Chang Youn Lee

Alzheimer’s disease (AD), an age-dependent, progressive neurodegenerative disorder, is the most common type of dementia, accounting for 50–70% of all dementia cases. Due to the increasing incidence and corresponding socioeconomic burden of dementia, it has rapidly emerged as a challenge to public health worldwide. The characteristics of AD include the development of extracellular amyloid-beta plaques and intracellular neurofibrillary tangles, vascular changes, neuronal inflammation, and progressive brain atrophy. However, the complexity of the biology of AD has hindered progress in elucidating the underlying pathophysiological mechanisms of AD, and the development of effective treatments. MicroRNAs (miRNAs, which are endogenous, noncoding RNAs of approximately 22 nucleotides that function as posttranscriptional regulators of various genes) are attracting attention as powerful tools for studying the mechanisms of diseases, as they are involved in several biological processes and diseases, including AD. AD is a multifactorial disease, and several reports have suggested that miRNAs play an important role in the pathological processes of AD. In this review, the basic biology of miRNAs is described, and the function and physiology of miRNAs in the pathological processes of AD are highlighted. In addition, the limitations of current pharmaceutical therapies for the treatment of AD and the development of miRNA-based next-generation therapies are discussed.

The acute demise of stem cells following transplantation significantly compromises the efficacy of stem cell-based cell therapeutics for infarcted hearts. As the stem cells transplanted into the damaged heart are readily exposed to the hostile environment, it can be assumed that the acute death of the transplanted stem cells is also inflicted by the same environmental cues that caused massive death of the host cardiac cells. Pyroptosis, a highly inflammatory form of programmed cell death, has been added to the list of important cell death mechanisms in the damaged heart. However, unlike the well-established cell death mechanisms such as necrosis or apoptosis, the exact role and significance of pyroptosis in the acute death of transplanted stem cells have not been explored in depth. In the present study, we found that M1 macrophages mediate the pyroptosis in the ischemia/reperfusion (I/R) injured hearts and identified miRNA-762 as an important regulator of interleukin 1β production and subsequent pyroptosis. Delivery of exogenous miRNA-762 prior to transplantation significantly increased the post-transplant survival of stem cells and also significantly ameliorated cardiac fibrosis and heart functions following I/R injury. Our data strongly suggest that suppressing pyroptosis can be an effective adjuvant strategy to enhance the efficacy of stem cell-based therapeutics for diseased hearts.

Osteoarthritis (OA) can be defined as degradation of articular cartilage of the joint, and is the most common degenerative disease. To regenerate the damaged cartilage, different experimental approaches including stem cell therapy have been tried. One of the major limitations of stem cell therapy is the poor post-transplantation survival of the stem cells. Anoikis, where insufficient matrix support and adhesion to extracellular matrix causes apoptotic cell death, is one of the main causes of the low post-transplantation survival rate of stem cells. Therefore, enhancing the initial interaction of the transplanted stem cells with chondrocytes could improve the therapeutic efficacy of stem cell therapy for OA. Previously, protein kinase C activator phorbol 12-myristate 13-acetate (PMA)-induced increase of mesenchymal stem cell adhesion via activation of focal adhesion kinase (FAK) has been reported. In the present study, we examine the effect PMA on the adipose-derived stem cells (ADSCs) adhesion and spreading to culture substrates, and further on the initial interaction between ADSC and chondrocytes. PMA treatment increased the initial adhesion of ADSC to culture substrate and cellular spreading with increased expression of adhesion molecules, such as FAK, vinculin, talin, and paxillin, at both RNA and protein level. Priming of ADSC with PMA increased the number of ADSCs attached to confluent layer of cultured chondrocytes compared to that of untreated ADSCs at early time point (4 h after seeding). Taken together, the results of this study suggest that priming ADSCs with PMA can increase the initial interaction with chondrocytes, and this proof of concept can be used to develop a non-invasive therapeutic approach for treating OA. It may also accelerate the regeneration process so that it can relieve the accompanied pain faster in OA patients. Further in vivo studies examining the therapeutic effect of PMA pretreatment of ADSCs for articular cartilage damage are required.

The paracrine effect is likely the major mechanism of the adipose-derived stem cell (ASC)-mediated cardioprotective effect. However, the exact composition and nature of ASC-released paracrine factors remain elusive. In the present study, we examined the effect of osteoprotegerin (OPG), a stem cell-released decoy receptor for death ligand, on the survival of cardiomyocytes exposed to oxidative stress. The production of OPG from ASCs under oxidative stress was determined by ELISA and immunohistochemistry. The effects of OPG and the OPG-containing conditioned media of ASCs on the survival of cardiomyocytes were determined using a cell viability assay. Hydrogen peroxide (H2O2) significantly increased OPG production from ASCs in vitro, and OPG production from the ASCs transplanted into the ischemia–reperfusion-injured heart was also observed. OPG significantly attenuated cardiomyocyte death in vitro. OPG-containing conditioned media of ASCs also significantly protected cardiomyocytes. Delivery of siRNA specific to OPG significantly decreased the OPG production of ASCs, and also offset the protective effect of the conditioned media of ASCs. Our study strongly suggests that OPG is one of the prosurvival factors released from ASCs that may contribute to the ASC-mediated cardioprotection and calls for further studies to elucidate detailed underlying mechanisms.

Because of their capability of differentiation into lineage-specific cells, stem cells are an attractive therapeutic modality in regenerative medicine. To develop an effective stem cell-based therapeutic strategy with predictable results, deeper understanding of the underlying molecular mechanisms of stem cell differentiation and/or pluripotency maintenance is required. Thus, reviewing the key factors involved in the transcriptional and epigenetic regulation of stem cell differentiation and maintenance is important. Accumulating data indicate that long noncoding RNAs (lncRNAs) mediate numerous biological processes, including stem cell differentiation and maintenance. Here, we review recent findings on the human lncRNA regulation of stem cell potency and differentiation. Although the clinical implication of these lncRNAs is only beginning to be elucidated, it is anticipated that lncRNAs will become important therapeutic targets in the near future.

Stem cell therapy using adult stem cells, such as mesenchymal stem cells (MSCs) has produced some promising results in treating the damaged heart. However, the low survival rate of MSCs after transplantation is still one of the crucial factors that limit the therapeutic effect of stem cells. In the damaged heart, oxidative stress due to reactive oxygen species (ROS) production can cause the death of transplanted MSCs. Apoptosis signal-regulating kinase 1 (ASK1) has been implicated in the development of oxidative stress-related pathologic conditions. Thus, we hypothesized that down-regulation of ASK1 in human MSCs (hMSCs) might attenuate the post-transplantation death of MSCs. To test this hypothesis, we screened microRNAs (miRNAs) based on a miRNA-target prediction database and empirical data and investigated the anti-apoptotic effect of selected miRNAs on human adipose-derived stem cells (hASCs) and on rat myocardial infarction (MI) models. Our data indicated that miRNA-301a most significantly suppressed ASK1 expression in hASCs. Apoptosis-related genes were significantly down-regulated in miRNA-301a-enriched hASCs exposed to hypoxic conditions. Taken together, these data show that miRNA-mediated down-regulation of ASK1 protects MSCs during post-transplantation, leading to an increase in the efficacy of MSC-based cell therapy.

Background: We previously reported that phorbol 12-myristate 13-acetate (PMA) treatment can induce the cardiac differentiation of mesenchymal stem cells (MSCs). In the present study, we investigated how PMA induces cardiac differentiation of MSCs, focusing on its effect on the transcription factors responsible for increased cardiac marker gene expression. Methods: Human MSCs (hMSCs) were treated with 1 μM PMA for 9 days. The expression of MSC markers and cardiac markers in the PMA-treated hMSC, as well as the nuclear translocation of transcription factors, nuclear factor of activated T cells (NFAT), and myogenic differentiation 1 (MyoD), was examined. Transcriptional activity of NFAT was examined by utilizing a green fluorescent protein (GFP) vector containing NFAT motif of human interleukin-2 promoter. The effect of PMA on the expression of key cell cycle regulators was examined. Results: PMA induces the transcriptional activity of NFAT and MyoD, which have been associated with increased expression of cardiac troponin T (cTnT) and myosin heavy chain (MHC), respectively. Our data suggested that protein kinase C (PKC) mediates the effect of PMA on NFAT activation. Furthermore, PMA treatment increased cell-cycle regulator p27^kip1 expression, suggesting that PMA triggers the cardiac differentiation program in MSCs by regulating key transcription factors and cell cycle regulators. Conclusions: The results of this study demonstrate the importance of NFAT activation during PMA-induced MSC differentiation and help us to better understand the underlying mechanisms of small molecule-mediated MSC differentiation so that we can develop a strategy for synthesizing novel and improved differentiation-inducing small molecules.

Background: Recently, cell-based therapeutic lymphangiogenesis has emerged and provided hope for lymphatic regeneration. Previous studies have demonstrated that secretomes of mesenchymal stem cells (MSCs) facilitate the regeneration of various damaged tissues. This study was conducted to evaluate the lymphangiogenic potential of hypoxic conditioned media (HCM) from MSCs. Methods: To investigate the effects of MSC-secreted factors in starved human lymphatic endothelial cells (hLEC), hLECs were treated with endothelial basal medium (EBM)-2 (control), normoxic conditioned media (NCM), or HCM in vitro and in vivo. Results: MSCs expressed lymphangiogenic factors including EGF, FGF2, HGF, IGF-1, and VEGF-A and -C. hLECs were treated with each medium. hLEC proliferation, migration, and tube formation were improved under HCM compared with NCM. Moreover, expression of mitochondrial-related factors, MFN1and 2, were improved in HCM-treated hLECs. Lymphedema mice injected with HCM showed markedly decreased lymphedema via increased lymphatic vessel formation when compared with EBM-2- or NCM-treated mice. Conclusions: This study suggested that HCM from MSCs contain high levels of secreted lymphangiogenic factors and promote lymphangiogenesis by regulating mitochondrial-related factors. Thus, treatment with HCM may be a therapeutic strategy for lymphedema.

Despite the success of interventional processes such as drug-eluting stents, complete prevention of restenosis is still hindered by impaired or delayed endothelialization or both. Here, we report that 1H-pyrrole-2,5-dione-based small molecule-generated mesenchymal stem cell-derived functional endothelial cells (MDFECs) facilitated rapid transmural coverage of injured blood vessels. Small molecules that induced CD31 expression were screened by principal component analysis (PCA). Rat mesenchymal stem cells (MSCs) were treated with selected small molecules for up to 16 days, and the expression levels of CD90 and CD31 were examined by immunocytochemistry. In vitro functional assays of MDFECs, including tube formation assays and nitric oxide production assays, were performed. After MDFECs (intravenous, 3×10⁶ cells per animal) were injected into balloon-injured rats, neointima formation was monitored for up to 21 days. The endothelial coverage of denuded blood vessels was evaluated by Evans Blue staining. The functionality of repaired blood vessels was evaluated by measuring vasorelaxation and hemodynamic changes. Additionally, derivatives of the selected small molecules were examined for their ability to induce endothelial markers. PCA indicated that 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione effectively induced MDFECs. MDFECs inhibited the neointima formation of denuded blood vessels by facilitating more rapid endothelialization. Further examination indicated that derivatives with a 1H-pyrrole-2,5-dione moiety are important for initiating the endothelial cell differentiation of MSCs. Small molecules with 1H-pyrrole-2,5-dione as a core structure have great potential to improve the efficacy of MSC-based cell therapy for vascular diseases, such as atherosclerosis and restenosis.

Introduction: Mesenchymal 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.Methods: A 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 MSCsin 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.Results: Here, 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.Conclusions: Taken together, these data indicate that let-7b may protect MSCs implanted into infarcted myocardium from apoptosis and autophagy by directly targeting caspase-3 signaling.