 ± 0.06 times higher than that in the control group, respectively, with statistical significance. Conclusions: hUCMSCs can be differentiated into myocardial cells by 5-aza induction in vitro. 5-Aza may affect this process by regulating the expression of GATA4 and Nkx2.5 genes. The DLL4-Notch signal pathway may be involved in this process.