Assessing systemic, developmental, and reproductive toxicity and estrogenicity of Korean red ginseng extract G1899 in juvenile Sprague-Dawley Rats.

Background: Korean red ginseng (KRG) is a product from ginseng roots, which is enriched with ginsenosides and has been utilized for a long time as an adaptogen to alleviate various physiological or disease conditions. While KRG is generally considered safe, conducting a thorough toxicological assessment of the spray-dried powder G1899 during the juvenile period is essential to establish its safety profile. This study aimed to assess the safety of G1899 during the juvenile period using Sprague-Dawley rats. Methods: Two studies were conducted separately: a juvenile toxicity study and a uterotrophic bioassay. To assess the potential toxicity at systemic, postnatal developmental, and reproductive levels, G1899 was orally gavaged once a day in post-weaning juvenile Sprague-Dawley (SD) rats at 0, 1250, 2500, or 5000 mg/kg/day. Estrogenicity was assessed by orally gavaging G1899 in immature female SD rats at 0, 2500, or 5000 mg/kg/day on postnatal days (PND) 19-21, followed by a uterotrophic bioassay. These studies were conducted in accordance with the Good Laboratory Practice (GLP) regulations and regulatory test guidelines. Results: Regarding juvenile toxicity, no abnormalities related to the G1899 treatment were observed in any group during the experiment. Moreover, no uterotrophic responses were observed in the dosed female group. Based on these results, the no observed adverse effect level (NOAEL) of G1899 was determined to be at least 5000 mg/kg/day for general systemic function, developmental/reproductive function, and estrogenic activity. Conclusion: Our results suggest that G1899 is not toxic to juveniles at doses of up to 5000 mg/kg/day.

Generation of telomeric repeat binding factor 1 (TRF1)-knockout human embryonic stem cell lines, KRIBBe010-A-95, KRIBBe010-A-96, and KRIBBe010-A-97, using CRISPR/Cas9 technology.

Telomeric repeat binding factor 1 (TRF1) plays an essential role in maintaining telomere length. Here, we established TRF1-knockout human pluripotent stem cells (hPSCs; hTRF1-KO) using the CRISPR/Cas9 technology. The hTRF1-KO cell lines expressed pluripotency markers and demonstrated a normal karyotype (46, XX) and DNA profile. In addition, hTRF1-KOcells spontaneously differentiated into all three germ layers in vitro. Thus, these cell lines could be useful models in various research fields.

Gut metabolite trimethylamine N-oxide induces aging-associated phenotype of midbrain organoids for the induced pluripotent stem cell-based modeling of late-onset disease.

Brain organoids are valuable research models for human development and disease since they mimic the various cell compositions and structures of the human brain; however, they have challenges in presenting aging phenotypes for degenerative diseases. This study analyzed the association between aging and the gut metabolite trimethylamine N-oxide (TMAO), which is highly found in the midbrain of elderly and Parkinson's disease (PD) patients. TMAO treatment in midbrain organoid induced aging-associated molecular changes, including increased senescence marker expression (P21, P16), p53 accumulation, and epigenetic alterations. In addition, TMAO-treated midbrain organoids have shown parts of neurodegeneration phenotypes, including impaired brain-derived neurotrophic factor (BDNF) signaling, loss of dopaminergic neurons, astrocyte activation, and neuromelanin accumulation. Moreover, we found TMAO treatment-induced pathophysiological phosphorylation of α-synuclein protein at Ser-129 residues and Tau protein at Ser202/Thr205. These results suggest a role of TMAO in the aging and pathogenesis of the midbrain and provide insight into how intestinal dysfunction increases the risk of PD. Furthermore, this system can be utilized as a novel aging model for induced pluripotent stem cell (iPSC)-based modeling of late-onset diseases.

Blood vessel formation in cerebral organoids formed from human embryonic stem cells.

Current cerebral organoid technology provides excellent in vitro models mimicking the structure and function of the developing human brain, which enables studies on normal and pathological brain; however, further improvements are necessary to overcome the problems of immaturity and dearth of non-parenchymal cells. Vascularization is one of the major challenges for recapitulating processes in the developing human brain. Here, we examined the formation of blood vessel-like structures in cerebral organoids induced by vascular endothelial growth factor (VEGF) in vitro. The results indicated that VEGF enhanced differentiation of vascular endothelial cells (ECs) without reducing neuronal markers in the embryonic bodies (EBs), which then successfully developed into cerebral organoids with open-circle vascular structures expressing an EC marker, CD31, and a tight junction marker, claudin-5, characteristic of the blood-brain barrier (BBB). Further treatment with VEGF and Wnt7a promoted the formation of the outer lining consisting of pericyte-like cells, which surrounded the vascular tubes. RNA sequencing revealed that VEGF upregulated genes associated with tube formation, vasculogenesis, and the BBB; it also changed the expression of genes involved in brain embryogenesis, suggesting a role of VEGF in neuronal development. These results indicate that VEGF treatment can be used to generate vessel-like structures with mature BBB characteristics in cerebral organoids in vitro.

Integrin-Linked Kinase Deficiency in Collecting Duct Principal Cell Promotes Necroptosis of Principal Cell and Contributes to Kidney Inflammation and Fibrosis.

BACKGROUND: Necroptosis is a newly discovered cell death pathway that plays a critical role in AKI. The involvement of integrin-linked kinase (ILK) in necroptosis has not been studied. METHODS: We performed experiments in mice with an Ilk deletion in collecting duct (CD) principal cells (PCs), and cultured tubular epithelial cells treated with an ILK inhibitor or ILK siRNA knockdown. RESULTS: Ilk deletion in CD PCs resulted in acute tubular injury and early mortality in mice. Progressive interstitial fibrosis and inflammation associated with the activation of the canonical TGF-ß signaling cascade were detected in the kidneys of the mice lacking ILK in the CD PCs. In contrast to the minimal apoptosis detected in the animals' injured CDs, widespread necroptosis was present in ILK-deficient PCs, characterized by cell swelling, deformed mitochondria, and rupture of plasma membrane. In addition, ILK deficiency resulted in increased expression and activation of necroptotic proteins MLKL and RIPK3, and membrane translocation of MLKL in CD PCs. ILK inhibition and siRNA knockdown reduced cell survival in cultured tubular cells, concomitant with increased membrane accumulation of MLKL and/or phospho-MLKL. Administration of a necroptosis inhibitor, necrostatin-1, blocked cell death in vitro and significantly attenuated inflammation, interstitial fibrosis, and renal failure in ILK-deficient mice. CONCLUSIONS: The study demonstrates the critical involvement of ILK in necroptosis through modulation of the RIPK3 and MLKL pathway and highlights the contribution of CD PC injury to the development of inflammation and interstitial fibrosis of the kidney.

Pathological cardiac remodeling occurs early in CKD mice from unilateral urinary obstruction, and is attenuated by Enalapril.

Cardiovascular disease constitutes the leading cause of mortality in patients with chronic kidney disease (CKD) and end-stage renal disease. Despite increasing recognition of a close interplay between kidney dysfunction and cardiovascular disease, termed cardiorenal syndrome (CRS), the underlying mechanisms of CRS remain poorly understood. Here we report the development of pathological cardiac hypertrophy and fibrosis in early stage non-uremic CKD. Moderate kidney failure was induced three weeks after unilateral urinary obstruction (UUO) in mice. We observed pathological cardiac hypertrophy and increased fibrosis in UUO-induced CKD (UUO/CKD) animals. Further analysis indicated that this cardiac fibrosis was associated with increased expression of transforming growth factor ß (TGF-ß) along with significant upregulation of Smad 2/3 signaling in the heart. Moreover early treatment of UUO/CKD animals with an angiotensin-converting-enzyme inhibitor (ACE I), Enalapril, significantly attenuated cardiac fibrosis. Enalapril antagonized activation of the TGF-ß signaling pathway in the UUO/CKD heart. In summary our study demonstrates the presence of pathological cardiac hypertrophy and fibrosis in mice early in UUO-induced CKD, in association with early activation of the TGF-ß/Smad signaling pathway. We also demonstrate the beneficial effect of ACE I in alleviating this early fibrogenic process in the heart in UUO/CKD animals.

Corrigendum: Small molecule-mediated up-regulation of microRNA targeting a key cell death modulator BNIP3 improves cardiac function following ischemic injury.

This corrects the article DOI: 10.1038/srep23472.

microRNA-133a attenuates cardiomyocyte hypertrophy by targeting PKCδ and Gq.

During the past decade, microRNAs have continuously been suggested as a promising therapeutic tool due to their beneficial effects, such as their multi-targets and multi-functions in pathologic conditions. As a pathologic phenotype is generally regulated by multiple signaling pathways, in this study we identified a microRNA regulating multiple target genes within cardiac hypertrophic signaling pathways. microRNA-133a is known to play a crucial role in cardiac hypertrophy. However, the role of microRNA-133a, which may regulate several signaling pathways in norepinephrine-induced cardiac hypertrophy via multi-targeting, has not been investigated. In the current study, we showed that microRNA-133a can protect cardiomyocyte hypertrophy against norepinephrine stimulation in neonatal rat ventricular cardiomyocytes via new targets, PKCδ and Gq, all of which are related to downstream signaling pathways of the α1-adrenergic receptor. Taken together, these results suggest the advantages of the therapeutic use of microRNAs as an effective potential drug regulating multiple signaling pathways under pathologic conditions.

Manganese promotes intracellular accumulation of AQP2 via modulating F-actin polymerization and reduces urinary concentration in mice.

Aquaporin-2 (AQP2) is a water channel protein expressed in principal cells (PCs) of the kidney collecting ducts (CDs) and plays a critical role in mediating water reabsorption and urine concentration. AQP2 undergoes both regulated trafficking mediated by vasopressin (VP) and constitutive recycling, which is independent of VP. For both pathways, actin cytoskeletal dynamics is a key determinant of AQP2 trafficking. We report here that manganese chloride (MnCl2) is a novel and potent regulator of AQP2 trafficking in cultured cells and in the kidney. MnCl2 treatment promoted internalization and intracellular accumulation of AQP2. The effect of MnCl2 on the intracellular accumulation of AQP2 was associated with activation of RhoA and actin polymerization without modification of AQP2 phosphorylation. Although the level of total and phosphorylated AQP2 did not change, MnCl2 treatment impeded VP-induced phosphorylation of AQP2 at its serine-256, -264, and -269 residues and dephosphorylation at serine 261. In addition, MnCl2 significantly promoted F-actin polymerization along with downregulation of RhoA activity and prevented VP-induced membrane accumulation of AQP2. Finally, MnCl2 treatment in mice resulted in significant polyuria and reduced urinary concentration, likely due to intracellular relocation of AQP2 in the PCs of kidney CDs. More importantly, the reduced urinary concentration caused by MnCl2 treatment in animals was not corrected by VP. In summary, our study identified a novel effect of MnCl2 on AQP2 trafficking through modifying RhoA activity and actin polymerization and uncovered its potent impact on water diuresis in vivo.

Small molecule-mediated induction of miR-9 suppressed vascular smooth muscle cell proliferation and neointima formation after balloon injury.

Pathologic proliferation and migration of vascular smooth muscle cells (VSMCs) exacerbate cardiovascular disease. MicroRNAs (miRNAs), as endogenous inhibitors of protein synthesis, are expected to modulate pathologic proliferation of VSMCs. Here we report that both platelet-derived growth factor receptor (PDGFR) targeting miR-9 and a small molecule that increases miR-9 can inhibit the serum-induced proliferation of VSMCs. First, based on miRNA-target prediction databases and empirical data, we have selected miR-9 as a potent anti-proliferative miRNA in VSMCs. Further examination indicated that miR-9 directly targets PDGFR disrupting downstream signaling cascades, and this resulted in inhibition of VSMC proliferation and migration. Exogenous delivery of miR-9 inhibited VSMC proliferation in vitro, and a small molecule that increased miR-9 expression also inhibited neointima formation following balloon injury in vivo. We provide evidence of miRNA-mediated modulation of VSMC proliferation and further demonstrate that small molecule-mediated regulation of miRNA targeting a key regulator of VSMC proliferation is a viable therapeutic strategy for treating vascular disease involving pathologic VSMC proliferation such as restenosis.

Therapeutic Potential of Stem Cells Strategy for Cardiovascular Diseases.

Despite development of medicine, cardiovascular diseases (CVDs) are still the leading cause of mortality and morbidity worldwide. Over the past 10 years, various stem cells have been utilized in therapeutic strategies for the treatment of CVDs. CVDs are characterized by a broad range of pathological reactions including inflammation, necrosis, hyperplasia, and hypertrophy. However, the causes of CVDs are still unclear. While there is a limit to the currently available target-dependent treatments, the therapeutic potential of stem cells is very attractive for the treatment of CVDs because of their paracrine effects, anti-inflammatory activity, and immunomodulatory capacity. Various studies have recently reported increased therapeutic potential of transplantation of microRNA- (miRNA-) overexpressing stem cells or small-molecule-treated cells. In addition to treatment with drugs or overexpressed miRNA in stem cells, stem cell-derived extracellular vesicles also have therapeutic potential because they can deliver the stem cell-specific RNA and protein into the host cell, thereby improving cell viability. Here, we reported the state of stem cell-based therapy for the treatment of CVDs and the potential for cell-free based therapy.

Hypoxic conditioned medium from mesenchymal stem cells promotes lymphangiogenesis by regulation of mitochondrial-related proteins.

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.

Small molecule-mediated up-regulation of microRNA targeting a key cell death modulator BNIP3 improves cardiac function following ischemic injury.

Genetic ablation of BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), an essential regulator of cardiac cell death, is an effective way to prevent cardiac cell death triggered by pathologic conditions. However, currently there exists no known means, such as inhibitors, to down-regulate BNIP3 in mature heart. Here, we report that a small molecule inducer of microRNA-182 (miR-182) suppressed ischemia/reperfusion (I/R)-induced cardiac cell death by down-regulating BNIP3. We first selected miR-182 as a potent BNIP3-targeting miRNA based on miRNA-target prediction databases and empirical data. The subsequent screening of small molecules for inducing miR-182 expression identified Kenpaullone as a hit compound. Both exogenous miR-182 and Kenpaullone significantly suppressed hypoxia-induced cardiomyocyte death in vitro. To investigate the effect of changing substituents of Kenpaullone on miR-182 expression, we synthesized 9 derivatives of Kenpaullone. Among these derivatives, compound 5 showed significantly improved ability to induce miR-182 expression. The results of the in vivo study showed that compound 5 significantly improved heart function following I/R-injury in rats. Our study provides strong evidence that the small molecule-mediated up-regulation of miRNAs is a viable strategy to down-regulate target proteins with no known chemical inhibitor and that compound 5 may have potential to prevent I/R-inflicted cardiac cell death.

TCF4-Targeting miR-124 is Differentially Expressed amongst Dendritic Cell Subsets.

Dendritic cells (DCs) are professional antigen-presenting cells that sample their environment and present antigens to naïve T lymphocytes for the subsequent antigen-specific immune responses. DCs exist in a range of distinct subpopulations including plasmacytoid DCs (pDCs) and classical DCs (cDCs), with the latter consisting of the cDC1 and cDC2 lineages. Although the roles of DC-specific transcription factors across the DC subsets have become understood, the posttranscriptional mechanisms that regulate DC development are yet to be elucidated. MicroRNAs (miRNAs) are pivotal posttranscriptional regulators of gene expression in a myriad of biological processes, but their contribution to the immune system is just beginning to surface. In this study, our in-house probe collection was screened to identify miRNAs possibly involved in DC development and function by targeting the transcripts of relevant mouse transcription factors. Examination of DC subsets from the culture of mouse bone marrow with Flt3 ligand identified high expression of miR-124 which was able to target the transcript of TCF4, a transcription factor critical for the development and homeostasis of pDCs. Further expression profiling of mouse DC subsets isolated from in vitro culture as well as via ex vivo purification demonstrated that miR-124 was outstandingly expressed in CD24(+) cDC1 cells compared to in pDCs and CD172α(+) cDC2 cells. These results imply that miR-124 is likely involved in the processes of DC subset development by posttranscriptional regulation of a transcription factor(s).

1H-pyrrole-2,5-dione-based small molecule-induced generation of mesenchymal stem cell-derived functional endothelial cells that facilitate rapid endothelialization after vascular injury.

INTRODUCTION: 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. METHODS: 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, 3×10(6) 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. RESULTS: 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. CONCLUSIONS: 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.

Suppression of miR-181a attenuates H2O2-induced death of mesenchymal stem cells by maintaining hexokinase II expression.

BACKGROUND: Low survival rate of transplanted cells compromises the efficacy of cell therapy. Hexokinase II (HKII) is known to have anti-apoptotic activity through its interaction with mitochondria. The objective was to identify miRNAs targeting HKII and investigate whether miRNA-mediated modulation of HKII could improve the survival of mesenchymal stem cells (MSCs) exposed to H2O2. The expression of HKII in MSCs exposed to H2O2 was evaluated, and HKII-targeting miRNA was screened based on miRNA-target prediction databases. The effect of H2O2 on the expression of the selected HKII-targeting miRNA was examined and the effect of modulation of the selected HKII-targeting miRNA using anti-miRNA on H2O2-induced apoptosis of MSC was evaluated. RESULTS: H2O2 (600 µM) induced cell death of MSCs and decreased mitochondrial HKII expression. We have identified miR-181a as a HKII-targeting miRNA and H2O2 increased the expression of miR-181a in MSCs. Delivery of anti-miR-181a, which neutralizes endogenous miR-181a, significantly attenuated H2O2-induced decrease of HKII expression and disruption of mitochondrial membrane potential, improving the survival of MSCs exposed to H2O2. CONCLUSIONS: These findings suggest that H2O2-induced up-regulation of miR-181a contributes to the cell death of MSCs by down-regulating HKII. Neutralizing miR-181a can be an effective way to prime MSCs for transplantation into ischemic tissues.

ROS-mediated bidirectional regulation of miRNA results in distinct pathologic heart conditions.

Under distinct pathological heart conditions, the expression of a single miRNA can display completely opposite patterns. However, the mechanism underlying the bidirectional regulation of a single miRNA and the clinical implications of this regulation remain largely unknown. To address this issue, we examined the regulation of miR-1, one of the most abundant miRNAs in the heart, during cardiac hypertrophy and ischemia/reperfusion (I/R). Our data indicated that different magnitudes and chronicities of ROS levels in cardiomyocytes resulted in differential expression of miR-1, subsequently altering the expression of myocardin. In animal models, the administration of a miR-1 mimic attenuated cardiac hypertrophy by suppressing the transverse aortic constriction-induced increase in myocardin expression, whereas the administration of anti-miR-1 ameliorated I/R-induced cardiac apoptosis and deterioration of heart function. Our findings indicated that a pathologic stimulus such as ROS can bidirectionally alter the expression of miRNA to contribute to the development of pathological conditions exhibiting distinct phenotypes and that the meticulous adjustment of the pathological miRNA levels is required to improve clinical outcomes.

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

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 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. 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.

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.

Enhanced Healing of Rat Calvarial Bone Defects with Hypoxic Conditioned Medium from Mesenchymal Stem Cells through Increased Endogenous Stem Cell Migration via Regulation of ICAM-1 Targeted-microRNA-221.

The use of conditioned medium from mesenchymal stem cells may be a feasible approach for regeneration of bone defects through secretion of various components of mesenchymal stem cells such as cytokines, chemokines, and growth factors. Mesenchymal stem cells secrete and accumulate multiple factors in conditioned medium under specific physiological conditions. In this study, we investigated whether the conditioned medium collected under hypoxic condition could effectively influence bone regeneration through enhanced migration and adhesion of endogenous mesenchymal stem cells. Cell migration and adhesion abilities were increased through overexpression of intercellular adhesion molecule-1 in hypoxic conditioned medium treated group. Intercellular adhesion molecule-1 was upregulated by microRNA-221 in mesenchymal stem cells because microRNAs are key regulators of various biological functions via gene expression. To investigate the effects in vivo, evaluation of bone regeneration by computed tomography and histological assays revealed that osteogenesis was enhanced in the hypoxic conditioned medium group relative to the other groups. These results suggest that behavioral changes of endogenous mesenchymal stem cells through microRNA-221 targeted-intercellular adhesion molecule-1 expression under hypoxic conditions may be a potential treatment for patients with bone defects.