• IMMUNE NETWORK
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• v.22 no.6
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• pp.49.1-49.15
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• 2022

Exosomes derived from mesenchymal stem cells (MSCs) could protect against myocardial infarction (MI). TLR4 is reported to play an important role in MI, while microRNA-182-5p (miR-182-5p) negatively regulates TLR4 expression. Therefore, we hypothesize that MSCs-derived exosomes overexpressing miR-182-5p may have beneficial effects on MI. We generated bone marrow mesenchymal stem cells (BM-MSCs) and overexpressed miR-182-5p in these cells for exosome isolation. H₂O₂-stimulated neonatal mouse ventricle myocytes (NMVMs) and MI mouse model were employed, which were subjected to exosome treatment. The expression of inflammatory factors, heart function, and TLR4 signaling pathway activation were monitored. It was found that miR-182-5p decreased TLR4 expression in BM-MSCs and NMVMs. Administration of exosomes overexpressing miR-182-5p to H₂O₂-stimulated NMVMs enhanced cell viability and suppressed the expression of inflammatory cytokines. In addition, they promoted heart function, suppressed inflammatory responses, and de-activated TLR4/NF-κB signaling pathway in MI mice. In conclusion, miR-182-5p transferred by the exosomes derived from BM-MSCs protected against MI-induced impairments by targeting TLR4.

• BMB Reports
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• v.57 no.5
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• pp.232-237
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• 2024

This study investigated how adipose tissue-derived mesenchymal stem cells (AT-MSCs) respond to chondrogenic induction using droplet-based single-cell RNA sequencing (scRNA-seq). We analyzed 37,219 high-quality transcripts from control cells and cells induced for 1 week (1W) and 2 weeks (2W). Four distinct cell clusters (0-3), undetectable by bulk analysis, exhibited varying proportions. Cluster 1 dominated in control and 1W cells, whereas clusters (3, 2, and 0) exclusively dominated in control, 1W, and 2W cells, respectively. Furthermore, heterogeneous chondrogenic markers expression within clusters emerged. Gene ontology (GO) enrichment analysis of differentially expressed genes unveiled cluster-specific variations in key biological processes (BP): (1) Cluster 1 exhibited up-regulation of GO-BP terms related to ribosome biogenesis and translational control, crucial for maintaining stem cell properties and homeostasis; (2) Additionally, cluster 1 showed up-regulation of GO-BP terms associated with mitochondrial oxidative metabolism; (3) Cluster 3 displayed up-regulation of GO-BP terms related to cell proliferation; (4) Clusters 0 and 2 demonstrated similar up-regulation of GO-BP terms linked to collagen fibril organization and supramolecular fiber organization. However, only cluster 0 showed a significant decrease in GO-BP terms related to ribosome production, implying a potential correlation between ribosome regulation and the differentiation stages of AT-MSCs. Overall, our findings highlight heterogeneous cell clusters with varying balances between proliferation and differentiation before, and after, chondrogenic stimulation. This provides enhanced insights into the single-cell dynamics of AT-MSCs during chondrogenic differentiation.

• IMMUNE NETWORK
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• v.21 no.4
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• pp.30.1-30.12
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• 2021

High expression of mitofusin-2 (MFN2), a mitochondrial fusion protein, has been frequently associated with poor prognosis of patients with cervical cancer. Here, we aimed to identify the function of MFN2 in cervical cancer to understand its influence on disease prognosis. To this end, from cervical adenocarcinoma, we performed an MTT assay and quantitative RT-PCR (qRT-PCR) analysis to assess the effects of MFN2 on the proliferation and of HeLa cells. Then, colony-formation ability and tumorigenesis were evaluated using a tumor xenograft mouse model. The migration ability related to MFN2 was also measured using a wound healing assay. Consequently, epithelial-mesenchymal transition (EMT) of MFN2-knockdowned HeLa cells originating from adenocarcinoma. markers related to MFN2 were assessed by qRT-PCR. Clinical data were analyzed using cBioPortal and The Cancer Genome Atlas. We found that MFN2 knockdown reduced the proliferation, colony formation ability, migration, and in vivo tumorigenesis of HeLa cells. Primarily, migration of MFN2-knockdowned HeLa cells decreased through the suppression of EMT. Thus, we concluded that MFN2 facilitates cancer progression and in vivo tumorigenesis in HeLa cells. These findings suggest that MFN2 could be a novel target to regulate the EMT program and tumorigenic potential in HeLa cells and might serve as a therapeutic target for cervical cancer. Taken together, this study is expected to contribute to the treatment of patients with cervical cancer.

• International Journal of Stem Cells
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• v.16 no.4
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• pp.415-424
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• 2023

Therapeutic efficacy of mesenchymal stem cells (MSCs) is determined by biodistribution and engraftment in vivo. Compared to intravenous infusion, biodistribution of locally transplanted MSCs are partially understood. Here, we performed a pharmacokinetics (PK) study of MSCs after local transplantation. We grafted human MSCs into the brains of immune-compromised nude mice. Then we extracted genomic DNA from brains, lungs, and livers after transplantation over a month. Using quantitative polymerase chain reaction with human Alu-specific primers, we analyzed biodistribution of the transplanted cells. To evaluate the role of residual immune response in the brain, MSCs expressing a cytosine deaminase (MSCs/CD) were used to ablate resident immune cells at the injection site. The majority of the Alu signals mostly remained at the injection site and decreased over a week, finally becoming undetectable after one month. Negligible signals were transiently detected in the lung and liver during the first week. Suppression of Iba1-positive microglia in the vicinity of the injection site using MSCs/CD prolonged the presence of the Alu signals. After local transplantation in xenograft animal models, human MSCs remain predominantly near the injection site for limited time without disseminating to other organs. Transplantation of human MSCs can locally elicit an immune response in immune compromised animals, and suppressing resident immune cells can prolong the presence of transplanted cells. Our study provides valuable insights into the in vivo fate of locally transplanted stem cells and a local delivery is effective to achieve desired dosages for neurological diseases.

• International Journal of Stem Cells
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• v.16 no.4
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• pp.438-447
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• 2023

Recently, ex-vivo gene therapy has emerged as a promising approach to enhance the therapeutic potential of mesenchymal stem cells (MSCs) by introducing functional genes in vitro. Here, we explored the need of using selection markers to increase the gene delivery efficiency and evaluated the potential risks associated with their use in the manufacturing process. We used MSCs/CD that carry the cytosine deaminase gene (CD) as a therapeutic gene and a puromycin resistance gene (PuroR) as a selection marker. We evaluated the correlation between the therapeutic efficacy and the purity of therapeutic MSCs/CD by examining their anti-cancer effect on co-cultured U87/GFP cells. To simulate in vivo horizontal transfer of the PuroR gene in vivo, we generated a puromycin-resistant E. coli (E. coli/PuroR) by introducing the PuroR gene and assessed its responsiveness to various antibiotics. We found that the anti-cancer effect of MSCs/CD was directly proportional to their purity, suggesting the crucial role of the PuroR gene in eliminating impure unmodified MSCs and enhancing the purity of MSCs/CD during the manufacturing process. Additionally, we found that clinically available antibiotics were effective in inhibiting the growth of hypothetical microorganism, E. coli/PuroR. In summary, our study highlights the potential benefits of using the PuroR gene as a selection marker to enhance the purity and efficacy of therapeutic cells in MSC-based gene therapy. Furthermore, our study suggests that the potential risk of horizontal transfer of antibiotics resistance genes in vivo can be effectively managed by clinically available antibiotics.

• International Journal of Stem Cells
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• v.16 no.2
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• pp.244-249
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• 2023

Background and Objectives: To examine whether ischemic retinal ganglion cells (RGCs) will be salvaged from cell death by human adipose-derived mesenchymal stem cells (ADSCs) in an organotypic retina model. Methods and Results: Deprived of arterial oxygen supply, whole mice retinas were cultured as an ex vivo organotypic cultures on an insert membrane in a 24-well plate. The therapeutic potential of ADSCs was examined by co-culture with organotypic retinas. ADSCs were seeded on top of the RGCs allowing direct contact, or at the bottom of the well, sharing the same culture media and allowing a paracrine activity. The number of surviving RGCs was assessed using Brn3a staining and confocal microscopy. Cytokine secretion of ADSCs to medium was analyzed by cytokine array. When co-cultured with ADSCs, the number of surviving RGCs was similarly significantly higher in both treatment groups compared to controls. Analysis of ADSCs cytokines secretion profile, showed secretion of anti-apoptotic and pro-proliferative cytokines (threshold>1.4). Transplantation of ADSCs in a co-culture system with organotypic ischemic retinas resulted in RGCs recovery. Since there was no advantage to direct contact of ADSCs with RGCs, the beneficial effect seen may be related to paracrine activity of ADSCs. Conclusions: These data correlated with secretion profile of ADSCs' anti-apoptotic and pro-proliferative cytokines.

• The Korean Journal of Physiology and Pharmacology
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• v.28 no.1
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• pp.83-91
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• 2024

Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor activated under hypoxic conditions, and it plays a crucial role in cellular stress regulation. While HIF-1α activity is essential in normal tissues, its presence in the tumor microenvironment represents a significant risk factor as it can induce angiogenesis and confer resistance to anti-cancer drugs, thereby contributing to poor prognoses. Typically, HIF-1α undergoes rapid degradation in normoxic conditions via oxygen-dependent degradation mechanisms. However, certain cancer cells can express HIF-1α even under normoxia. In this study, we observed an inclination toward increased normoxic HIF-1α expression in cancer cell lines exhibiting increased HDAC6 expression, which prompted the hypothesis that HDAC6 may modulate HIF-1α stability in normoxic conditions. To prove this hypothesis, several cancer cells with relatively higher HIF-1α levels under normoxic conditions were treated with ACY-241, a selective HDAC6 inhibitor, and small interfering RNAs for HDAC6 knockdown. Our data revealed a significant reduction in HIF-1α expression upon HDAC6 inhibition. Moreover, the downregulation of HIF-1α under normoxic conditions decreased zinc finger E-box-binding homeobox 1 expression and increased E-cadherin levels in lung cancer H1975 cells, consequently suppressing cell invasion and migration. ACY-241 treatment also demonstrated an inhibitory effect on cell invasion and migration by reducing HIF-1α level. This study confirms that HDAC6 knockdown and ACY-241 treatment effectively decrease HIF-1α expression under normoxia, thereby suppressing the epithelial-mesenchymal transition. These findings highlight the potential of selective HDAC6 inhibition as an innovative therapeutic strategy for lung cancer.

• Journal of Veterinary Science
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• v.25 no.2
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• pp.31.1-31.8
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• 2024

Background: Recently, there has been a growing interest in stem cells for human medicine. Limited feline endometrial mesenchymal stem cell (fEM-MSC) research in veterinary medicine necessitates reporting for future feline disease research and therapy. Objectives: This study aimed to isolate fEM-MSCs from feline endometrial tissues and evaluate their morphology, proliferative ability, differentiation ability, and immunophenotype. Methods: Feline endometrial tissues were obtained from the ovariohysterectomies of healthy cats and isolated using an enzymatic method. The morphology and proliferative ability of the isolated cells were assessed using a doubling time (DT) assay from passages 3 to 6 (P3 - P6). We measured pluripotency gene expressions of cells in P2 using quantitative real-time polymerase chain reaction (qRT-PCR). To investigate MSC characteristics, a trilineage differentiation assay was conducted in P4, and cells in P4 were immunophenotyped using flow cytometry. Results: fEM-MSCs showed a typical spindle-shaped morphology under a microscope, and the DT was maintained from P3 to P6. fEM-MSCs could differentiate into adipocytes, osteoblasts, and chondrocytes, and expressed three pluripotency markers (OCT4, SOX2, and NANOG) by qRT-PCR. Immunophenotypic analysis showed that the fEM-MSCs were CD14 ^-, CD34 ^-, CD45 ^-, CD9⁺, and CD44⁺. Conclusions: In this study, the feline endometrium was a novel source of MSCs, and to the best of our knowledge, this is the first report on the isolation method and characteristics of fEM-MSCs.

• International Journal of Stem Cells
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• v.15 no.2
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• pp.217-226
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• 2022

Background and Objectives: Stroke is the most common cause of human death and functional disability, resulting in more than 5 million deaths worldwide each year. Bone marrow mesenchymal stem cells (BMSCs) are a kind of stem cell that are able to self-renew and differentiate into many types of tissues. Therefore, BMSCs have the potential to replace damaged neurons and promote the reconstruction of nerve conduction pathways and connective tissue. However, it remains unknown whether transplanted BMSCs promote angiogenesis or improve the tissue microenvironment directly or indirectly through paracrine interactions. This study aimed to determine the therapeutic effect of BMSCs on ischemic stroke with hypertension in a rodent model and to explore the possible mechanisms underlying any benefits. Methods and Results: Middle cerebral artery occlusion was used to establish the experimental stroke model. The area of cerebral infarction, expression of vascular endothelial growth factor (VEGF) and glial cell line-derived neurotrophic factor (GDNF), and increment of astrocyte were measured by TTC staining, western blot, real-time quantitative polymerase chain reaction (RT-qPCR) and immunocytochemistry. The results showed a smaller area of cerebral infarction and improved neurological function scores in animals treated with BMSCs compared to controls. The results of RT-qPCR and western blot assays showed higher expression of VEGF and GDNF in BMSC-treated animals compared with controls. Our study also showed that one round of BMSCs transplantation significantly promoted the proliferation of subventricular zone and cortical cells, especially astrocytes, on the ischemic side following cerebral ischemia. Conclusions: Above findings support that BMSCs have therapeutic effects for ischemic stroke complicated with hypertension, which may occur via up-regulated expression of VEGF and GDNF and reduction of neuronal apoptosis, thereby promoting the recovery of nerve function.

• International Journal of Stem Cells
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• v.15 no.2
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• pp.164-172
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• 2022

Background and Objectives: Despite advances in wound treatments, chronic diabetic wounds remain a significant medical challenge. Exosomes from mesenchymal stem cells (MSCs) and small molecule activators of nuclear factor erythroid 2-related factor 2 (Nrf2) have emerged as potential therapies for nonhealing diabetic wounds. This study aimed to evaluate the effects of exosomes from bone marrow-derived MSCs (BMSCs) alone, or in combination with a small molecule activator of Nrf2 on diabetic wound healing. Methods and Results: BMSCs and endothelial progenitor cells (EPCs) were isolated from the femur and tibia bone marrow of Sprague-Dawley (SD) rats and culture-expanded. Exosomes were harvested from the BMSC culture supernatants through ultracentrifugation. The effects of the exosomes and Nrf2 knockdown, alone or in combination, on EPC tube formation were evaluated. Streptozotocin-induced diabetic rats bearing a fresh full-thickness round wound were treated with the exosomes alone, or in combination with a lentiviral shRNA targeting Nrf2 (Lenti-sh-Nrf2) or tert-butylhydroquinone (tBHQ), a small molecule activator of Nrf2. Two weeks later, wound closure, re-epithelization, collagen deposition, neovascularization, and local inflammation were evaluated. BMSC exosomes promoted while Nrf2 knockdown inhibited EPC tube formation. BMSC exosomes accelerated wound closure, re-epithelization, collagen deposition, and neovascularization, and reduced wound inflammation in diabetic rats. These regenerative and anti-inflammatory effects of the exosomes were inhibited by Lenti-sh-Nrf2 but enhanced by tBHQ administration. Conclusions: BMSC exosomes in combination with a small molecule Nrf2 activator hold promise as a new therapeutic option for chronic diabetic wounds.