Cardiomyogenic Differentiation Research Articles

Abstract 534: Modulatory Effect of Nkx2.5+ Cardiomyoblast Secreted Exosomes in Cardiometabolism

It was found cardiac precursors secreted exosomes (CPC-exo) may have anti-apoptotic and anti-fibrotic actions to facilitate cardiac repair in the damaged hearts. It remains unclear the effect of CPC-exo in cardiometabolism. The aim of the study was to assess the modulatory action of CPC-exo in cardiometabolism of cardiomyopathic cardiomyocytes. Nkx2.5+ cardiomyoblasts were enzymatically isolated and were purified by FACS sorter from mouse embryonic stem cells under cardiomyogenic differentiation or transgenic mice hearts (2 weeks old) carrying GFP reporter driven by Nkx2.5-enhancer for the purification of the exosomes (mESC-Nkx2.5+exo and postnatal-Nkx2.5+exo) from the culture medium by ExoQuick-TC. The vesicle size was further measured by Nanosight. The exosomal miRNA profile was characterized by miRNA microarray. It was further compared the different effect of mESC-Nkx2.5+exo and postnatal-Nkx2.5+exo in the mitochondrial function of human cardiomyocytes derived from iPSC of left ventricle non compaction (LVNC) patients (LVNC-hiPSC-CM) by Agilent seahorse XF24 analyzer. Cell shortening was measured by Ionoptix system. It was found the defect of cardiac mitochondrial function associated with the weak cell shortening in LVNC-hiPSC-CM on day 70 after differentiation. With the treatment of mESC-Nkx2.5+exo and postnatal-Nkx2.5+exo during day 50-70, the mitochondrial function and cell shortening of LVNC-hiPSC-CM were significantly improved on day 70. The functional exosomal miRNAs were characterized in advance in this study. In conclusion, CPC-exo may exert the benefit in the improvement of cardiometabolism in cardiomyopathic cardiomyocytes.

Pre-Conditioning Stem Cells in a Biomimetic Environment for Enhanced Cardiac Tissue Repair: In Vitro and In Vivo Analysis.

Stem cell-based therapies represent a valid approach to restore cardiac function due to their beneficial effect in reducing scar area formation and promoting angiogenesis. However, their translation into the clinic is limited by the poor differentiation and inability to secrete sufficient therapeutic factors. To address this issue, several strategies such as genetic modification and biophysical preconditioning have been used to enhance the efficacy of stem cells for cardiac tissue repair. In this study, a biomimetic approach was used to mimic the natural mechanical stimulation of the myocardium tissue. Specifically, human adipose-derived stem cells (hASCs) were cultured on a thin gelatin methacrylamide (GelMA) hydrogel disc and placed on top of a beating cardiomyocyte layer. qPCR studies and metatranscriptomic analysis of hASCs gene expression were investigated to confirm the correlation between mechanical stimuli and cardiomyogenic differentiation. In vivo intramyocardial delivery of pre-conditioned hASCs was carried out to evaluate their efficacy to restore cardiac function in mice hearts post-myocardial infarction. The cyclic strain generated by cardiomyocytes significantly upregulated the expression of both mechanotransduction and cardiomyogenic genes in hASCs as compared to the static control group. The inherent angiogenic secretion profile of hASCs was not hindered by the mechanical stimulation provided by the designed biomimetic system. Finally, in vivo analysis confirmed the regenerative potential of the pre-conditioned hASCs by displaying a significant improvement in cardiac function and enhanced angiogenesis in the peri-infarct region. Overall, these findings indicate that cyclic strain provided by the designed biomimetic system is an essential stimulant for hASCs cardiomyogenic differentiation, and therefore can be a potential solution to improve stem-cell based efficacy for cardiovascular repair.

Quantitative Secretomics Reveals Extrinsic Signals Involved in Human Pluripotent Stem Cell Cardiomyogenesis.

Human pluripotent stem cells can be differentiated in vitro into cardiomyocytes (CMs) but the molecular mechanisms behind this process are still not fully understood. In particular, the identification of morphogens remained elusive because the mass spectrometry-based identification of secreted proteins from cell culture supernatants is impeded by high levels of albumin present in common differentiation media. An albumin-free cardiomyogenic differentiation medium is established in this study and applied for secretomics at seven different time points during in vitro differentiation. By this analysis 4832 proteins are identified with 1802 being significantly altered during differentiation and 431 of these are annotated as secreted. Numerous extrinsic components of Wnt, TGFβ, Activin A, Nodal, BMP, or FGF signaling pathways are quantitatively assessed during differentiation. Notably, the abundance of pathway agonists is generally lower compared to the respective antagonists but their curves of progression over timer were rather similar. It is hypothesized that TGFβ, Activin A, and Nodal signaling are turned down shortly upon the initiation of cardiac differentiation whereas BMP signaling is switched on. Wnt and FGF signaling peaks between d0 and d3 of differentiation, and interestingly, Activin A and TGFβ signaling seem to be reactivated at the cardiac progenitor stages and/or in early CMs.

Generation and primary characterization of iAM-1, a versatile new line of conditionally immortalized atrial myocytes with preserved cardiomyogenic differentiation capacity.

AimsThe generation of homogeneous cardiomyocyte populations from fresh tissue or stem cells is laborious and costly. A potential solution to this problem would be to establish lines of immortalized cardiomyocytes. However, as proliferation and (terminal) differentiation of cardiomyocytes are mutually exclusive processes, their permanent immortalization causes loss of electrical and mechanical functions. We therefore aimed at developing conditionally immortalized atrial myocyte (iAM) lines allowing toggling between proliferative and contractile phenotypes by a single-component change in culture medium composition.Methods and resultsFreshly isolated neonatal rat atrial cardiomyocytes (AMs) were transduced with a lentiviral vector conferring doxycycline (dox)-controlled expression of simian virus 40 large T antigen. Under proliferative conditions (i.e. in the presence of dox), the resulting cells lost most cardiomyocyte traits and doubled every 38 h. Under differentiation conditions (i.e. in the absence of dox), the cells stopped dividing and spontaneously reacquired a phenotype very similar to that of primary AMs (pAMs) in gene expression profile, sarcomeric organization, contractile behaviour, electrical properties, and response to ion channel-modulating compounds (as assessed by patch-clamp and optical voltage mapping). Moreover, differentiated iAMs had much narrower action potentials and propagated them at >10-fold higher speeds than the widely used murine atrial HL-1 cells. High-frequency electrical stimulation of confluent monolayers of differentiated iAMs resulted in re-entrant conduction resembling atrial fibrillation, which could be prevented by tertiapin treatment, just like in monolayers of pAMs.ConclusionThrough controlled expansion and differentiation of AMs, large numbers of functional cardiomyocytes were generated with properties superior to the differentiated progeny of existing cardiomyocyte lines. iAMs provide an attractive new model system for studying cardiomyocyte proliferation, differentiation, metabolism, and (electro)physiology as well as to investigate cardiac diseases and drug responses, without using animals.

Comparative effectiveness of three-dimensional scaffold, differentiation media and co-culture with native cardiomyocytes to trigger in vitro cardiogenic differentiation of menstrual blood and bone marrow stem cells

The main purpose of this study was to find effectiveness of 3D silk fibroin scaffold in comparison with co-culturing in presence of native cardiomyocytes on cardiac differentiation propensity of menstural blood(MenSCs)-versus bone marrow-derived stem-cells (BMSCs). We showed that both 3D fibroin scaffold and co-culture system supported efficient cardiomyogenic differentiation of MenSCs and BMSCs, as judged by the expression of cardiac-specific genes and proteins, Connexin-43, Connexin-40, alpha Actinin (ACTN-2), Tropomyosin1 (TPM1) and Cardiac Troponin T (TNNT2). No significant difference (except for higher expression of ACTN-2 in co-cultured MenSCs) was found between differentiation potential of the cells cultured in 3D fibroin scaffold and co-culture system. Collectively, our results imply that inductive signals served by biological factors of native cardiomyocytes to trigger cardiogenic differentiation of stem-cells may be efficiently provided by natural and biocompatible 3D fibroin scaffold suggesting the usefulness of this construct for cardiac tissue engineering.

Preinduction with bone morphogenetic protein-2 enhances cardiomyogenic differentiation of c-kit+ mesenchymal stem cells and repair of infarcted myocardium

BackgroundPreclinical and clinical trails show that c-kit+ cardiac stem cells can differentiate towards cardiovascular cells and improve cardiac function after myocardial infarction (MI). However, survival and differentiation of the engrafted stem cells within ischemic and inflammatory microenvironment are poor. Methodsc-Kit+ cells were isolated from mesenchymal stem cells (MSCs) of rat bone marrow. Reliability of preinduction with bone morphogenetic protein-2 (BMP-2) in promotion of survival and differentiation of c-kit+ MSCs was assessed in vitro and after transplantation. Resultsc-Kit+ MSCs have a potential to differentiate towards cardiomyocytes. BMP-2 promotes proliferation, migration and paracrine of the cells, and protects the cells to survive in the hypoxic condition. After induction with 10 ng/mL BMP-2 for 24 h, the cells can differentiate into cardiomyocytes at four weeks. The electrophysiological characteristics of the differentiated cells are same as adult ventricular cardiomyocytes. In rat MI models, cardiac function was improved, the size of scar tissue was reduced, and regeneration of the myocardium and microvessels was enhanced significantly at four weeks after transplantation of BMP-2-preinduced cells. The survived cells and cardiomyocytes differentiated from the engrafted cells were increased greatly. ConclusionThe results suggest that transient treatment with BMP-2 can induce c-kit+ MSCs to differentiate into functional cardiomyocytes. Preinduction with BMP-2 enhances survival and differentiation of the cells. BMP-2-primed cells promote repair of the infarcted myocardium and improvement of cardiac function. Transplantation of BMP-2-preinduced c-kit+ MSCs is a feasible strategy for MI therapy.

Photolithographic-stereolithographic-tandem fabrication of 4D smart scaffolds for improved stem cell cardiomyogenic differentiation

4D printing is a highly innovative additive manufacturing process for fabricating smart structures with the ability to transform over time. Significantly different from regular 4D printing techniques, this study focuses on creating novel 4D hierarchical micropatterns using a unique photolithographic-stereolithographic-tandem strategy (PSTS) with smart soybean oil epoxidized acrylate (SOEA) inks for effectively regulating human bone marrow mesenchymal stem cell (hMSC) cardiomyogenic behaviors. The 4D effect refers to autonomous conversion of the surficial-patterned scaffold into a predesigned construct through an external stimulus delivered immediately after printing. Our results show that hMSCs actively grew and were highly aligned along the micropatterns, forming an uninterrupted cellular sheet. The generation of complex patterns was evident by triangular and circular outlines appearing in the scaffolds. This simple, yet efficient, technique was validated by rapid printing of scaffolds with well-defined and consistent micro-surface features. A 4D dynamic shape change transforming a 2-D design into flower-like structures was observed. The printed scaffolds possessed a shape memory effect beyond the 4D features. The advanced 4D dynamic feature may provide seamless integration with damaged tissues or organs, and a proof of concept 4D patch for cardiac regeneration was demonstrated for the first time. The 4D-fabricated cardiac patch showed significant cardiomyogenesis confirmed by immunofluorescence staining and qRT-PCR analysis, indicating its promising potential in future tissue and organ regeneration applications.

TGF-β1 combined with Sal-B promotes cardiomyocyte differentiation of rat mesenchymal stem cells.

Transforming growth factor β1 (TGF-β1) and salvianolic acid B (Sal-B) are key signaling factors for stem cell differentiation into cardiomyocytes (CMs). The present study compared the biological effect of TGF-β1 and Sal-B, alone or in combination, on bone marrow mesenchymal stromal cells (BMSCs) that differentiate into myocardial-like cells in a simulated myocardial microenvironment in vitro. BMSCs were isolated from bones of limbs of 10 male Sprague Dawley rats and cultured. The 2nd-generation BMSCs were co-incubated with TGF-β1 and Sal-B, alone or in combination, for 72 h. The control group was BMSCs cultured without any inductive substance. The levels GATA binding protein 4 (GATA4) and homeobox protein NKx2.5 were determined by reverse-transcription quantitative polymerase chain reaction and immunofluorescence staining was used to evaluate α-sarcomeric actin and cardiac troponin I (cTNI) as cardiomyogenic differentiation markers. The ultrastructure of BMSCs in each group was also observed. BMSCs were initially spindle-shaped with irregular processes. The cells gradually increased in number 24 h post-inoculation and proliferated 7 days later. Compared with the control group, BMSCs in the treatment groups had fusiform shapes, orientating with one accord and were connected with adjoining cells forming myotube-like structures on day 28. The morphology and architecture/myotubes of BMSCs was similar among the treatment groups, but the amount of cells in the combined group was comparatively higher. The results of immunofluorescence staining revealed the expression of the CM-specific proteins α-sarcomeric actin and cTNI in these cells. The expression of these cardiac-specific markers in the combined group was significantly higher than that in the other groups (P<0.01 or P<0.05). In addition, the transcriptional expression of GATA4 and NKx2.5 in the treatment groups was stable and significantly higher than that in the control group on day 7. Transmission electron microscopy showed that BMSCs in the treatment groups all had myofilaments, rough endoplasmic reticulum and mitochondria in the cytoplasm when compared with the control group. Taken together, these results indicated that the combination of TGF-β1 and Sal-B effectively promotes cardiomyogenic differentiation of BMSCs in vitro and their application may represent a therapeutic strategy for the treatment of ischemic heart disease.

Cardiomyogenic differentiation of human adipose-derived mesenchymal stem cells transduced with Tbx20-encoding lentiviral vectors.

Ischemic heart disease often results in myocardial infarction and is the leading cause of mortality and morbidity worldwide. Improvement in the function of infarcted myocardium is a main purpose of cardiac regenerative medicine. One possible way to reach this goal is via stem cell therapy. Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into a variety of cell types but display limited cardiomyogenic differentiation potential. Members of the T-box family of transcription factors including Tbx20 play important roles in heart development and cardiomyocyte homeostasis. Therefore, in the current study, we investigated the potential of Tbx20 to enhance the cardiomyogenic differentiation of human adipose-derived MSCs (ADMSCs). Human ADMSCs were transduced with a bicistronic lentiviral vector encoding Tbx20 (murine) and the enhanced green fluorescent protein (eGFP) and analyzed 7 and 14 days post transduction. Transduction of human ADMSCs with this lentiviral vector increased the expression of the cardiomyogenic differentiation markers ACTN1, TNNI3, ACTC1, NKX2.5, TBX20 (human), and GATA4 as revealed by RT-qPCR. Consistently, immunocytological results showed elevated expression of α-actinin and cardiac troponin I in these cells in comparison to the cells transduced with control lentiviral particles coding for eGFP alone. Accordingly, forced expression of Tbx20 exerts cardiomyogenic effects on human ADMSCs by increasing the expression of cardiomyogenic differentiation markers at the RNA and protein level.

MicroRNA-499a-5p Promotes Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells to Cardiomyocytes.

Since the adult mammalian heart has limited regenerative capacity, cardiac trauma, disease, and aging cause permanent loss of contractile tissue. This has fueled the development of stem cell-based strategies to provide the damaged heart with new cardiomyocytes. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are capable of self-renewal and differentiation into cardiomyocytes, albeit inefficiently. MicroRNAs (miRNAs, miRs) are non-coding RNAs that have the potential to control stem cell fate decisions and are employed in cardiac regeneration and repair. In this study, we tested the hypothesis that overexpression of miR-499a induces cardiomyogenic differentiation in BM-MSCs. Human BM-MSCs (hBM-MSCs) were transduced with lentiviral vectors encoding miR-499a-3p or miR-499a-5p and analyzed by immunostaining and western blotting methods 14days post-transduction. MiR-499a-5p-transduced cells adopted a polygonal/rod-shaped (myocyte-like) phenotype and showed an increase in the expression of the cardiomyocyte markers α-actinin and cTnI, as cardiogenic differentiation markers. These results indicate that miR-499a-5p overexpression promotes the cardiomyogenic differentiation of hBM-MSCs and may thereby increase their therapeutic efficiency in cardiac regeneration.

Elucidating molecular events underlying topography mediated cardiomyogenesis of stem cells on 3D nanofibrous scaffolds

Toward engineering a cardiac patch, the objective of this work was to assess stem cell response to a three-dimensional (3D) nanofibrous scaffold and probe the underlying molecular mechanisms including both cell signaling and epigenetic changes. Cardiomyogenesis of human mesenchymal stem cells (hMSCs) in 3D poly(ε-caprolactone) (PCL) nanofibers and macroporous scaffolds was compared with two-dimensional (2D) PCL films. In addition, nanofiber mats of PCL and its blend with gelatin (PCL-Gel) were prepared with fibers of random or unidirectional alignment to assess the roles of topography (fibrous architecture and its alignment) and biochemical cue (cell-adhesive sites) in directing cell functions. Cells on 3D random nanofibers, exhibited elevated expression of known cardiac markers such as cardiac actinin, cardiac troponin and β-myocardial heavy chain compared to cells on 2D films suggesting enhanced differentiation that was further accentuated on the aligned fibers. 3D macroporous scaffolds did not enhance the cardiomyogenic differentiation. However, minimal differences were noted between cells on PCL and PCL-Gel fibers, irrespective of alignment. Co-culture with neonatal rat cardiomyocytes induced beating in the differentiated cells. The use of small molecule inhibitors revealed that cytoskeletal elements F-actin, microtubules and downstream ROCK protein are essential for the cardiomyogenesis of hMSCs on the nanofibers. The activation of ERK, AKT and mTOR was observed during cardiomyogenesis. Interestingly, enhanced differentiation on the aligned nanofibers was associated with increased level of the histone deacytelase SIRT6 and decreased level of the acetylated histone H3K9 suggesting a role for epigenetic regulation. This study demonstrates that aligned nanofibrous scaffolds augment cardiomyogenic differentiation wherein topography plays a critical role in driving stem cell function. In addition, this study offers insight into molecular pathways driving the cellular response.

Fibrin scaffold could promote survival of the human adipose-derived stem cells during differentiation into cardiomyocyte-like cells.

Human adipose-derived stem cells (hADSCs) are capable of differentiation into many cells including cardiac cells. Different types of scaffolds are used for cell differentiation but the best is yet to be determined. In this study, fibrin scaffold (3D) was fabricated using human plasma fibrinogen and compared with culture plates (2D) for the growth and differentiation of hADSCs into cardiomyocyte-like cells. For this purpose, after obtaining the properties of the isolated hADSCs and fibrin scaffold, four biochemical tests were employed to determine the relative growth rate of hADSCs in 2D and 3D cultures. To examine the effects of two different culture systems on cardiomyogenic differentiation, hADSCs were treated with 10 or 50μM 5-azacytidine (5-Aza) for 24h and followed until 10weeks. The results indicated that the growth of hADSCs in 3D significantly increased after the seventh day (P < 0.05). Western blot, qRT-PCR and immunochemistry assays were used to evaluate the rate of cardiac differentiation, which showed significantly higher expression of special cardiac genes such as NKX2.5, Cx43, MLC2v, βMHC, HAND1, HAND2 and cTnI (P < 0.05) in the treated hADSCs with 50μM 5-Aza in the 3D group. However, the expression level of the specific cardiac proteins in 3D was not significant using western blot and immunofluorescence staining. In conclusion, this study suggests that the fibrin scaffold with a compressive stress of 107.74kPa can keep the cells alive for 10weeks and also allows a higher and sooner differentiation of hADSCs into cardiomyocyte-like cells treated with 50μM 5-Aza.

CD90+ cardiac fibroblasts reduce fibrosis of acute myocardial injury in rats.

To explore the differentiation tendency of CD90+ cardiac fibroblast (CFs) into cardiomyogenic cells in vitro and repair functions in acute myocardial infarction rats. CD90+ subpopulation was sorted from rat CFs by flow cytometry. 10 μmoL/L of 5-Azacytosine (5-aza) was used to induce differentiation of CFs into cardiomyogenic cells. An acute myocardial infarction model was prepared by ligation of the rat left anterior descending coronary artery. After nuclei were labeled by DAPI, induced CD90+ CFs were injected into the infarction marginal zone. Before coronary ligation, 40 min after ligation, and at 7 and 14 days after cell transplantation, cardiac function changes were detected by ultrasound imaging system respectively. cTnT and endothelial cell marker VIII factor were detected by immunofluorescence staining. Infarct size was examined by TTC staining. Fibrosis was evaluated with masson's trichrome staining, vimentin, type I and type III collagen staining. CD90+ CFs sorted by flow cytometry was 34.9%. On day 28 after induction, the cTnT positive rate was 61.17 ± 9.75%. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) at days 7 and 14 after transplantation were significantly increased compared with those before transplantation (P < 0.05). LVEF and LVFS at the 14th day after transplantation were also significantly increased compared with those at the 7th day (LVEF: 61.40 ± 2.45% vs. 56.25 ± 2.9%, LVFS: 33.21 ± 0.68% vs. 30.26 ± 2.06%, P < 0.01). Additionally, small numbers of CD90+ CFs differentiated into cardiomyocytes and became involved in neovascularization. CD90+ CFs and CFs reduced myocardial infarct size at days 14. It was significantly smaller in rats with CFs transplantation group than those in MI group(24.78 ± 2.28% vs. 31.28 ± 2.83%, P < 0.05), and it was also significantly smaller in rats with CD90+CFs transplantation group than those in CFs transplantation group (17.47 ± 4.15% vs. 24.78 ± 2.28%, P < 0.05). Meanwhile, The percentage of fibrotic area and vimentin, type I and type III collagen in the infarct border zone and infarct area were both significantly reduced in CD90 + CFs. CD90+ CFs is the preponderant subpopulation of cardiomyogenic differentiation, with potential use as seed cells in basic and clinical research of heart regeneration and repair.

Transduced Mesenchymal Stem Cells and Cardiomyogenic Differentiation

Transduced Mesenchymal Stem Cells and Cardiomyogenic Differentiation

Intramyocardial angiogenetic stem cells and epicardial erythropoietin save the acute ischemic heart.

ABSTRACTIschemic heart failure is the leading cause of mortality worldwide. An early boost of intracardiac regenerative key mechanisms and angiogenetic niche signaling in cardiac mesenchymal stem cells (MSCs) could improve myocardial infarction (MI) healing. Epicardial erythropoietin (EPO; 300 U kg−1) was compared with intraperitoneal and intramyocardial EPO treatments after acute MI in rats (n=156). Real-time PCR and confocal microscopy revealed that epicardial EPO treatment enhanced levels of intracardiac regenerative key indicators (SDF-1, CXCR4, CD34, Bcl-2, cyclin D1, Cdc2 and MMP2), induced transforming growth factor β (TGF-β)/WNT signaling in intramyocardial MSC niches through the direct activation of AKT and upregulation of upstream signals FOS and Fzd7, and augmented intracardiac mesenchymal proliferation 24 h after MI. Cardiac catheterization and tissue analysis showed superior cardiac functions, beneficial remodeling and increased capillary density 6 weeks after MI. Concomitant fluorescence-activated cell sorting, co-cultures with neonatal cardiomyocytes, angiogenesis assays, ELISA, western blotting and RAMAN spectroscopy demonstrated that EPO could promote cardiomyogenic differentiation that was specific of tissue origin and enhance paracrine angiogenetic activity in cardiac CD45−CD44+DDR2+ MSCs. Epicardial EPO delivery might be the optimal route for efficient upregulation of regenerative key signals after acute MI. Early EPO-mediated stimulation of mesenchymal proliferation, synergistic angiogenesis with cardiac MSCs and direct induction of TGF-β/WNT signaling in intramyocardial cardiac MSCs could initiate an accelerated healing process that enhances cardiac recovery.

Zwitterionic starch-based hydrogel for the expansion and “stemness” maintenance of brown adipose derived stem cells

Brown adipose derived stem cells (BADSCs) have become a promising stem cell treatment candidate for myocardial infarction because of their efficiently spontaneous differentiation capacity towards cardiomyocytes. The lack of existing cell passage protocols motivates us to develop a neotype 3D cell expansion technique for BADSCs. In this study, “clickable” zwitterionic starch based hydrogels are developed using methacrylate modified sulfobetaine derived starch with dithiol-functionalized poly (ethylene glycol) as crosslinker via the “thiol-ene” Michael addition reaction. Moreover, CGRGDS peptide is immobilized into the hydrogel via a similar “clickable” approach. Their Young's moduli range from 22.28 to 74.81 kPa depending on the concentration of precursor solutions. Excellent anti-fouling property is also presented owing to the introduction of zwitterionic moieties. BADSCs are homogeneously encapsulated in the hydrogels and then routinely cultured for 10 days. Results suggest a capacious cell proliferation and the extent increases with either the decrease of mechanical strength or the introduction of CGRGDS. More excitingly, the cell “stemness” is well maintained during this period and the expanded cells released from the hydrogels well keep the efficiently spontaneous cardiomyogenic differentiation capacity. Therefore, it is suggested that zwitterionic starch based hydrogel is able for the expansion and “stemness ” maintenance of BADSCs.

Effective component of Salviamiltiorrhiza in promoting cardiomyogenic differentiation of human placenta‑derived mesenchymal stem cells.

Our previous study indicated that Salviamiltiorrhiza (SM) induced human placenta‑derived mesenchymal stem cells (hPDMSCs) to differentiate into cardiomyocytes, however, the effective component of SM in promoting cardiomyogenic differentiation remained to be elucidated. In the present study, the most commonly examined components of SM, including danshensu, salvianolic acidB, protocatechuic aldehyde, tanshinoneI (TSI), TSIIA and cryptotanshinone, were used to determine the effective components of SM in promoting cardiomyogenic differentiation. The above components of SM slowed cell growth rate and altered cell morphology with a spindle or irregular shape to different degrees. The cells treated with the above components of SM showed increasing of cardiac protein expression to differing degrees, including GATA‑binding protein 4, atrial natriuretic factor, α‑sarcomeric actin and cardiac troponin‑I. Among the components of SM, TSIIA induced the most marked effects. In addition, the above components of SM increased the expression of phosphorylated glycogen synthase kinase‑3β, but decreased the expression of β‑catenin to different degrees, with TSIIA also having the most marked effects. In conclusion, the results of the present study suggested that TSIIA was the most effective active component of SM in inducing hPDMSCs to differentiate into cardiomyocytes, and that Wnt/β‑catenin signaling was important in the process of TSIIA promoting cardiomyogenic differentiation.

CREG1 Interacts with Sec8 to Promote Cardiomyogenic Differentiation and Cell-Cell Adhesion.

CREG1 Interacts with Sec8 to Promote Cardiomyogenic Differentiation and Cell-Cell Adhesion.

Collagen scaffold enhances the regenerative properties of mesenchymal stromal cells.

MSCs are widely applied to regenerate heart tissue in myocardial diseases but when grown in standard two-dimensional (2D) cultures exhibit limited potential for cardiac repair and develop fibrogenic features with increasing culture time. MSCs can undergo partial cardiomyogenic differentiation, which improves their cardiac repair capacity. When applied to collagen patches they may improve cardiac tissue regeneration but the mechanisms remain elusive. Here, we investigated the regenerative properties of MSCs grown in a collagen scaffold as a three-dimensional (3D) culture system, and performed functional analysis using an engineered heart tissue (EHT) model. We showed that the expression of cardiomyocyte-specific proteins by MSCs co-cultured with rat neonatal cardiomyocytes was increased in collagen patches versus conventional cultures. MSCs in 3D collagen patches were less fibrogenic, secreted more cardiotrophic factors, retained anti-apoptotic and immunomodulatory function, and responded less to TLR4 ligand lipopolysaccharide (LPS) stimulation. EHT analysis showed no effects by MSCs on cardiomyocyte function, whereas control dermal fibroblasts abrogated the beating of cardiac tissue constructs. We conclude that 3D collagen scaffold improves the cardioprotective effects of MSCs by enhancing the production of trophic factors and modifying their immune modulatory and fibrogenic phenotype. The improvement in myocardial function by MSCs after acquisition of a partial cardiac cell-like phenotype is not due to enhanced MSC contractility. A better understanding of the mechanisms of MSC-mediated tissue repair will help to further enhance the therapeutic potency of MSCs.

Subtype-specific differentiation of cardiac pacemaker cell clusters from human induced pluripotent stem cells

BackgroundHuman induced pluripotent stem cells (hiPSC) harbor the potential to differentiate into diverse cardiac cell types. Previous experimental efforts were primarily directed at the generation of hiPSC-derived cells with ventricular cardiomyocyte characteristics. Aiming at a straightforward approach for pacemaker cell modeling and replacement, we sought to selectively differentiate cells with nodal-type properties.MethodshiPSC were differentiated into spontaneously beating clusters by co-culturing with visceral endoderm-like cells in a serum-free medium. Subsequent culturing in a specified fetal bovine serum (FBS)-enriched cell medium produced a pacemaker-type phenotype that was studied in detail using quantitative real-time polymerase chain reaction (qRT-PCR), immunocytochemistry, and patch-clamp electrophysiology. Further investigations comprised pharmacological stimulations and co-culturing with neonatal cardiomyocytes.ResultshiPSC co-cultured in a serum-free medium with the visceral endoderm-like cell line END-2 produced spontaneously beating clusters after 10–12 days of culture. The pacemaker-specific genes HCN4, TBX3, and TBX18 were abundantly expressed at this early developmental stage, while levels of sarcomeric gene products remained low. We observed that working-type cardiomyogenic differentiation can be suppressed by transfer of early clusters into a FBS-enriched cell medium immediately after beating onset. After 6 weeks under these conditions, sinoatrial node (SAN) hallmark genes remained at high levels, while working-type myocardial transcripts (NKX2.5, TBX5) were low. Clusters were characterized by regular activity and robust beating rates (70–90 beats/min) and were triggered by spontaneous Ca2+ transients recapitulating calcium clock properties of genuine pacemaker cells. They were responsive to adrenergic/cholinergic stimulation and able to pace neonatal rat ventricular myocytes in co-culture experiments. Action potential (AP) measurements of cells individualized from clusters exhibited nodal-type (63.4%) and atrial-type (36.6%) AP morphologies, while ventricular AP configurations were not observed.ConclusionWe provide a novel culture media-based, transgene-free approach for targeted generation of hiPSC-derived pacemaker-type cells that grow in clusters and offer the potential for disease modeling, drug testing, and individualized cell-based replacement therapy of the SAN.