Dong Suk Yoon

SIRT1 directly regulates SOX2 to maintain self-renewal and multipotency in bone marrow-derived mesenchymal stem cells.

SOX2 is crucial for the maintenance of the self‐renewal capacity and multipotency of mesenchymal stem cells (MSCs); however, the mechanism by which SOX2 is regulated remains unclear. Here, we report that RNA interference of sirtuin 1 (SIRT1) in human bone marrow (BM)‐derived MSCs leads to a decrease of SOX2 protein, resulting in the deterioration of the self‐renewal and differentiation capacities of BM‐MSCs. Using immunoprecipitation, we demonstrated direct binding between SIRT1 and SOX2 in HeLa cells overexpressing SOX2. We further discovered that the RNA interference of SIRT1 induces the acetylation, nuclear export, and ubiquitination of SOX2, leading to proteasomal degradation in BM‐MSCs. SOX2 suppression by trichostatin A (TSA), a known histone deacetylase inhibitor, was reverted by treatment with resveratrol (0.1 and 1 µM), a known activator of SIRT1 in BM‐MSCs. Furthermore, 0.1 and 1 µM resveratrol reduced TSA‐mediated acetylation and ubiquitination of SOX2 in BM‐MSCs. SIRT1 activation by resveratrol enhanced the colony‐forming ability and differentiation potential to osteogenic and adipogenic lineages in a dose‐dependent manner. However, the enhancement of self‐renewal and multipotency by resveratrol was significantly decreased to basal levels by RNA interference of SOX2. These results strongly suggest that the SIRT1‐SOX2 axis plays an important role in maintaining the self‐renewal capability and multipotency of BM‐MSCs. In conclusion, our findings provide evidence for positive SOX2 regulation by post‐translational modification in BM‐MSCs through the inhibition of nuclear export and subsequent ubiquitination, and demonstrate that SIRT1‐mediated deacetylation contributes to maintaining SOX2 protein in the nucleus. Stem Cells 2014;32:3219–3231

miR-449a regulates the chondrogenesis of human mesenchymal stem cells through direct targeting of lymphoid enhancer-binding factor-1

microRNAs are small molecules, about 17-23 nucleotides in length, that act as translational regulators of their target gene. By binding to a target, microRNAs are known to either inhibit translation or induce degradation of the target. Despite the great interest in microRNAs, however, the exact targets of each individual microRNA in different processes remain largely unknown. In this study, we determined that the lymphoid enhancer-binding factor-1 (LEF-1) was expressed during the chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and sought to identify a novel microRNA targeting this gene. Through subsequent studies, we have identified, for the first time, one particular microRNA, miR-449a, that recognizes and regulates the expression of LEF-1 in a dose-dependent and sequence-specific manner. In addition, we observed that the inhibition of LEF-1 via miR-449a led to the subsequent repression of Sox 9, which is a well-established regulator of chondrogenesis. Collectively, this study demonstrated that miR-449a directly targets LEF-1, which in turn affects the expression of Sox 9, ultimately leading to the proper regulation of the differentiation and chondrogenesis of human MSCs (hBM-MSCs).

Characterization of Different Subpopulations from Bone Marrow-Derived Mesenchymal Stromal Cells by Alkaline Phosphatase Expression

Multiple surface markers have been utilized for the enrichment of bone marrow mesenchymal stromal cells (MSCs) and to define primitive stem cells. We classified human bone marrow-derived MSC populations according to tissue nonspecific alkaline phosphatase (TNAP) activity. TNAP expression varied among unexpanded primary MSCs, and its level was not related to colony-forming activity or putative surface markers, such as CD105 and CD29, donor age, or gender. TNAP levels were increased in larger cells, and a colony-forming unit-fibroblast assay revealed that the colony size was decreased during in vitro expansion. TNAP-positive (TNAP+) MSCs showed limited multipotential capacity, whereas TNAP-negative (TNAP-) MSCs retained the differentiation potential into 3 lineages (osteogenic-, adipogenic-, and chondrogenic differentiation). High degree of calcium mineralization and high level of osteogenic-related gene expression (osteopontin, dlx5, and cbfa1) were found in TNAP+ cells. In contrast, during chondrogenic differentiation, type II collagen was successfully induced in TNAP- cells, but not in TNAP+ cells. TNAP+ cells showed high levels of the hypertrophic markers, type X collagen and cbfa1. Mesenchymal stem cell antigen-1 (MSCA-1) is identical to TNAP. Therefore, TNAP+ cells were sorted by using antibody targeting MSCA-1. MSCA-1-positive cells sorted for TNAP+ cells exhibited low proliferation rates. Expression of cell cycle-related genes (cyclin A2, CDK2, and CDK4) and pluripotency marker genes (rex1 and nanog) were higher in TNAP- MSC than in TNAP+ MSC. Therefore, TNAP- cells can be described as more primitive bone marrow-derived cells and TNAP levels in MSCs can be used to predict chondrocyte hypertrophy or osteogenic capacity.

Cell recruiting chemokine-loaded sprayable gelatin hydrogel dressings for diabetic wound healing.

UNLABELLED In this study, we developed horseradish peroxidase (HRP)-catalyzed sprayable gelatin hydrogels (GH) as a bioactive wound dressing that can deliver cell-attracting chemotactic cytokines to the injured tissues for diabetic wound healing. We hypothesized that topical administration of chemokines using GH hydrogels might improve wound healing by inducing recruitment of the endogenous cells. Two types of chemokines (interleukin-8; IL-8, macrophage inflammatory protein-3α; MIP-3α) were simply loaded into GH hydrogels during in situ cross-linking, and then their wound-healing effects were evaluated in streptozotocin-induced diabetic mice. The incorporation of chemokines did not affect hydrogels properties including swelling ratio and mechanical stiffness, and the bioactivities of IL-8 and MIP-3α released from hydrogel matrices were stably maintained. In vivo transplantation of chemokine-loaded GH hydrogels facilitated cell infiltration into the wound area, and promoted wound healing with enhanced re-epithelialization/neovascularization and increased collagen deposition, compared with no treatment or the GH hydrogel alone. Based on our results, we suggest that cell-recruiting chemokine-loaded GH hydrogel dressing can serve as a delivery platform of various therapeutic proteins for wound healing applications. STATEMENT OF SIGNIFICANCE Despite development of materials combined with therapeutic agents for diabetic wound treatment, impaired wound healing by insufficient chemotactic responses still remain as a significant problem. In this study, we have developed enzyme-catalyzed gelatin (GH) hydrogels as a sprayable dressing material that can deliver cell-attracting chemokines for diabetic wound healing. The chemotactic cytokines (IL-8 and MIP-3α) were simply loaded within hydrogel during in situ gelling, and wound healing efficacy of chemokine-loaded GH hydrogels was investigated in STZ-induced diabetic mouse model. These hydrogels significantly promoted wound-healing efficacy with faster wound closure, neovascularization, and thicker granulation. Therefore, we expect that HRP-catalyzed in situ forming GH hydrogels can serve as an injectable/sprayable carrier of various therapeutic agents for wound healing applications.

Zinc inhibits osteoclast differentiation by suppression of Ca2+-Calcineurin-NFATc1 signaling pathway

BackgroundZinc, an essential trace element, inhibits osteoclast differentiation in vitro and in vivo. The molecular mechanism for the inhibitory effect of zinc, however, is poorly understood. The purpose of this study was to investigate the effect of zinc and determine its molecular mechanism on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in mouse bone marrow-derived monocyte cells (BMMs) and RAW264.7 cells.ResultsIn BMMs, zinc treatment during osteoclast differentiation decreased RANKL-induced osteoclast formation in a dose-dependent manner. We show that zinc suppressed the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1). Zinc also accumulated phospho-Nfatc1 (p-Nfatc1) in the cytosol in a dose-dependent manner and inhibited the translocation of Nfatc1 to the nucleus in RAW264.7 cells. Zinc suppressed the activities of Nfatc1 in the nucleus without changing the activities of NF-κB in RAW264.7 cells. In contrast, calcineurin activity decreased in response to zinc but its protein level was unchanged. RANKL-induced Ca2+ oscillations were inhibited by zinc treatment, but phospho-phospholipase Cγ1 (p-PLCγ1), the upstream signaling molecule of Ca2+ oscillations, was unaffected. Moreover, a constitutively active form of Nfatc1 obviously rescued suppression of osteoclastogenesis by zinc.ConclusionsTaken together, these results demonstrate for the first time that the inhibitory effect of zinc during osteoclastogesis is caused by suppressing the Ca2+-Calcineurin-NFATc1 signaling pathway. Thus, zinc may be a useful therapeutic candidate for the prevention of bone loss caused by NFATc1 activation in osteoclasts.

Interleukin-6 induces the lineage commitment of bone marrow-derived mesenchymal multipotent cells through down-regulation of Sox2 by osteogenic transcription factors

Bone marrow‐derived mesenchymal stromal cells (BM‐MSCs) are a heterogeneous population of cells that differ in size and morphology. BM‐MSCs become committed to the osteogenic lineage as senescence approaches and lose multipotency. Nevertheless, little is known about the effects of cell‐cell interaction between different populations on stemness loss and lineage commitment. The current study aimed to identify mechanisms by which cell‐cell interactions between heterogeneous BM‐MSCs affect stemness and lineage commitment of multipotent subpopulation. The lineage commitment of primitive multipotent cells was strongly induced in the presence of cytokines secreted by senescent‐like cells in a cell culture insert system. Senescent‐like cells secreted higher levels of interleukin‐6 (IL‐6) than primitive multipotent cells in a human cytokine array. IL‐6 induced the lineage commitment and stemness loss in multipotent cells by decreasing Sox2 expression. Furthermore, we confirmed that IL‐6 decreased the transcriptional activity of Sox2 through up‐regulation of Runx2 and Dlx5. We suggest a mechanism by which IL‐6 modulates the expression of Sox2, resulting in decreased multipotency and causing primitive multipotent cells to undergo osteogenic lineage commitment. This is the first study to identify mechanisms in which the cell‐cell interactions between the different populations play important roles in the stemness loss and lineage commitment of multipotent populations.—Yoon, D. S., Kim, Y. H., Lee, S., Lee, K.‐M., Park, K. H., Jang, Y., Lee, J. W. Interleukin‐6 induces the lineage commitment of bone marrow‐derived mesenchymal multipotent cells through down‐regulation of Sox2 by osteogenic transcription factors. FASEB J. 28, 3273–3286 (2014). www.fasebj.org

Characterization of adipose tissue-derived stromal vascular fraction for clinical application to cartilage regeneration.

Bone marrow concentration (BMC) is the most recognized procedure to prepare mesenchymal stem cells for cartilage regeneration. However, bone marrow aspiration is highly invasive and results in low stem cell numbers. Recently, adipose tissue-derived stromal vascular fraction (AT-SVF) was studied as an alternate source of stem cells for cartilage regeneration. However, AT-SVF is not fully characterized in terms of functional equivalence to BMC. Therefore, in this study, we characterized AT-SVF and assessed its suitability as a one-step surgical procedure for cartilage regeneration, as an alternative to BMC. AT-SVF contained approximately sixfold less nucleated cells than BMC. However, adherent cells in AT-SVF were fourfold greater than BMC. Additionally, the colony-forming unit frequency of AT-SVF was higher than that of BMC, at 0.5 and 0.01%, respectively. The mesenchymal stem cell (MSC) population (CD45−CD31−CD90+CD105+) was 4.28% in AT-SVF and 0.42% in BMC, and the adipose-derived stromal cell (ASC) population (CD34+CD31−CD146−) was 32% in AT-SVF and 0.16% in BMC. In vitro chondrogenesis demonstrated that micromass was not formed in BMC, whereas it was clearly formed in AT-SVF. Taken together, uncultured AT-SVF could be used in one-step surgery for cartilage regeneration as a substitute for BMC.

Different effects of resveratrol on early and late passage mesenchymal stem cells through β-catenin regulation.

Resveratrol is a sirtuin 1 (SIRT1) activator and can function as an anti-inflammatory and antioxidant factor. In mesenchymal stem cells (MSCs), resveratrol enhances the proliferation and differentiation potential and has an anti-aging effect. However, contradictory effects of resveratrol on MSC cultures have been reported. In this study, we found that resveratrol had different effects on MSC cultures according to their cell passage and SIRT1 expression. Resveratrol enhanced the self-renewal potential and multipotency of early passage MSCs, but accelerated cellular senescence of late passage MSCs. In early passage MSCs expressing SIRT1, resveratrol decreased ERK and GSK-3β phosphorylation, suppressing β-catenin activity. In contrast, in late passage MSCs, which did not express SIRT1, resveratrol increased ERK and GSK-3β phosphorylation, activating β-catenin. We confirmed that SIRT1-deficient early passage MSCs treated with resveratrol lost their self-renewal potential and multipotency, and became senescent due to increased β-catenin activity. Sustained treatment with resveratrol at early passages maintained the self-renewal potential and multipotency of MSCs up to passage 10. Our findings suggest that resveratrol can be effectively applied to early passage MSC cultures, whereas parameters such as cell passage and SIRT1 expression must be taken into consideration before applying resveratrol to late passage MSCs.

Synergistic Action of IL-8 and Bone Marrow Concentrate on Cartilage Regeneration Through Upregulation of Chondrogenic Transcription Factors

The objective of this study was to determine whether a biphasic scaffold loaded with a combination of a chemokine and bone marrow concentrate (BMC) could improve tissue regeneration in knee articular cartilage of beagles with cylindrical osteochondral defects. For this investigation, an osteochondral defect (6 mm in diameter and 8 mm deep) was created in the weight-bearing articular surface of the femoral medial condyle in beagles. Bone marrow was aspirated from the posterior iliac crests of beagles to obtain mesenchymal stem cells (MSCs) for in vitro assay. Hematoxylin and eosin (HE), Massons trichrome (MT), safranin O/fast green staining, and immunohistochemistry were performed for histological analysis. Quantitative real-time polymerase chain reaction was performed to understand the roles of BMC in chondrogenic differentiation of MSCs. At 12 weeks after transplantation of biphasic scaffolds, we observed that interleukin-8 (IL-8) or the combination of IL-8 and BMC induced massive bone regeneration compared to saline, BMC only, and MSCs. In gross appearance, the osteochondral defect site was nearly completely filled with repair tissue in the group that received the combination of IL-8 and BMC but not in the other groups. Moreover, histological analysis showed obvious differences in cartilage regeneration among groups. HE and MT staining showed that the cartilage defect sites of the group receiving the combination of IL-8 and BMC were regenerated with cartilage-like tissues showing chondrocyte morphology. Safranin O staining showed hyaline cartilage regeneration in the group receiving IL-8 and BMC, whereas fibrous-like tissues were formed in the other groups. Furthermore, immunostaining revealed the presence of type II collagen and aggrecan in regenerated cartilage tissue of the group receiving IL-8 and BMC, whereas regenerated cartilage tissues of the other groups weakly expressed type II collagen and aggrecan. These results indicate that the combination of a chemokine IL-8 and BMC has significant positive effects on osteochondral regeneration in a beagle model through enhancing expression of the chondrogenic transcription factors and markers such as Sox9 and type II collagen.

Triclosan Disrupts SKN-1/Nrf2-Mediated Oxidative Stress Response in C . elegans and Human Mesenchymal Stem Cells

Triclosan (TCS), an antimicrobial chemical with potential endocrine-disrupting properties, may pose a risk to early embryonic development and cellular homeostasis during adulthood. Here, we show that TCS induces toxicity in both the nematode C. elegans and human mesenchymal stem cells (hMSCs) by disrupting the SKN-1/Nrf2-mediated oxidative stress response. Specifically, TCS exposure affected C. elegans survival and hMSC proliferation in a dose-dependent manner. Cellular analysis showed that TCS inhibited the nuclear localization of SKN-1/Nrf2 and the expression of its target genes, which were associated with oxidative stress response. Notably, TCS-induced toxicity was significantly reduced by either antioxidant treatment or constitutive SKN-1/Nrf2 activation. As Nrf2 is strongly associated with aging and chemoresistance, these findings will provide a novel approach to the identification of therapeutic targets and disease treatment.