Enhanced Osteocyte Differentiation: Cathepsin D and L Secretion by Human Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ASCs) have the potential to differentiate into bone, cartilage, fat, and neural cells and promote tissue regeneration and healing. It is known that they can have variable responses to hypoxic conditions. In the present study, we aimed to explore diverse changes in the cells and secretome of ASCs under a hypoxic environment over time and to present the possibility of ASCs as therapeutic agents from a different perspective. The expression differences of proteins between normoxic and hypoxic conditions (6, 12, or 24 h) were specifically investigated in human ASCs using 2-DE combined with MALDI-TOF MS analysis, and secreted proteins in ASC-derived conditioned media (ASC-derived CM) were examined by an adipokine array. In addition, genetic and/or proteomic interactions were assessed using a DAVID and miRNet functional annotation bioinformatics analysis. We found that 64 and 5 proteins were differentially expressed in hypoxic ASCs and in hypoxic ASC-derived CM, respectively. Moreover, 7 proteins among the 64 markedly changed spots in hypoxic ASCs were associated with bone-related diseases. We found that two proteins, cathepsin D (CTSD) and cathepsin L (CTSL), identified through an adipokine array independently exhibited significant efficacy in promoting osteocyte differentiation in bone-marrow-derived mesenchymal stem cells (BM-MSCs). This finding introduces a promising avenue for utilizing hypoxia-preconditioned ASC-derived CM as a potential therapeutic approach for bone-related diseases.

Ultraefficient extracellular vesicle-guided direct reprogramming of fibroblasts into functional cardiomyocytes.

Direct lineage conversion holds great promise in the regenerative medicine field for restoring damaged tissues using functionally engineered counterparts. However, current methods of direct lineage conversion, even those using virus-mediated transgenic expression of tumorigenic factors, are extremely inefficient (~25%). Thus, advanced methodologies capable of revolutionizing efficiency and addressing safety concerns are key to clinical translation of these technologies. Here, we propose an extracellular vesicle (EV)-guided, nonviral, direct lineage conversion strategy to enhance transdifferentiation of fibroblasts to induced cardiomyocyte-like cells (iCMs). The resulting iCMs have typical cardiac Ca2+ transients and electrophysiological features and exhibit global gene expression profiles similar to those of cardiomyocytes. This is the first demonstration of the use of EVs derived from embryonic stem cells undergoing cardiac differentiation as biomimetic tools to induce cardiac reprogramming with extremely high efficiency (>60%), establishing a general, more readily accessible platform for generating a variety of specialized somatic cells through direct lineage conversion.

Brite Adipocyte FGF21 Attenuates Cardiac Ischemia/Reperfusion Injury in Rat Hearts by Modulating NRF2.

Although the optimal therapy for myocardial infarction includes reperfusion to restore blood flow to the ischemic area, myocardial injury after ischemia/reperfusion usually leads to an inflammatory response, oxidative stress, and cardiomyocyte apoptosis. In this study, rat adipose-derived stem cells were differentiated into low-thermogenic beige adipocytes (LBACs) and high-thermogenic beige adipocytes (HBACs) to study the different cardioprotective effects of heterogeneous expression of brown adipocytes. We found that antioxidant and antiapoptotic factors in H9c2 cardiomyocytes were upregulated by high levels of secreted FGF21 in HBAC conditioned medium (HBAC-CM), whereas FGF21 in HBAC-CM did not affect antioxidative or antiapoptotic cell death in H9c2 cardiomyocytes with Nrf2 knockdown. These results show that NRF2 mediates antioxidative and antiapoptotic effects through the HBAC-secreted factor FGF21. Consistent with this finding, the expression of antioxidant and antiapoptotic genes was upregulated by highly secreted FGF21 after HBAC-CM treatment compared to LBAC-CM treatment in H9c2 cardiomyocytes via NRF2 activation. Furthermore, HBAC-CM significantly attenuated ischemic rat heart tissue injury via NRF2 activation. Based on these findings, we propose that HBAC-CM exerts beneficial effects in rat cardiac ischemia/reperfusion injury by modulating NRF2 and has potential as a promising therapeutic agent for myocardial infarction.

Cold-pressed oil from Citrus aurantifolia inhibits the proliferation of vascular smooth muscle cells via regulation of PI3K/MAPK signaling pathways.

Vascular occlusive disease is a chronic disease with significant morbidity and mortality. Although a variety of therapies and medications have been developed, the likelihood of disease re-emergence is high and this can be life-threatening. Based on a previous screening experiment related to vascular obstructive diseases using 34 types of essential oils, cold-pressed oil (CpO) from Citrus aurantifolia (lime) has been demonstrated to have the best effect for the inhibition of vascular smooth muscle cells (VSMCs) proliferation. The aim of the present study was to evaluate the effect of lime CpO on the pathological changes of VSMCs. To determine this, the effect of lime CpO on VSMC proliferation, a major cause of vascular disease, was investigated. To determine the safe concentration interval for toxicity of CpO during VSMC culture, a dilution of 1x10-5 was determined using Cell Counting Kit-8 assay, which was confirmed to be non-toxic using a lactate dehydrogenase assay. To examine the effect of lime CpO in cellular signaling pathways, changes in phosphorylation of both the PI3K/AKT/mTOR and extracellular signal-regulated MEK/ERK signaling pathways with serum were investigated. Furthermore, lime CpO with FBS also significantly decreased the expression levels of the cell cycle regulators cyclin D1 and proliferating cell nuclear antigen. Additionally, lime CpO with FBS significantly inhibited the sprouting of VSMCs in an ex vivo culture system. These results suggested that lime CpO inhibited the abnormal proliferation of VSMCs and can be developed as a nature-based therapeutic agent for obstructive vascular disease.

Recent advances in stem cell therapy for neurodegenerative disease: Three dimensional tracing and its emerging use.

Neurodegenerative disease is a brain disorder caused by the loss of structure and function of neurons that lowers the quality of human life. Apart from the limited potential for endogenous regeneration, stem cell-based therapies hold considerable promise for maintaining homeostatic tissue regeneration and enhancing plasticity. Despite many studies, there remains insufficient evidence for stem cell tracing and its correlation with endogenous neural cells in brain tissue with three-dimensional structures. Recent advancements in tissue optical clearing techniques have been developed to overcome the existing shortcomings of cross-sectional tissue analysis in thick and complex tissues. This review focuses on recent progress of stem cell treatments to improve neurodegenerative disease, and introduces tissue optical clearing techniques that can implement a three-dimensional image as a proof of concept. This review provides a more comprehensive understanding of stem cell tracing that will play an important role in evaluating therapeutic efficacy and cellular interrelationship for regeneration in neurodegenerative diseases.

ADSC-Based Cell Therapies for Musculoskeletal Disorders: A Review of Recent Clinical Trials.

Recently published clinical trials involving the use of adipose-derived stem cells (ADSCs) indicated that approximately one-third of the studies were conducted on musculoskeletal disorders (MSD). MSD refers to a wide range of degenerative conditions of joints, bones, and muscles, and these conditions are the most common causes of chronic disability worldwide, being a major burden to the society. Conventional treatment modalities for MSD are not sufficient to correct the underlying structural abnormalities. Hence, ADSC-based cell therapies are being tested as a form of alternative, yet more effective, therapies in the management of MSDs. Therefore, in this review, MSDs subjected to the ADSC-based therapy were further categorized as arthritis, craniomaxillofacial defects, tendon/ligament related disorders, and spine disorders, and their brief characterization as well as the corresponding conventional therapeutic approaches with possible mechanisms with which ADSCs produce regenerative effects in disease-specific microenvironments were discussed to provide an overview of under which circumstances and on what bases the ADSC-based cell therapy was implemented. Providing an overview of the current status of ADSC-based cell therapy on MSDs can help to develop better and optimized strategies of ADSC-based therapeutics for MSDs as well as help to find novel clinical applications of ADSCs in the near future.

MicroRNA-26a/b-5p promotes myocardial infarction-induced cell death by downregulating cytochrome c oxidase 5a.

Myocardial infarction (MI) damage induces various types of cell death, and persistent ischemia causes cardiac contractile decline. An effective therapeutic strategy is needed to reduce myocardial cell death and induce cardiac recovery. Therefore, studies on molecular and genetic biomarkers of MI, such as microRNAs (miRs), have recently been increasing and attracting attention due to the ideal characteristics of miRs. The aim of the present study was to discover novel causative factors of MI using multiomics-based functional experiments. Through proteomic, MALDI-TOF-MS, RNA sequencing, and network analyses of myocardial infarcted rat hearts and in vitro functional analyses of myocardial cells, we found that cytochrome c oxidase subunit 5a (Cox5a) expression is noticeably decreased in myocardial infarcted rat hearts and myocardial cells under hypoxic conditions, regulates other identified proteins and is closely related to hypoxia-induced cell death. Moreover, using in silico and in vitro analyses, we found that miR-26a-5p and miR-26b-5p (miR-26a/b-5p) may directly modulate Cox5a, which regulates hypoxia-related cell death. The results of this study elucidate the direct molecular mechanisms linking miR-26a/b-5p and Cox5a in cell death induced by oxygen tension, which may contribute to the identification of new therapeutic targets to modulate cardiac function under physiological and pathological conditions.

Suppressing Pyroptosis Augments Post-Transplant Survival of Stem Cells and Cardiac Function Following Ischemic Injury.

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 1ß 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.

IGF-1 Facilitates Cartilage Reconstruction by Regulating PI3K/AKT, MAPK, and NF-kB Signaling in Rabbit Osteoarthritis.

PURPOSE: The pathogenesis of osteoarthritis (OA) is characterized by joint degeneration. The pro-inflammatory cytokine interleukin (IL)-1ß plays a vital role in the pathogenesis of OA by stimulation of specific signaling pathways like NF-κB, PI3K/Akt, and MAPKs pathways. The catabolic role of growth factors in the OA may be inhibited cytokine-activated pathogen. The purpose of this study was to investigate the potential effects of insulin-like growth factor-1 (IGF-1) on IL-1ß-induced apoptosis in rabbit chondrocytes in vitro and in an in vivo rabbit knee OA model. METHODS: In the present study, the OA developed in chondrocyte with the treatment of IL-1ß and articular cartilage ruptures by removal of cartilage from the rabbit knee femoral condyle. After IGF-1 treatment, immunohistochemistry and qRT-PCR were identified OA expression with changes in MMPs (matrix metalloproteinases). The production of ROS (intracellular reactive oxygen species) in the OA was detected by flow cytometry. Further, the disease progression was microscopically investigated and pathophysiological changes were analyzed using histology. The NF-κB, PI3K/Akt and P38 (MAPK) specific pathways that are associated with disease progression were also checked using the Western blot technique. RESULTS: The expression of MMPs and various apoptotic markers are down-regulated following administration of IGF-1 in a dose-dependent fashion while significantly up-regulation of TIMP-1. The results showed that higher levels of ROS were observed upon treatment of chondrocytes and chondral OA with IL-1ß. Collectively, our results indicated that IGF-1 protected NF-κB pathway by suppression of PI3K/Akt and MAPKs specific pathways. Furthermore, the macroscopic and pathological investigation showed that it has a chondroprotective effect by the formation of hyaline cartilage. CONCLUSION: Our results indicate a protective effect of IGF-1 against OA pathogenesis by inhibition of NF-κB signaling via regulation of the MAPK and PI3K/Akt signaling pathways and prevention of apoptosis by suppression of ROS production.

Ginsenoside Rb1 inhibits monoiodoacetate-induced osteoarthritis in postmenopausal rats through prevention of cartilage degradation.

BACKGROUND: Ginsenoside Rb1 (G-Rb1), one of the major active compounds in Panax ginseng, has already been shown to reduce inflammation in various diseases. Osteoarthritis (OA) has traditionally been considered a degenerative disease with degradation of joint articular cartilage. However, recent studies have shown the association of inflammation with OA. In the present study, we investigated whether Rb1 had an antiinflammatory effect on monoiodoacetate (MIA)-induced OA in ovariectomized rats as a model of postmenopausal arthritis. METHODS: G-Rb1 at a dosage of 3 and 10 µg/kg body weight was administered every 3 days intraarticularly for a period of 4 weeks to observe antiarthritic effects. Diclofenac (10 mg/kg) served as a positive control. RESULTS: The administration of Rb1 significantly ameliorated OA inflammatory symptoms and reduced serum levels of inflammatory cytokines. Furthermore, G-Rb1 administration considerably enhanced the expression of bone morphogenetic protein-2 and collagen 2A and reduced the levels of matrix metalloproteinase-13 genes, indicating a chondroprotective effect of G-Rb1. G-Rb1 also significantly reduced the expression of several inflammatory cytokines/chemokines (interferon gamma (IFN-γ), monocyte chemoattractant protein-1 (MCP-1)/CCL-2, interleukin [IL]-1ß, and IL-6). Histological analysis demonstrated that G-Rb1 significantly attenuated the pathological changes in MIA-induced OA in ovariectomized rats. Safranin O and toluidine blue staining further demonstrated that G-Rb1 effectively prevented the degradation of cartilage and glycosaminoglycans, respectively. CONCLUSION: Overall, our results suggest that G-Rb1 exerts cartilage protective effect on MIA-induced ovariectomized OA rats, by inhibiting inflammatory mediators such as IL-6, IL-1ß, MCP-1/CCL-2, cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2). These results shed a light on possible therapeutic application of G-Rb1 in OA.

Neutralization of hexokinase 2-targeting miRNA attenuates the oxidative stress-induced cardiomyocyte apoptosis.

Hexokinase 2 (HK2) is a metabolic sensor that couples glycolysis and oxidative phosphorylation of mitochondria by binding to the outer mitochondrial membrane (OMM), and it also has been implicated in induction of apoptotic process by regulating the integrity of OMM. When HK2 detaches from the mitochondria, it triggers permeability increase of the OMM and subsequently facilitates the cytosolic release of cytochrome c, a major apoptosis-inducing factor. According to previous studies, a harsh microenvironment created by ischemic heart disease such as low tissue oxygen and nutrients, and increased reactive oxygen species (ROS) can cause cardiomyocyte apoptosis. Under these conditions, the expression of HK2 in heart significantly decrease and such down-regulation of HK2 was correlated to the increased apoptosis of cardiomyocytes. Therefore, prevention of HK2 down-regulation may salvage cardiomyocytes from apoptosis. MicroRNAs are short, non-coding RNAs that either inhibit transcription of target mRNAs or degrade the targeted mRNAs via complementary binding to the 3'UTR (untranslated region) of the targeted mRNAs. Since miRNAs are known to be involved in virtually every biological processes, it is reasonable to assume that the expression of HK2 is also regulated by miRNAs. Currently, to my best knowledge, there is no previous study examined the miRNA-mediated regulation of HK2 in cardiomyocytes. Thus, in the present study, miRNA-mediated modulation of HK2 during ROS (H2O2)-induced cardiomyocyte apoptosis was investigated. First, the expression of HK2 in cardiomyocytes exposed to H2O2 was evaluated. H2O2 (500 µM) induced cardiomyocyte apoptosis and it also decreased the mitochondrial expression of HK2. Based on miRNA-target prediction databases and empirical data, miR-181a was identified as a HK2-targeting miRNA. To further examine the effect of negative regulation of the selected HK2-targeting miRNA on cardiomyocyte apoptosis, anti-miR-181a, which neutralizes endogenous miR-181a, was utilized. Delivery of anti-miR-181a significantly abrogated the H2O2-induced suppression of HK2 expression and subsequent disruption of mitochondrial membrane potential, improving the survival of cardiomyocytes exposed to H2O2. These findings suggest that miR-181a-mediated down-regulation of HK2 contributes to the apoptosis of cardiomyocytes exposed to ROS. Neutralizing miR-181a can be a viable and effective means to prevent cardiomyocyte from apoptosis in ischemic heart disease.

Isoliquiritigenin Enhances the Beige Adipocyte Potential of Adipose-Derived Stem Cells by JNK Inhibition.

Human adipose-derived stem cells (hASCs) can be isolated from fat tissue and have attracted interest for their potential therapeutic applications in metabolic disease. hASCs can be induced to undergo adipogenic differentiation in vitro by exposure to chemical agents or inductive growth factors. We investigated the effects and mechanism of differentiating hASC-derived white adipocytes into functional beige and brown adipocytes with isoliquiritigenin (ILG) treatment. Here, we showed that hASC-derived white adipocytes could promote brown adipogenesis by expressing both uncoupling protein 1 (UCP1) and PR/SET Domain 16 (PRDM16) following low-dose ILG treatments. ILG treatment of white adipocytes enhanced the expression of brown fat-specific markers, while the expression levels of c-Jun N-terminal kinase (JNK) signaling pathway proteins were downregulated. Furthermore, we showed that the inhibition of JNK phosphorylation contributed to white adipocyte differentiation into beige adipocytes, which was validated by the use of SP600125. We identified distinct regulatory effects of ILG dose responses and suggested that low-dose ILG induced the beige adipocyte potential of hASCs via JNK inhibition.

Proteome Analysis of Human Natural Killer Cell Derived Extracellular Vesicles for Identification of Anticancer Effectors.

Cancer immunotherapy is a clinically validated therapeutic modality for cancer and has been rapidly advancing in recent years. Adoptive transfer of immune cells such as T cells and natural killer (NK) cells has emerged as a viable method of controlling the immune system against cancer. Recent evidence indicates that even immune-cell-released vesicles such as NK-cell-derived exosomes also exert anticancer effect. Nevertheless, the underlying mechanisms remain elusive. In the present study, the anticancer potential of isolated extracellular vesicles (EVs) from expanded and activated NK-cell-enriched lymphocytes (NKLs) prepared by house-developed protocol was evaluated both in vitro and in vivo. Moreover, isolated EVs were characterized by using two-dimensional electrophoresis (2-DE)-based proteome and network analysis, and functional study using identified factors was performed. Our data indicated that the EVs from expanded and active NKLs had anticancer properties, and a number of molecules, such as Fas ligand, TRAIL, NKG2D, ß-actin, and fibrinogen, were identified as effector candidates based on the proteome analysis and functional study. The results of the present study suggest the possibility of NK-cell-derived EVs as a viable immunotherapeutic strategy for cancer.

Novel Therapeutic Effects of Pterosin B on Ang II-Induced Cardiomyocyte Hypertrophy.

Pathological cardiac hypertrophy is characterized by an abnormal increase in cardiac muscle mass in the left ventricle, resulting in cardiac dysfunction. Although various therapeutic approaches are being continuously developed for heart failure, several studies have suggested natural compounds as novel potential strategies. Considering relevant compounds, we investigated a new role for Pterosin B for which the potential life-affecting biological and therapeutic effects on cardiomyocyte hypertrophy are not fully known. Thus, we investigated whether Pterosin B can regulate cardiomyocyte hypertrophy induced by angiotensin II (Ang II) using H9c2 cells. The antihypertrophic effect of Pterosin B was evaluated, and the results showed that it reduced hypertrophy-related gene expression, cell size, and protein synthesis. In addition, upon Ang II stimulation, Pterosin B attenuated the activation and expression of major receptors, Ang II type 1 receptor and a receptor for advanced glycation end products, by inhibiting the phosphorylation of PKC-ERK-NF-κB pathway signaling molecules. In addition, Pterosin B showed the ability to reduce excessive intracellular reactive oxygen species, critical mediators for cardiac hypertrophy upon Ang II exposure, by regulating the expression levels of NAD(P)H oxidase 2/4. Our results demonstrate the protective role of Pterosin B in cardiomyocyte hypertrophy, suggesting it is a potential therapeutic candidate.

Isoliquiritigenin Derivatives Inhibit RANKL-Induced Osteoclastogenesis by Regulating p38 and NF-κB Activation in RAW 264.7 Cells.

Bone diseases may not be imminently life-threatening or a leading cause of death such as heart diseases or cancers. However, as aging population grows in almost every part of the world, they surely impose significant socioeconomic burden on the society, not to mention the patients and their families. Osteoporosis is the most common type of bone disease, which frequently develops in seniors, especially in postmenopausal women. Although currently several anti-osteoclastic drugs designed to suppress excessive osteoclast activation, a major cause of osteoporosis, are commercially available, accompanying adverse effects ranging from mild to severe have been reported as well. Natural products have become increasingly popular because of their effectiveness with fewer side effects. Isoliquiritigenin (ILG), a natural flavonoid from licorice, has been reported to suppress osteoclast differentiation and activation. In the present study, newly synthesized ILG derivatives were screened for their anti-osteoporotic activity as more potent substitute candidates to ILG. Out of the 12 ILG derivatives tested, two compounds demonstrated significantly improved bone loss in vitro by inhibiting both osteoclastogenesis and osteoclast activity. The results of the present study indicate that these compounds may serve as a potential drug for osteoporosis and warrant further studies to evaluate their in vivo efficacy.

Adipose tissue derived stromal vascular fraction as an adjuvant therapy in stroke rehabilitation: Case reports.

INTRODUCTION: Stroke often causes residual hemiparesis, and upper extremity motor impairment is usually more disabling than lower extremity in those who are suffering from post-stroke hemiparesis. Cell therapy is one of the promising therapies to reduce post-stroke disability. PATIENT CONCERNS: Three male participants were included in the study to investigate the feasibility and tolerability of autologous adipose tissue derived stromal vascular fraction. DIAGNOSIS: All participants had hemiparesis after 1st-ever stroke longer than 6 months previously. INTERVENTIONS: Under general anesthesia, liposuction of abdominal subcutaneous fat was performed. Stromal vascular fraction freshly isolated from the adipose tissue extract was injected into the muscles of paretic upper extremity. All participants received inpatient stroke rehabilitation consisted of physical and occupational therapy more than 3 hours a day for 2 months or more. OUTCOMES: The whole procedure did not produce any significant adverse event in all participants. Adipose tissue extracts yielded sufficient stromal cells. One participant showed clinically important change in upper extremity Fugl-Meyer assessment after the injection and it lasted up to 6 months. Functional magnetic resonance imaging showed concomitant increase in ipsilesional cortical activity. The other 2 participants did not show remarkable changes. LESSONS: Intramuscular injection of autologous adipose tissue derived stromal vascular fraction seems to be a safe and tolerable procedure in subjects with chronic stroke, and its utility in rehabilitation needs further investigation.

Targeted Delivery of Recombinant Heat Shock Protein 27 to Cardiomyocytes Promotes Recovery from Myocardial Infarction.

Ischemic heart disease, especially myocardial infarction (MI), is the leading cause of death worldwide. Apoptotic mechanisms are thought to play a significant role in cardiomyocyte death after MI. Increased production of heat shock proteins (Hsps) in cardiomyocytes is a normal response to promote tolerance and to reduce cell damage. Hsp27 is considered to be a therapeutic option for the treatment of ischemic heart disease due to its protective effects on hypoxia-induced apoptosis. Despite its antiapoptotic effects, the lack of strategies to deliver Hsp27 to the heart tissue in vivo limits its clinical applicability. In this study, we utilized an antibody against the angiotensin II type 1 (AT1) receptor, which is expressed immediately after ischemia/reperfusion in the heart of MI rats. To achieve cardiomyocyte-targeted Hsp27 delivery after ischemia/reperfusion, we employed the immunoglobulin-binding dimer ZZ, a modified domain of protein A, in conjunction with the AT1 receptor antibody. Using the AT1 receptor antibody, we achieved systemic delivery of ZZ-TAT-GFP fusion protein into the heart of MI rats. This approach enabled selective delivery of Hsp27 to cardiomyocytes, rescued cells from apoptosis, reduced the area of fibrosis, and improved cardiac function in the rat MI model, thus suggesting its applicability as a cardiomyocyte-targeted protein delivery system to inhibit apoptosis induced by ischemic injury.

Differentiation of adipose-derived stem cells into functional chondrocytes by a small molecule that induces Sox9.

Osteoarthritis (OA) is a common joint disease that results from the disintegration of joint cartilage and the underlying bone. Because cartilage and chondrocytes lack the ability to self-regenerate, efforts have been made to utilize stem cells to treat OA. Although various methods have been used to differentiate stem cells into functional chondrocytes, the currently available methods cannot induce stem cells to undergo differentiation into chondrocyte-like cells without inducing characteristics of hypertrophic chondrocytes, which finally lead to cartilage disintegration and calcification. Therefore, an optimized method to differentiate stem cells into chondrocytes that do not display undesired phenotypes is needed. This study focused on differentiating adipose-derived stem cells (ASCs) into functional chondrocytes using a small molecule that regulated the expression of Sox9 as a key factor in cartilage development and then explored its ability to treat OA. We selected ellipticine (ELPC), which induces chondrocyte differentiation of ASCs, using a GFP-Sox9 promoter vector screening system. An in vivo study was performed to confirm the recovery rate of cartilage regeneration with ASC differentiation into chondrocytes by ELPC in a collagenase-induced animal model of OA. Taken together, these data indicate that ellipticine induces ASCs to differentiate into mature chondrocytes without hypertrophic chondrocytes in vitro and in vivo, thus overcoming a problem encountered in previous studies. These results indicate that ELPC is a novel chondrocyte differentiation-inducing drug that shows potential as a cell therapy for OA.

A Combinational Therapy of Articular Cartilage Defects: Rapid and Effective Regeneration by Using Low-Intensity Focused Ultrasound After Adipose Tissue-Derived Stem Cell Transplantation.

BACKGROUND: Although low-intensity pulsed ultrasound has been reported to be potential cartilage regeneration, there still unresolved treatment due to cartilage fibrosis and degeneration by a lack of rapid and high-efficiency treatment. The purpose of this study was to investigate the effect of a combination therapy of focused acoustic force and stem cells at site for fast and efficient healing on cartilage regeneration. METHODS: Using a rat articular cartilage defects model, one million adipose tissue-derived stem cells (ASCs) were injected into the defect site, and low-intensity focused ultrasound (LOFUS) in the range of 100-600 mV was used for 20 min/day for 2 weeks. All experimental groups were sacrificed after 4 weeks in total. The gross appearance score and hematoxylin and eosin (H&E), Alcian blue, and Safranin O staining were used for measuring the chondrogenic potential. The cartilage characteristics were observed, and type II collagen, Sox 9, aggrecan, and type X collagen were stained with immunofluorescence. The results of the comprehensive analysis were calculated using the Mankin scoring method. RESULTS: The gross appearance scores of regenerated cartilage and chondrocyte-like cells in H&E images were higher in LOFUS-treated groups compared to those in negative control or ASC-treated groups. Safranin O and Alcian blue staining demonstrated that the 100 and 300 mV LOFUS groups showed greater synthesis of glycosaminoglycan and proteoglycan. The ASC + LOFUS 300 mV group showed positive regulation of type II collagen, Sox 9 and aggrecan and negative regulation of type X collagen, which indicated the occurrence of cartilage regeneration based on the Mankin score result. CONCLUSION: The combination therapy, which involved treatment with ASC and 300 mV LOFUS, quickly and effectively reduced articular cartilage defects.

Small G protein signaling modulator 3 (SGSM3) knockdown attenuates apoptosis and cardiogenic differentiation in rat mesenchymal stem cells exposed to hypoxia.

Connexin 43 (Cx43) may be important in cell death and survival due to cell-to-cell communication-independent mechanisms. In our previous study, we found that small G protein signaling modulator 3 (SGSM3), a partner of Cx43, contributes to myocardial infarction (MI) in rat hearts. Based on these previous results, we hypothesized that SGSM3 could also play a role in bone marrow-derived rat mesenchymal stem cells (MSCs), which differentiate into cardiomyocytes and/or cells with comparable phenotypes under low oxygen conditions. Cx43 and Cx43-related factor expression profiles were compared between normoxic and hypoxic conditions according to exposure time, and Sgsm3 gene knockdown (KD) using siRNA transfection was performed to validate the interaction between SGSM3 and Cx43 and to determine the roles of SGSM3 in rat MSCs. We identified that SGSM3 interacts with Cx43 in MSCs under different oxygen conditions and that Sgsm3 knockdown inhibits apoptosis and cardiomyocyte differentiation under hypoxic stress. SGSM3/Sgsm3 probably has an effect on MSC survival and thus therapeutic potential in diseased hearts, but SGSM3 may worsen the development of MSC-based therapeutic approaches in regenerative medicine. This study was performed to help us better understand the mechanisms involved in the therapeutic efficacy of MSCs, as well as provide data that could be used pharmacologically.