Effect of Raldh2 silence on the differentiation of P19 cells into cardiomyocyte-like cells / 复旦学报（医学版）

Objective To study the effect of retinaldehyde dehydrogenase 2 (Raldh2) on the differentiation of P19 cell to cardiomyocyte-like cells and explore the potential mechanism.Methods A miRNA expression plasmid specific to Raldh2 was packaged and constructed by RNA interference (RNAi) method.The P19 stable cell line carried Raldh2 miRNA expression was selected by adding blasticidin and induced to differentiate towards cardiomyocyte-like cells.The mRNA levels of myocardium development-related markers were determined by qPCR at different stages during the differentiation process.Results The miRNA expression plasmid specific to Raldh2 could effectively suppress Raldh2 expression,and the MiRaldh2 group,a P19 stable cell line was established successfully in which the knockdown efficiency on Raldh2 was 91％ (t =25.52,P＜0.000 1,95％ CI:0.81-1.01).When compared with P19 group,the mRNA levels of cardiac transcription factors were generally decreased in the MiRaldh2 group during the whole differentiation process.In detail,on the 7th day,the relatively low expression rates of these cardiac markers including Gata4,Tef-1,N-myc,α-mhc and Ctnt was0.16±0.01 (t=17.29,P＜0.000 1),0.51 ±0.02 (t=3.564,P=0.023 5),0.23 ±0.01 (t=13.17,P =0.000 2),0.20 ± 0.02 (t=17.76,P＜0.000 1) and 0.59 ± 0.06 (t =3.642,P =0.021 9) in MiRaldh2 group when compared with the P19 group.Conversely,the mRNA levels of Nkx2.5 and Hand2 were dramatically increased in MiRaldh2 group on day 2 to 7 and the expression rates on the 7th day was 2.25 ± 0.35 (t =3.526,P =0.024 3) compared with the P19 group while Hand2 was 3.58 ± 0.20 (t =9.214,P =0.011 6).Conclusions Knockdown of Raldh2 inhibits the P19 cells differentiated into cardiomyocyte-like cells,which suggests that Raldh2 may play a potential role in early development of heart.The low expression of Raldh2 might be an explanation of the cardiac malformations associated with retinoic acid deficiency.

Optimal concentration of 1, 25-vitamin-D3 to induce the differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells in vitro / 解剖学报

Objective To explore the optimal concentration of 1, 25-vitamin-D3 for inducing the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cardiomyocyte-like cells in vitro. Methods BMSCs of SD rats were isolated and cultured by whole bone marrow adherent method combined with density gradient centrifugations. Depending on the final concentration of 1, 25-vitamin-D3, the 2nd-generation BMSCs were divided into five groups: 3 nmol/L group, 6 nmol/L group, 12 nmol/L group, 24 nmol/L group and the control group. The adherent cells were observed dynamically under the inverted phase contrast microscope, including their morphology and growth status. The surface antigens, morphological characteristics, protein expression and mRNA expression of the cells in each group were assessed. Results 1. Under the inverted phase contrast microscope, most of the primary cells showed a short spindle shape after 72 hours of culture. After 1 week of culture, the cells showed diversified morphology. The adjacent cells of BMSCs, induced after 4 weeks, were closely connected with each other, and the arrangement had obvious directivity. There were differences in the number and morphology of BMSCs, induced by 1, 25-vitamin-D3 at different concentrations. 2. The result of flow cytometry showed that the positive expression rates of CD29, CD45 and CD90 were 97. 4%, 3. 3% and 91. 4%, respectively, 3. The results of immunofluorescence, immunocytochemistry and Western blotting revealed that the expressions of tropomyosin (TPM), connexin43 (Cx43) and cardiac troponin T(cTnT) in 6 nmol/L group were significantly higher than that of the other groups, while that in control group were weak or negative (P<0. 05). 4. Transmission electron microscope(TEM) observation showed that the induced cells had a cardiomyocyte-like ultrastructure: there were many parallel arranged myofilaments, mitochondria, ribosomes, rough endoplasmic reticulum and other organelles in the cytoplasm. 5. The result of Real-time PCR showed that the induced cells could express GATA binding protein 4(GATA 4) and Nkx2. 5 at the 1st week, and then the expression of them decreased at the 2nd week, but then increased at the 4th week. The 6 nmol/L group was superior to the other three groups in gene expression (P<0. 05). Conclusion 1, 25-vitamin-D3 can induce BMSCs to obtain myocardial differentiation phenotype, and the optimum concentration of inducing differentiation is 6 nmol/L.

Cardiac extracellular matrix hydrogel together with or without inducer cocktail improves human adipose tissue-derived stem cells differentiation into cardiomyocyte-like cells.

Studies have demonstrated that differentiation of stem cells into cardiomyocytes is a complex phenomenon that requires sufficient inducing factors at various time points. Cardiac extracellular matrix (cECM) could provide tissue specific microenvironment and act as an inductive template for efficient cell differentiation. The aim of this study was to investigate the effect of cECM on differentiation of human adipose tissue-derived stem cells (hADSCs) into cardiomyocytes using cECM hydrogel in combination with a cardiac inductive cocktail. hADSCs were cultured on ECM-coated plates with and without inductive cocktail for 3weeks. qRT-PCR and western blot analysis were used to evaluate the expression pattern of special cardiac genes and proteins. When hADSCs were cultured in the presence of cECM cardiac genes including GATA4, HAND1, HAND2, NKX2.5, Troponin I, ßMHC, Connexin43 were highly expressed in differentiated cells. Also Connexin43, cTnI and ßMHC proteins were expressed as well. We could show that cECM by itself could affect viability, proliferation and differentiation of hADSCs. However, combination of cECM with a cardiac inducing cocktail could improve the results.

Inducing bone marrow mesenchymal stem cells to differentiate into cardiomyocyte-like cells via Wnt-11 in vitro / 西安交通大学学报(医学版)

Objective To probe into the optimal concentration of Wnt-11 to induce the differentiation of bone marrow mesenchymal stem cells (BMMSCs)into cardiomyocyte-like cells in vitro.Methods BMMSCs were isolated from the bone marrow of SD rats using whole bone marrow culture method.After cultured for 48 h, BMMSCs of the second generation were utilized for directed induction.Based on the final concentration of Wnt-11 , BMMSCs were divided up into Group A (100 ng/mL),Group B (200 ng/mL),Group C (400 ng/mL)and Group D (blank control).After 72-hour induction,the cells were cultured in complete medium for 4 weeks while cells in Group D were cultured only in the complete medium.The morphological changes were observed under the phase contrast microscope.Surface antigen expression of BMMSCs was identified by flow cytometry.When cells were cultured for 4 weeks,the expressions of Desmin,Connexin43 and cTnI were detected by immunocytochemistry. Meanwhile, the ultrastructural changes were observed using transmission electron microscope. The mRNA expressions of cardiac transcription factors GATA-4,Nkx2.5 andα-MHC in BMMSCs were detected by RT-qPCR at 1,2 and 4 weeks after induction.Results Primary BMMSCs formed cell colonies at 2 weeks;the cells were mainly fusiform or star-shape,and a few irregularly-shaped ones were also visible.The passaged cells were larger than those of primary culture.After induction,the cells exhibited long shuttle-shape and were aligned in parallel. Flow cytometery displayed that the positive rate of the surface antigens of BMMSCs CD29,CD45,and CD90 was 97.9%,0.4% and 99.5%,respectively.When BMMSCs-induced via Wnt-11 were cultured for 4 weeks,Desmin, cTnI and Connexin43 were all positively expressed in induction groups.Whereas in the blank control group they were slightly positive or negative;the positive rate in Group B was the highest (P<0 .05 ).Transmission electron microscopy exhibited that organelles such as rough endoplasmic reticulum,mitochondria,as well as some ribosomes were visible in the cytoplasm of these cells in each induction group.In addition,myofilaments were arranged in parallel in the cytoplasm.The cells in induction groups could express GATA-4 and Nkx2 .5 in the first week,and then the expression of them decreased in the second week,but then increased in the fourth week;gene expression in induction Group B was significantly higher than in the other two induction groups (P<0 .05 ).The expression of GATA-4 and Nkx2 .5 in Group D was 1 ,α-MHC was not expressed in the four groups during the culture period. Conclusion Wnt-11 can induce the differentiation of BMMSCs into cardiomyocyte-like cells in vitro,and the optimal concentration of Wnt-11 is 200 ng/mL.

A xanthine-derivative K(+)-channel opener protects against serotonin-induced cardiomyocyte hypertrophy via the modulation of protein kinases.

This study investigated whether KMUP-1, a xanthine-derivative K(+) channel opener, could prevent serotonin-induced hypertrophy in H9c2 cardiomyocytes via L-type Ca(2+) channels (LTCCs). Rat heart-derived H9c2 cells were incubated with serotonin (10 µM) for 4 days. The cell size increased by 155.5%, and this was reversed by KMUP-1 (≥1 µM), and attenuated by the LTCC blocker verapamil (1 µM) and the 5-HT2A antagonist ketanserin (0.1 µM), but unaffected by the 5-HT2B antagonist SB206553. A perforated whole-cell patch-clamp technique was used to investigate Ca(2+) currents through LTCCs in serotonin-induced H9c2 hypertrophy, in which cell capacitance and current density were increased. The LTCC current (ICa,L) increased ~2.9-fold in serotonin-elicited H9c2 hypertrophy, which was attenuated by verapamil and ketanserin, but not affected by SB206553 (0.1 µM). Serotonin-increased ICa,L was reduced by KMUP-1, PKA and PKC inhibitors (H-89, 1 µM and chelerythrine, 1 µM) while the current was enhanced by the PKC activator PMA, (1 µM) but not the PKA activator 8-Br-cAMP (100 µM), and was abolished by KMUP-1. In contrast, serotonin-increased ICa,L was blunted by the PKG activator 8-Br-cGMP (100 µM), but unaffected by the PKG inhibitor KT5823 (1 µM). Notably, KMUP-1 blocked serotonin-increased ICa,L but this was partially reversed by KT5823. In conclusion, serotonin-increased ICa,L could be due to activated 5-HT2A receptor-mediated PKA and PKC cascades, and/or indirect interaction with PKG. KMUP-1 prevents serotonin-induced H9c2 cardiomyocyte hypertrophy, which can be attributed to its PKA and PKC inhibition, and/or PKG stimulation.

Differentiation of Human Amniotic Mesenchymal Cells into Cardiomyocyte-like Cells in Vitro / 中国生物工程杂志

To explore the plasticity of human amniotic mesenchymal cells(hAMCs) into cardiomyocyte-like cells,hAMCs were isolated from human amnion with collagenase digestion.Phenotype of the isolated cells was analyzed by flow cytometry(FCM).hAMCs were treated with 5-azacytidine and basic fibroblast growth factor(bFGF) to investigate their ability of differentiation into cardiomyocytes.The induced differentiated cells were evaluated by immunofluorescence for desmin and ?-actin expression and by RT-PCR for Nkx2.5,GATA-4 and alpha-myosin heavy chain(?-MHC) mRNA expression.The results showed that,after primary culture,hAMCs could reach a confluence of 80% with swirl like growth at 6 days.The cells proliferated rapidly after passages with a 100% confluence at 3~4 d.hAMCs were positive expression of CD44 and vimentin,but negative for CK19.After induced differentiation at 8~10d,the differentiated cells have close-up arranged with long spindle-shape.At 2 weeks and 4 weeks,induced cells expressed ?-actinin and cardiac-specific transcription factor Nkx2.5.Expressions of GATA-4 and desmin can be detected but ?-MHC can not in the hAMCs both before and after the induction.In conclusion,hAMCs have the ability of differentiation into cardiomyocyte-like cells,which means that hAMCs can be regarded as candidate cells for cellular cardiomyoplasty(CCM).