Chang Youn Lee

The role of microRNA-23b in the differentiation of MSC into chondrocyte by targeting protein kinase A signaling

Chondrogenic differentiation of mesenchymal stem cells (MSCs) is critical for successful cartilage regeneration. Several methods have been developed to attempt to chondrogenic differentiation, because chondrogenic differentiated cells can form stable cartilage and induce expression of a cartilage-specific phenotype. In this study, we found that both H-89 and microRNA-23b induced differentiation into chondrocyte of hMSCs through down-regulation of protein kinase A (PKA) signaling. The small molecule, H-89, was identified by PCA analysis as a potential mediator of chondrogenic differentiation. H-89 induced the expression of the chondrocyte marker, aggrecan, as well as miR-23b. We searched that miR-23b regulates protein level of PKA. When miR-23b was transfected into hMSCs, chondrogenic differentiation was induced. We confirmed the target of miR-23b using a reporter gene assay. Furthermore, not only H-89 or miR-23b-treated cells, but also cell co-treated with H-89 and miR-23b differentiated into chondrocytes. Our results indicate that H-89 induces the expression of endogenous miR-23b, thereby inducing chondrogenic differentiation by negatively inhibition of PKA signaling.

Survival of hypoxic human mesenchymal stem cells is enhanced by a positive feedback loop involving miR-210 and hypoxia-inducible factor 1

The use of mesenchymal stem cells (MSCs) has emerged as a potential new treatment for myocardial infarction. However, the poor viability of MSCs after transplantation critically limits the efficacy of this new strategy. The expression of microRNA-210 (miR-210) is induced by hypoxia and is important for cell survival under hypoxic conditions. Hypoxia increases the levels of hypoxia inducible factor-1 (HIF-1) protein and miR-210 in human MSCs (hMSCs). miR-210 positively regulates HIF-1α activity. Furthermore, miR-210 expression is also induced by hypoxia through the regulation of HIF-1α. To investigate the effect of miR-210 on hMSC survival under hypoxic conditions, survival rates along with signaling related to cell survival were evaluated in hMSCs over-expressing miR-210 or ones that lacked HIF-1α expression. Elevated miR-210 expression increased survival rates along with Akt and ERK activity in hMSCs with hypoxia. These data demonstrated that a positive feedback loop involving miR-210 and HIF-1α was important for MSC survival under hypoxic conditions.

Cardiomyocytes from phorbol myristate acetate-activated mesenchymal stem cells restore electromechanical function in infarcted rat hearts

Despite the safety and feasibility of mesenchymal stem cell (MSC) therapy, an optimal cell type has not yet emerged in terms of electromechanical integration in infarcted myocardium. We found that poor to moderate survival benefits of MSC-implanted rats were caused by incomplete electromechanical integration induced by tissue heterogeneity between myocytes and engrafted MSCs in the infarcted myocardium. Here, we report the development of cardiogenic cells from rat MSCs activated by phorbol myristate acetate, a PKC activator, that exhibited high expressions of cardiac-specific markers and Ca2+ homeostasis-related proteins and showed adrenergic receptor signaling by norepinephrine. Histological analysis showed high connexin 43 coupling, few inflammatory cells, and low fibrotic markers in myocardium implanted with these phorbol myristate acetate-activated MSCs. Infarct hearts implanted with these cells exhibited restoration of conduction velocity through decreased tissue heterogeneity and improved myocardial contractility. These findings have major implications for the development of better cell types for electromechanical integration of cell-based treatment for infarcted myocardium.

Therapeutic Potential of Differentiated Mesenchymal Stem Cells for Treatment of Osteoarthritis

Osteoarthritis (OA) is a chronic, progressive, and irreversible degenerative joint disease. Conventional OA treatments often result in complications such as pain and limited activity. However, transplantation of mesenchymal stem cells (MSCs) has several beneficial effects such as paracrine effects, anti-inflammatory activity, and immunomodulatory capacity. In addition, MSCs can be differentiated into several cell types, including chondrocytes, osteocytes, endothelia, and adipocytes. Thus, transplantation of MSCs is a suggested therapeutic tool for treatment of OA. However, transplanted naïve MSCs can cause problems such as heterogeneous populations including differentiated MSCs and undifferentiated cells. To overcome this problem, new strategies for inducing differentiation of MSCs are needed. One possibility is the application of microRNA (miRNA) and small molecules, which regulate multiple molecular pathways and cellular processes such as differentiation. Here, we provide insight into possible strategies for cartilage regeneration by transplantation of differentiated MSCs to treat OA patients.

MicroRNA‐365 Inhibits the Proliferation of Vascular Smooth Muscle Cells by Targeting Cyclin D1

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a common feature of disease progression in atherosclerosis. Cell proliferation is regulated by cell cycle regulatory proteins. MicroRNAs (miR) have been reported to act as important gene regulators and play essential roles in the proliferation and migration of VSMCs in a cardiovascular disease. However, the roles and mechanisms of miRs in VSMCs and neointimal formation are far from being fully understood. In this study, cell cycle‐specific cyclin D1 was found to be a potential target of miR‐365 by direct binding. Through an in vitro experiment, we showed that exogenous miR‐365 overexpression reduced VSMC proliferation and proliferating cell nuclear antigen (PCNA) expression, while miR‐365 was observed to block G1/S transition in platelet‐derived growth factor‐bb (PDGF‐bb)‐induced VSMCs. In addition, the proliferation of VSMCs by various stimuli, including PDGF‐bb, angiotensin II (Ang II), and serum, led to the downregulation of miR‐365 expression levels. The expression of miR‐365 was confirmed in balloon‐injured carotid arteries. Taken together, our results suggest an anti‐proliferative role for miR‐365 in VSMC proliferation, at least partly via modulating the expression of cyclin D1. Therefore, miR‐365 may influence neointimal formation in atherosclerosis patients. J. Cell. Biochem. 115: 1752–1761, 2014.

let-7b suppresses apoptosis and autophagy of human mesenchymal stem cells transplanted into ischemia/reperfusion injured heart 7by targeting caspase-3

IntroductionMesenchymal stem cells (MSCs) have therapeutic potential for the repair of myocardial injury. The efficacy of MSC therapy for myocardial regeneration mainly depends on the survival of cells after transplantation into the infarcted heart. In the transplanted regions, reactive oxygen species (ROS) can cause cell death, and this process depends on caspase activation and autophagosome formation.MethodsA Software TargetScan was utilized to search for microRNAs (miRNAs) that target caspase-3 mRNA. Six candidate miRNAs including let-7b were selected and transfected into human MSCs in vitro. Expression of MEK-EKR signal pathways and autophagy-related genes were detected. Using ischemia/reperfusion model (I/R), the effect of MSCs enriched with let-7b was determined after transplantation into infarcted heart area. Miller catheter was used to evaluate cardiac function.ResultsHere, we report that let-7b targets caspase-3 to regulate apoptosis and autophagy in MSCs exposed to ROS. Let-7b-transfected MSCs (let-7b-MSCs) showed high expression of survival-related proteins, including p-MEK, p-ERK and Bcl-2, leading to a decrease in Annexin V/PI- and TUNEL-positive cells under ROS-rich conditions. Moreover, autophagy-related genes, including Atg5, Atg7, Atg12 and beclin-1, were significantly downregulated in let-7b-MSCs. Using a rat model of acute myocardial infarction, we found that intramyocardial injection of let-7b-MSCs markedly enhanced left ventricular (LV) function and microvessel density, in accordance with a reduced infarct size and the expression of caspase-3.ConclusionsTaken together, these data indicate that let-7b may protect MSCs implanted into infarcted myocardium from apoptosis and autophagy by directly targeting caspase-3 signaling.

Upregulation of miR-23b enhances the autologous therapeutic potential for degenerative arthritis by targeting PRKACB in synovial fluid-derived mesenchymal stem cells from patients.

The use of synovial fluid-derived mesenchymal stem cells (SFMSCs) obtained from patients with degenerative arthropathy may serve as an alternative therapeutic strategy in osteoarthritis (OA) and rheumatoid arthritis (RA). For treatment of OA and RA patients, autologous transplantation of differentiated MSCs has several beneficial effects for cartilage regeneration including immunomodulatory activity. In this study, we induced chondrogenic differentiation of SFMSCs by inhibiting protein kinase A (PKA) with a small molecule and microRNA (miRNA). Chondrogenic differentiation was confirmed by PCR and immunocytochemistry using probes specific for aggrecan, the major cartilaginous proteoglycan gene. Absorbance of alcian blue stain to detect chondrogenic differentiation was increased in H-89 and/or miRNA-23btransfected cells. Furthermore, expression of matrix metalloproteinase (MMP)-9 and MMP-2 was decreased in treated cells. Therefore, differentiation of SFMSCs into chondrocytes through inhibition of PKA signaling may be a therapeutic option for OA or RA patients.

The promotion of cardiogenic differentiation of hMSCs by targeting epidermal growth factor receptor using microRNA-133a.

Human bone marrow-derived mesenchymal stem cells (hMSCs) are an attractive candidate for cell therapy in heart disease. Low survival and incomplete electromechanical integration between resident cardiomyocytes and transplanted hMSCs remain unsolved. In order for an infarcted heart to tolerate transplantation, differentiation capacity in stem cells must be reinforced. In this study, we found that compound 56, an epidermal growth factor receptor (EGFR) inhibitor, promotes cardiogenic differentiation of hMSCs and the transplantation of hMSCs treated with compound 56 resulted in enhancement of heart functions. Furthermore, hMSCs transfected with microRNA-133a (miR-133a), which targets EGFR, were observed to express cardiac-specific markers. We also discovered that luciferase activity is exclusively decreased by targeting EGFR in hMSCs transfected with miR-133a mimic. These results suggest that EGFR plays a key role in the regulation of cardiogenic differentiation in hMSCs.

Phorbol myristate acetate differentiates human adipose-derived mesenchymal stem cells into functional cardiogenic cells.

To achieve effective regeneration of injured myocardium, it is important to find physiological way of improving the cardiogenic differentiation of stem cells. Previous studies demonstrated that cardiomyocytes from bone marrow-derived mesenchymal stem cells (BMSCs) activated with phorbolmyristate acetate (PMA), a protein kinase C (PKC) activator, restore electromechanical function in infarcted rat hearts. In this study, we investigated the effect of PMA on cardiogenic differentiation of adipose-derived MSCs (ASCs) for clinical applications. To confirm the effect of PMA, ASCs treated with 1μM PMA were grown for nine days. The expression of cardiac-specific markers (cardiac troponin T, myosin light chain, myosin heavy chain) in PMA-treated MSCs was demonstrated by immunocytochemistry. Alhough few α(1A) receptors exist in ASCs, α(1)-adrenergic receptor subtypes were preferentially expressed in PMA-treated ASCs. Moreover, expression of the β-adrenergic and muscarinic receptors increased in PMA-treated ASCs compared to normal cells. The mRNA levels of Ca(2+)-related factors (SERCA 2a; sarcoplasmic reticulum Ca(2+)-ATPase, LTCC; L-type Ca(2+) channel) in treated ASCs were similar to the levels in cardiomyocytes. Following the transplantation of chemically activated cardiogenic ASCs into infarcted myocardium, histological analysis showed that infarct size, interstitial fibrosis, and apoptotic index were markedly decreased and cardiac function was restored. In conclusion, PMA might induce the cardiogenic differentiation of human ASCs as well as BMSCs. This result suggests successful use of human ASCs in cardiac regeneration therapy.

Protein kinase C activation stimulates mesenchymal stem cell adhesion through activation of focal adhesion kinase.

Emerging evidence suggests that cell therapy with mesenchymal stem cells (MSCs) has beneficial effects on the injured heart. However, the decreased survival and/or adhesion of MSCs under ischemic conditions limits the application of cell transplantation as a therapeutic modality. We investigated a potential method of increasing the adhesion ability of MSCs to improve their efficacy in the ischemic heart. Treatment of MSCs with PKC activator, phorbol 12-myristate 13-acetate (PMA), increased cell adhesion and spreading in a dose-dependent method and significantly decreased detachment. When MSCs were treated with PKC inhibitor, that is, rottlerin, adhesion of MSCs was slightly diminished, and detachment was also decreased compared to the treatment with PMA. MSCs treated with both PMA and rottlerin behaved similarly to normal controls. In 3D matrix cardiogel, treatment with PMA increased the number of MSCs compared to the control group and MSCs treated with rottlerin. Expressions of focal adhesion kinase, cytoskeleton-associated proteins, and integrin subunits were clearly demonstrated in PMA-treated MSCs by immunoblotting and/or immunocytochemistry. The effect of PKC activator treatment on MSCs was validated in vivo. Following injection into rat hearts, the PMA-treated MSCs exhibited significantly higher retention in infarcted myocardium compared to the MSC group. Infarct size, fibrosis area, and apoptotic cells were reduced, and cardiac function was improved in rat hearts injected with PMA-treated MSCs compared to sham and/or MSC-implanted group. These results indicate that PKC activator is a potential target for niche manipulation to enhance adhesion of MSCs for cardiac regeneration.