Normal Bone Marrow-derived Mesenchymal Stem Cells Research Articles

A novel LncRNA SPIRE1/miR-181a-5p/PRLR axis in mandibular bone marrow-derived mesenchymal stem cells regulates the Th17/Treg immune balance through the JAK/STAT3 pathway in periodontitis.

Periodontitis is a microbial-related chronic inflammatory disease associated with imbalanced differentiation of Th17 cells and Treg cells. Bone marrow-derived mesenchymal stem cells (BM-MSCs) possess wide immunoregulatory properties. Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) contribute to the immunomodulation in the pathological mechanisms of inflammatory diseases. However, critical lncRNAs/miRNAs involved in immunomodulation of mandibular BM-MSCs largely remain to be identified. Here, we explored the molecular mechanisms behind the defective immunomodulatory ability of mandibular BM-MSCs under the periodontitis settings. We found that mandibular BM-MSCs from P. gingivalis-induced periodontitis mice had significantly reduced expression of LncRNA SPIRE1 than that from normal control mice. LncRNA SPIRE1 knockdown in normal BM-MSCs caused Th17/Treg cell differentiation imbalance during the coculturing of BM-MSCs and CD4 T cells. In addition, LncRNA SPIRE1 was identified as a competitive endogenous RNA that sponges miR-181a-5p in BM-MSCs. Moreover, miR-181a-5p inhibition attenuated the impact of LncRNA SPIRE1 knockdown on the ability of BM-MSCs in modulating Th17/Treg balance. Prolactin receptor (PRLR) was validated as a downstream target of miR-181a-5p. Notably, targeted knockdown of LncRNA SPIRE1 or PRLR or transfection of miR-181a-5p mimics activated the JAK/STAT3 signaling in normal BM-MSCs, while treatment with STAT3 inhibitor C188-9 restored the immunomodulatory properties of periodontitis-associated BM-MSCs. Furthermore, BM-MSCs with miR-181a-5p inhibition or PRLR-overexpression showed enhanced in vivo immunosuppressive properties in the periodontitis mouse model. Our results indicate that the JAK/STAT3 pathway is involved in the immunoregulation of BM-MSCs, and provide critical insights into the development of novel targeted therapies against periodontitis.

Persistent Abnormal Immunocytes Induced Systemic Bone Loss in Locally Irradiated Rats.

Chronic and systemic bone complications frequently occur in patients who undergo radiotherapy; however, the pathological mechanisms underlying these complications remain unclear. This study aimed to observe persistent and systemic changes in locally irradiated rats and to determine the systemic pathological changes that persistently affect bone metabolism. We examined the inflammatory and oxidative stress responses that occurred after local irradiation using enzyme immunoassays and biochemical analyses. Lymphocytes obtained from the blood, spleen, thymus, and bone marrow were evaluated using flow cytometry. The proliferation and apoptosis characteristics of co-cultured bone marrow-derived mesenchymal stem cells (BMSCs) were detected by MTT assay and PI/Annexin V-FITC staining, respectively, and the differentiation of BMSCs was measured according to alkaline phosphatase (ALP) staining, alizarin red staining, and Oil Red O staining and by evaluating the mRNA expression of ALP, osteocalcin (OCN), osteopontin (OPN), collagen I, Runx2, and PPARγ. Our results revealed that no significant or continuous differences were present in the inflammatory response or the oxidative stress response throughout the body after local irradiation. B lymphocyte levels increased continuously in the blood, spleen, and bone marrow after local irradiation. T lymphocyte levels were decreased at 2weeks after local irradiation, and CD8+T lymphocyte levels were increased in the blood, thymus, and bone marrow at 12weeks after local irradiation. The ratio of CD4+/CD8+T lymphocytes began to decrease during the early phase after local irradiation and became significantly decreased at 12weeks after local irradiation. Normal BMSCs co-cultured with lymphocytes derived from irradiated rats exhibited decreased proliferation and increased apoptosis, and the ALP staining intensity, alizarin red staining intensity, and mRNA expression of related genes were all also decreased. Oil Red O staining intensity and mRNA expression of PPARγ were both increased. Lymphocyte levels contribute to chronic and systemic bone complications after radiotherapy by inhibiting the proliferation and osteoblastogenesis of BMSCs.

Reprogrammed mesenchymal stem cells derived from iPSCs promote bone repair in steroid-associated osteonecrosis of the femoral head

BackgroundCellular therapy based on mesenchymal stem cells (MSCs) is a promising novel therapeutic strategy for the osteonecrosis of the femoral head (ONFH), which is gradually becoming popular, particularly for early-stage ONFH. Nonetheless, the MSC-based therapy is challenging due to certain limitations, such as limited self-renewal capability of cells, availability of donor MSCs, and the costs involved in donor screening. As an alternative approach, MSCs derived from induced pluripotent stem cells (iPSCs), which may lead to further standardized-cell preparations.MethodsIn the present study, the bone marrow samples of patients with ONFH (n = 16) and patients with the fracture of the femoral neck (n = 12) were obtained during operation. The bone marrow-derived MSCs (BMSCs) were isolated by density gradient centrifugation. BMSCs of ONFH patients (ONFH-BMSCs) were reprogrammed to iPSCs, following which the iPSCs were differentiated into MSCs (iPSC-MSCs). Forty adult male rats were randomly divided into following groups (n = 10 per group): (a) normal control group, (b) methylprednisolone (MPS) group, (c) MPS + BMSCs treated group, and (d) MPS + iPSC-MSC-treated group. Eight weeks after the establishment of the ONFH model, rats in BMSC-treated group and iPSC-MSC-treated group were implanted with BMSCs and iPSC-MSCs through intrabone marrow injection. Bone repair of the femoral head necrosis area was analyzed after MSC transplantation.ResultsThe morphology, immunophenotype, in vitro differentiation potential, and DNA methylation patterns of iPSC-MSCs were similar to those of normal BMSCs, while the proliferation of iPSC-MSCs was higher and no tumorigenic ability was exhibited. Furthermore, comparing the effectiveness of iPSC-MSCs and the normal BMSCs in an ONFH rat model revealed that the iPSC-MSCs was equivalent to normal BMSCs in preventing bone loss and promoting bone repair in the necrosis region of the femoral head.ConclusionReprogramming can reverse the abnormal proliferation, differentiation, and DNA methylation patterns of ONFH-BMSCs. Transplantation of iPSC-MSCs could effectively promote bone repair and angiogenesis in the necrosis area of the femoral head.

The long noncoding RNA FTX promotes a malignant phenotype in bone marrow mesenchymal stem cells via the miR-186/c-Met axis

Gliomas are the most common and aggressive primary tumours of the central nervous system in adults. Bone marrow-derived mesenchymal stem cells (BMSCs) are an important component of the glioma microenvironment. Our previous study indicated that BMSCs in the glioma microenvironment could be induced to malignantly transform by glioma stem cells (GSCs). The malignant transformation of BMSCs is closely related to glioma progression; however, the underlying mechanism of this transformation has not been fully clarified. In this study, we found that compared with the levels in normal BMSCs, the levels of the long noncoding RNA FTX transcript XIST regulator (lncRNA-FTX) were increased in malignantly transformed BMSCs (tBMSCs), which was associated with the proliferation, migration and invasion of tBMSCs. Next, by using a luciferase reporter assay and an RNA pull-down assay, we found that lncRNA-FTX acted as a sponge for miR-186 in tBMSCs. Further research revealed that miR-186 could bind to the 3ʹ-UTR (untranslated region) of c-Met, which acts as an oncogene in gliomas. Through functional assays, we showed that lncRNA-FTX could regulate c-Met expression in tBMSCs in a miR-186-dependent manner. Based on these data, we concluded that lncRNA-FTX plays a key role in the GSC-mediated malignant transformation of BMSCs in the glioma microenvironment, which is of great significance for further understanding the pathogenesis of glioma.

Enhanced recruitment and hematopoietic reconstitution of bone marrow-derived mesenchymal stem cells in bone marrow failure by the SDF-1/CXCR4.

Aplastic anemia (AA) is a bone marrow failure disease. It is difficult to treat AA, and in addition, relapses are common because of its complex disease pathogenesis. Allogeneic bone marrow-derived mesenchymal stem cells (BMSCs) infusion is an effective and safe treatment option for the AA patients. However, it found that BMSCs infusion in AA patients is less than 30% effective. Therefore, the key to improve the efficacy of BMSCs treatment in these patients is to enhance their homing efficiency to the target sites. Studies have shown that stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) axis plays an important role in promoting BMSCs homing. In this study, human BMSCs were transduced with lentivirus stably expressing CXCR4-BMSCs. Transduced BMSCs resemble normal BMSCs in many ways. Migration ability of CXCR4-BMSCs toward SDF-1 was increased because of the overexpression of CXCR4. In the mice with bone marrow failure, the migration and colonization ability of CXCR4-BMSCs to the bone marrow was significantly improved as seen by IVIS imaging and FACS. The SDF-1 level in the bone marrow failure mice was significantly higher than in the normal mice. Thus, from our study, it is clear that after CXCR4-BMSCs were infused into mice with bone marrow failure, SDF-1 interacted with CXCR4 receptor, leading cells to migrate and colonize to bone marrow. Because of the high SDF-1 expression in mouse bone marrow and CXCR4 receptor expression in cells, BMSCs homing was increased.

NMDA receptor activation inhibits the antifibrotic effect of BM-MSCs on bleomycin-induced pulmonary fibrosis.

Endogenous glutamate (Glu) release and N-methyl-d-aspartate (NMDA) receptor (NMDAR) activation are associated with lung injury in different animal models. However, the underlying mechanism is unclear. Bone marrow-derived mesenchymal stem cells (BM-MSCs), which show potential use for immunomodulation and tissue protection, play a protective role in pulmonary fibrosis (PF) process. Here, we found the increased Glu release from the BM cells of bleomycin (BLM)-induced PF mice in vivo. BLM stimulation also increased the extracellular Glu in BM-MSCs via the antiporter system xc− in vitro. The gene expression of each subunit of NMDAR was detected in BM-MSCs. NMDAR activation inhibited the proliferation, migration, and paracrine function of BM-MSCs in vitro. BM-MSCs were derived from male C57BL/6 mice, transfected with lentiviral vectors carrying the enhanced green fluorescence protein gene, pretreated with NMDA, and transplanted into the female recipient mice that were intratracheally injected with BLM to induce PF. Transplantation of NMDA-pretreated BM-MSCs significantly aggravated PF as compared with that in the normal BM-MSCs transplantation group. The sex determination gene Y chromosome and green fluorescence protein genes of BM-MSCs were detected to observe BM-MSCs homing in the fibrotic lungs. Moreover, NMDAR activation inhibited BM-MSC migration by downregulating the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 signaling axis. NMDAR activation aggravated the transforming growth factor-β1-induced extracellular matrix production in alveolar epithelial cells and fibroblasts through the paracrine effects of BM-MSCs. In summary, these findings suggested that NMDAR activation-mediated Glu excitotoxicity induced by BLM in BM-MSCs abolished the therapeutic effects of normal BM-MSCs transplantation on BLM-induced PF.

Critical role of SDF-1/CXCR4 signaling pathway in stem cell homing in the deafened rat cochlea after acoustic trauma.

Previous animal studies have shown that stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor-4 (CXCR4) signaling pathway plays an important role in the targeted migration of bone marrow-derived mesenchymal stem cells (BMSCs) to the injured area. In the present study, we aimed to investigate the potential role of chemotactic SDF-1/CXCR4 signaling pathway in the homing of transplanted BMSCs to the injured cochlea after noise-induced hearing loss (NIHL) in a rat model. White noise exposure (110 dB) paradigm was used for hearing loss induction in male rats for 6 hours in 5 days. Distortion-product otoacoustic emission (DPOAE) responses were recorded before the experiment and post noise exposure. Hoechst 33342-labeled BMSCs and CXCR4 antagonist (AMD3100)-treated BMSCs were injected into the rat cochlea through the round window. SDF-1 protein expression in the cochlear tissue was assayed using western blot assay. The number of labeled BMSCs reaching the endolymph was determined after 24 hours. SDF-1 was significantly increased in the cochlear tissue of rats in the noise exposure group than in the control group. The number of Hoechst 33342-labeled BMSCs reaching the endolymph of the cochlea was significantly smaller in the AMD3100-treated BMSCs group than in the normal BMSCs group. Our present findings suggest that the SDF-1/CXCR4 signaling pathway has a critical role in BMSCs migration to the injured cochlea in a rat model of noise-induced hearing loss.

TLR4 plays a crucial role in MSC-induced inhibition of NK cell function

Mesenchymal stem cells (MSC) are a kind of stromal cell within the tumor microenvironment. In our research, MSC derived from acute myeloid leukemia patients' bone marrow (AML-MSC) and lung cancer tissues (LC-MSC) as well as normal bone marrow-derived MSC (BM-MSC) cultured in conditioned medium of HeLa cells were found to have higher expressions of Toll-like receptor (TLR4) mRNA compared with BM-MSC. The sorted TLR4-positive MSC (TLR4+ MSC) differed in cytokine (interleukin-6, interleukin-8, and monocyte chemoattractant protein-1) secretion from those of unsorted MSC. MSC was reported to inhibit natural killer (NK) cell proliferation and function. In this research, we confirmed that TLR4+ MSC aggravate this suppression. Furthermore, when TLR4 in the sorted cells were stimulated by LPS or following blocked by antibody, the suppression on NK cell proliferation and cytotoxicity were more intensive or recovered respectively. Compared to unsorted MSC, NKG2D receptor expression on NK cells were also inhibited by TLR4+ MSC. These findings suggest that activation of TLR4 pathway is important for TLR4+ MSC and MSC to obstruct anti-tumor immunity by inhibiting NK cell function, which may provide a potential stroma-targeted tumor therapy.

In Situ Recruitment of Human Bone Marrow-Derived Mesenchymal Stem Cells Using Chemokines for Articular Cartilage Regeneration.

Bone marrow-derived mesenchymal stem cells (BMSCs) are a good cell source for regeneration of cartilage as they can migrate directly to the site of cartilage injury and differentiate into articular chondrocytes. Articular cartilage defects do not heal completely due to the lack of chondrocytes or BMSCs at the site of injury. In this study, the chemotaxis of BMSCs toward chemokines, which may give rise to a complete regeneration of the articular cartilage, was investigated. CCR2, CCR4, CCR6, CXCR1, and CXCR2 were expressed in normal BMSCs and were increased significantly upon treatment with proinflammatory cytokines. BMSC migration was increased by MIP-3α and IL-8 more than by MCP-1 or SDF-1α. IL-8 and MIP-3α significantly enhanced the chemotaxis of BMSCs compared with MCP-1, SDF-1α, or PBS. Human BMSC recruitment to transplanted scaffolds containing either IL-8 or MIP-3α significantly increased in vivo compared to scaffolds containing PBS. Furthermore, IL-8- and MIP-3α-containing scaffolds enhanced tissue regeneration of an osteochondral defect site in beagle knee articular cartilage. Therefore, this study suggests that IL-8 and MIP-3α are the candidates that induce the regeneration of damaged articular cartilage.

GW24-e1260 Overexpressing cellular repressor of E1A-stimulated genes promote hypoxia-induced VEGF paracrine via HIF-1 in mesenchymal stem cells

ObjectivesHypoxia inducible factor-1α (HIF-1α) is the key transcription regulator for multiple angiogenic factors, including vascular endothelial growth factor (VEGF). Cellular repressor of E1A-stimulated genes (CREG) has also been identified a...

ASSA13-10-9 Overexpressing Cellular Repressor of E1A-Stimulated Genes Promote Hypoxia-Induced VEGF Paracrine Via HIF-1α in Mesenchymal Stem Cells

BackgroundHypoxia inducible factor-1α (HIF-1α) is the key transcription regulator for multiple angiogenic factors, including vascular endothelial growth factor (VEGF). Cellular repressor of E1A-stimulated genes (CREG) has also been identified a...

Topical application of hPDGF-A-modified porcine BMSC and keratinocytes loaded on acellular HAM promotes the healing of combined radiation-wound skin injury in minipigs

Purpose: To evaluate the efficacy of cultured cutaneous substitute (CCS) in accelerating the healing of combined radiation-skin wound injury (CRWI) in minipigs.Material and methods: Autologous porcine bone marrow-derived mesenchymal stem cells (BMSC) and skin-derived keratinocytes (SK) were infected by recombinant retrovirus expressing human (h) platelet-derived growth factor-A (hPDGF-A). CCS was constructed by loading acellular human amniotic membrane (HAM) with normal porcine BMSC and SK (BMSC−/SK−CCS) or with hPDGF-A modified counterparts (BMSC+/SK+CCS). The expression of exogenous hPDGF-A in cells and CCS was assessed by reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). The CCS or HAM were grafted to the dorsal CRWI sites (20 Gy local irradiation plus full-thickness skin removal, diameter = 40 mm) of minipigs. Wound healing rate and pathological changes were observed.Results: High levels of hPDGF-A expression were confirmed in gene-modified cells (3780 pg/ml), cultured CCS (506 pg/ml) and transplanted CCS (250 pg/ml). The transplantation of the BMSC+/SK+CCS resulted in a shorter healing time (16–18, days) (P < 0.05 vs. other groups). The healing rates ranked as BMSC+/SK+CCS > BMSC−/SK−CCS > HAM > wound control. Pathologically, there were better granulation formation and re-epithelialisation, and collagen deposition in BMSC+/SK+CCS-treated wound than those in other groups. The angiogenesis ability followed the same order as healing rate of different groups. At day 7, the area densities of vasculature in granulation tissue of group BMSC+/SK+CCS, BMSC−/SK−CCS, HAM, wound only were 15.4, 10.3, 6.0 and 5.7%, respectively, while the number densities of vasculature was 767, 691, 126 and 109 (number/mm2), respectively.Conclusions: Topical transplantation of hPDGF-A modified CCS may be applicable to the management of refractory wounds.

Transplantation of BMSCs expressing hPDGF-A/hBD2 promotes wound healing in rats with combined radiation-wound injury

The aim of this study was to test the efficacy of transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) expressing human platelet-derived growth factor A (hPDGF-A) and human beta-defensin2 (hBD2) in accelerating wound healing of combined radiation-wound injury. Recombinant adenovirus vector simultaneously expressing hPDGF-A and hBD2 was constructed and packaged into virus particles that were used to infect rat BMSCs. The expressions of the exogenous in BMSCs were determined by reverse transcriptase (RT)-PCR and western -blot, whereas the functions were determined by cell counting kit (CCK), wound-healing assay on monolayer cells and Kleihauer-Betke (K-B) test. The recombinant adenovirus-infected BMSCs (1 x 10(7)) were subcutaneously transplanted into the wound bed and wound healing was observed for the indicated duration. Rats with combined total body ionizing radiation (6 Gy) and full-thickness skin excision (2% of total body surface area) wound injury were treated with normal BMSCs (group N), BMSCs infected with recombinant adenovirus expressing hPDGF-A and hBD2 (group T) or phosphate-buffered saline (PBS) (group S). The mean wound healing time, percentage of residual wound area (n=8), blind pathological observation (n=3 per time point for each group) and the amount of bacteria under the scar (the same sample was used in the pathological study, n=3) were used for evaluating wound healing. Collagen was visualized by Sirius red staining. Exogenous hPDGF-A and hBD2 were expressed in BMSCs as indicated by RT-PCR and western blot. Faster wound healing of scratched monolayer cells was demonstrated in hPDGF-A/hBD2 gene-modified BMSCs (T-MSCs) when compared with the corresponding control (P<0.01), and conditioned culture medium from T-MSCs showed stimulative effect on BMSC proliferation and in vitro antibiotic effect in the presence of trypsin. Neutralizing antibody interfering in vitro demonstrated that secreted hPDGF-A was the main factor stimulating cell proliferation. In an in vivo test, the radiation-wound combined injury exhibited shorter healing time (21 days). Histologically, there was better granulation formation/maturation and skin-dependent regeneration, as well as more collagen deposition (P<0.01) in rats of group T than in other groups. The deposition and remodeling of collagen in wounds were ranked in the following order: group T>group N>group S. Significantly less bacterial colony formation in the cultured under-scar samples in the rats of group T was observed (P<0.01) at day 7 and thereafter when compared with control. After transplantation, the BMSCs expressed exogenous genes in the wound for at least 2 weeks, as indicated by the reporter gene. Topical transplantation of gene-modified BMSCs promoted wound healing, which may be the benefit of the secretion of antibacterial hBD2 and mitogenic hPDGF-A, resulting in better granulation formation/maturation and skin appendage regeneration in wound. These data demonstrated the potential application of this combination of cell therapy and gene therapy on refractory wound healing.