Human Bone Marrow Mesenchymal Stem Cells Research Articles

Therapeutic effect of low-dose BMSCs-Loaded 3D microscaffold on early osteonecrosis of the femoral head.

The early treatment of Osteonecrosis of Femoral Head (ONFH) remains a clinical challenge. Conventional Bone Marrow Mesenchymal Stem Cell (BMSC) injection methods often result in unsatisfactory outcomes due to mechanical cell damage, low cell survival and retention rates, inadequate cell matrix accumulation, and poor intercellular interaction. In this study, we employed a novel cell carrier material termed "3D Microscaffold" to deliver BMSCs, addressing these issues and enhancing the therapeutic effects of cell therapy for ONFH. We injected 3D microscaffold loaded with low-dose BMSCs or free high-dose BMSCs into the femoral heads of ONFH rats and assessed therapeutic effects using imaging, serology, histology, and immunohistochemistry. To understand the mechanism of efficacy, we established a co-culture model of human osteoblasts and BMSCs, followed by cell proliferation and activity detection, flow cytometry analysis, Quantitative RT-PCR, and Western blotting. Additionally, RNA sequencing was performed on femoral head tissues. Results showed that the 3D microscaffold with low-dose BMSCs had a therapeutic effect comparable to high-dose free BMSCs. Osteoblasts in the 3D microscaffold group exhibited superior phenotypes compared to the non-3D microscaffold group. Furthermore, we have, for the first time, preliminarily validated that the low-dose BMSCs-loaded 3D microscaffolds may promote the repair of femoral head necrosis through the synergistic action of the MAPK and Hippo signaling pathways.

NEAT1 regulates BMSCs aging through disruption of FGF2 nuclear transport

BackgroundThe aging of bone marrow mesenchymal stem cells (BMSCs) impairs bone tissue regeneration, contributing to skeletal disorders. LncRNA NEAT1 is considered as a proliferative inhibitory role during cellular senescence, but the relevant mechanisms remain insufficient. This study aims to elucidate how NEAT1 regulates mitotic proteins during BMSCs aging.MethodsBMSCs were isolated from alveolar bone of human volunteers aged 26–33 (young) and 66–78 (aged). NEAT1 expression and distribution changes during aging process were observed using fluorescence in situ hybridization (FISH) in young (3 months) and aged (18 months) mice or human BMSCs. Subsequent RNA pulldown and proteomic analyses, along with single-cell analysis, immunofluorescence, RNA immunoprecipitation (RIP), and co-immunoprecipitation (Co-IP), were conducted to investigate that NEAT1 impairs the nuclear transport of mitotic FGF2 and contributes to BMSCs aging.ResultsWe reveal that NEAT1 undergoes significant upregulated and shifts from nucleus to cytoplasm in bone marrow and BMSCs during aging process. In which, the expression correlates with nuclear DNA content during karyokinesis, suggesting a link to mitogenic factor. Within NEAT1 knockdown, hallmarks of cellular aging, including senescence-associated secretory phenotype (SASP), p16, and p21, were significantly downregulated. RNA pulldown and proteomic analyses further identify NEAT1 involved in osteoblast differentiation, mitotic cell cycle, and ribosome biogenesis, highlighting its role in maintaining BMSCs differentiation and proliferation. Notably, as an essential growth factor of BMSCs, Fibroblast Growth Factor 2 (FGF2) directly abundant binds to NEAT1 and the sites enriched with nuclear localization motifs. Importantly, NEAT1 decreased the interaction between FGF2 and Karyopherin Subunit Beta 1 (KPNB1), influencing the nuclear transport of mitogenic FGF2.ConclusionsOur findings position NEAT1 as a critical regulator of mitogenic protein networks that govern BMSC aging. Targeting NEAT1 might offer novel therapeutic strategies to rejuvenate aged BMSCs.

Mangiferin Protects Mesenchymal Stem Cells Against DNA Damage and Cellular Aging via SIRT1 Activation.

The protective effects of mangiferin (MAG) against etoposide- and high glucose (HG)-induced DNA damage and aging were investigated in human bone marrow-mesenchymal stem cells (hBM-MSCs). Etoposide, a topoisomerase II inhibitor, was used to induce double-strand breaks (DSBs) in hBM-MSCs, resulting in increased genotoxicity, elevated levels of the DNA damage sensor ATM and CDKN1A, and decreased levels of the aging markers H3 and H4. MAG activated AMPK and SIRT1, thus protecting against DSB-induced damage. Following long-term exposure to HG, MAG significantly mitigated DNA damage and delayed cellular aging, as evidenced by the preservation of H3, H4, LMNB1, and SIRT1 mRNA levels and reduction in γ-H2AX foci and DSBs. Furthermore, MAG improved genome stability, as indicated by decreased LINE1 expression and increased levels of the heterochromatin marker TRIM28, thereby maintaining H3K9me3 levels. MAG and metformin treatment enhanced cell proliferation, reduced senescence-associated β-galactosidase staining, and lowered the levels of the senescence-associated secretory phenotype factors IL-1A, IL-1B, IL-6, IL-8, CCL2, and CCL20 and senescence marker CDKN1A, CDKN2A and p53. MAG may reduce DNA damage and delay aging in hBM-MSCs under HG conditions, highlighting their potential as therapeutic agents for aging-related diseases.

Functional Injectable Hydrogel for Bone Regeneration: Regulation of the circSRPK1/miR‐320a Axis and Targeting Multiple Osteogenic Pathways via CDH2 and Osterix Genes

ABSTRACTHydrogels loaded with microRNA (miRNA) have shown promise in bone‐defect repair. Here, we present the first report of miRNA‐loaded hydrogels containing bioactivities to treat steroid‐induced osteonecrosis of the femoral head (SONFH), based on the mechanism of competing endogenous RNAs. Transcriptome sequencing of human bone marrow mesenchymal stem cells (HBMSCs) extracted from the proximal femoral bone marrow and subsequent functional assays revealed that the circSRPK1/miR‐320a axis promotes HBMSCs osteogenic differentiation. By incorporating antagomir‐320a (a miR‐320a inhibitor) encapsulated in liposomes into injectable hyaluronic acid (HA) hydrogels, we constructed an injectable hydrogel, HA@antagomir‐320a. This hydrogel demonstrated exceptional osteogenic properties, targeting multiple osteogenic pathways via CDH2 and Osterix and exhibited excellent in vitro biocompatibility. In vivo, it substantially enhanced bone formation in the osteonecrotic area of the femoral head. This injectable HA@antagomir‐320a hydrogel, which exhibited exceptional biocompatibility and osteogenic properties in vivo and in vitro, offers a promising and minimally invasive solution for the treatment of SONFH.

Global proteomics reveals pathways of mesenchymal stem cells altered by Mycobacterium tuberculosis

Mycobacterium tuberculosis (M. tb) has a remarkable ability to persist inside host cells. Several studies showed that M. tb infects and survives inside bone marrow mesenchymal stem cells (BM-MSCs) escaping the host immune system. Here, we have identified various cellular pathways that are modulated in human BM-MSCs upon infection with virulent M. tb and the proteomic profile of these cells varies from that of avirulent M. tb infected cells. We found that virulent M. tb infection reshapes host pathways such as stem cell differentiation, alternative splicing, cytokine production, mitochondrial function etc., which might be modulated by M. tb to persist inside this unconventional niche of human BM-MSCs. Additionally, we observed that virulent M. tb infection suppresses various cellular processes. This study uncovers the differences in the host proteomic profiles resulting from the virulent versus avirulent M. tb infection that can pave the way to identify host-directed therapeutic targets for the treatment of tuberculosis.

Fluorinated Porcine Bone-Derived Hydroxyapatite Promotes Vascularized Osteogenesis by Coordinating Human Bone Marrow Mesenchymal Stem Cell/Human Umbilical Vein Endothelial Cell Complexes.

Biogenic hydroxyapatite is known for its osteoinductive potential due to its similarity to human bone and biocompatibility, but insufficient vascularization compared to autogenous bone during early implantation limits bone integration and osteogenesis. Fluorine has been shown to improve hydroxyapatite's mechanical properties and the coupling of osteogenic and angiogenic cells. In this study, fluorine-modified biogenic hydroxyapatite (FPHA) with varying fluorine concentrations was prepared and tested for its ability to promote vascularized osteogenesis. FPHA prepared in this study retained the natural porous structure of biological cancellous bone and released F- ions when immersed in cell culture medium. The extraction solutions of FPHA0.25 and FPHA0.50 promoted the formation of capillary-like tubes by human umbilical vein endothelial cells (HUVECs), with FPHA0.25 significantly upregulating vegf mRNA and VEGF protein levels in co-cultured human bone marrow mesenchymal stem cells (HBMSCs). Additionally, FPHA0.25 and FPHA0.50 upregulated pdgf-bb mRNA and PDGF-BB protein levels in HUVECs. In vivo experiments using a rabbit cranial defect model demonstrated that FPHA0.25 promoted early bone formation and angiogenesis in the defect area, enhanced VEGF secretion, and increased PDGFR-β expression in endothelial and mesenchymal cells. These findings suggest that fluorine-modified biogenic hydroxyapatite with an optimal fluorine concentration (FPHA0.25) may offer a promising strategy to enhance the body's innate bone-healing potential by accelerating vascularization.

Gold Kiwi-Derived Nanovesicles Mitigate Ultraviolet-Induced Photoaging and Enhance Osteogenic Differentiation in Bone Marrow Mesenchymal Stem Cells.

Bone marrow mesenchymal stem cells (BM-MSCs) play a crucial role in bone formation through their ability to differentiate into osteoblasts. Aging, however, detrimentally affects the differentiation and proliferation capacities of BM-MSCs, consequently impairing bone regeneration. Thus, mitigating the aging effects on BM-MSCs is vital for addressing bone-related pathologies. In this study, we demonstrate that extracellular nanovesicles isolated from gold kiwi (GK-NVs) protect human BM-MSCs from ultraviolet (UV)-induced photoaging, thereby alleviating aging-related impairments in cellular functions that are crucial for bone homeostasis. Notably, GK-NVs were efficiently taken up by BM-MSCs without causing cytotoxicity. GK-NVs reduced intracellular reactive oxygen species (ROS) levels upon UV irradiation, restoring impaired proliferation and migration capabilities. Furthermore, GK-NVs corrected the skewed differentiation capacities of UV-irradiated BM-MSCs by enhancing osteoblast differentiation, as evidenced by the increased expression in osteoblast-specific genes and the calcium deposition, and by reducing adipocyte differentiation, as indicated by the decreased lipid droplet formation. These findings position GK-NVs as a promising biomaterial for the treatment of bone-related diseases such as osteoporosis.

Bone marrow mesenchymal stem cell-derived exosomes improve cancer drug delivery in human cell lines and a mouse osteosarcoma model.

Osteosarcoma is the most common primary bone tumor. Patients require chemotherapy drugs with high-targeting ability and low off-target toxicity to improve their survival. Exosomes are biological vesicles that mediate long-distance communication between cells and naturally target their source sites. Exosomes derived from bone marrow mesenchymal stem cells (BMSCs) naturally target bone tumor sites, suggesting their potential as effective anti-tumor therapy vectors. In this study, we evaluated the potential of BMSC-derived exosomes in targeting osteosarcoma and serving as a carrier for doxorubicin (DOX). We isolated exosomes from human BMSCs and synthesized hybrid exosomes (HEs) by fusing these exosomes with liposomes. These HEs were loaded with DOX to produce a novel drug, HE/DOX. We confirmed the successful synthesis of HE/DOX using fluorescence spectroscopy and estimated its size to be 151.1 ± 10.2 nm. HEs expressed the known exosomal proteins ALIX, CD63, and TSG101. Under acidic conditions similar to those observed in the tumor microenvironment, the drug release from HE/DOX was enhanced. In osteosarcoma cell lines and in a mouse osteosarcoma model, HE/DOX exhibited stronger tumor-inhibitory effects than free DOX. Our study demonstrates that BMSC-derived exosomes could effectively target osteosarcoma. Furthermore, HEs can serve as effective carriers of DOX, enabling the treatment of osteosarcoma. These findings highlight a promising direction for tumor-targeted therapy.

Abstract 4147630: Towards transplantation of hearts from donation after circulatory death - stem cell derived therapy for ameliorating donor organ damage

The use of heart transplantation (HTx), a key life-saving therapy for end-stage heart failure, is limited by the shortage of acceptable donor hearts. Although donation after circulatory death (DCD) offers a potentially promising resource for clinical transplantation, its adoption for HTx is concerned by warm ischemic damage during the death process. Mesenchymal stem cell (MSC)-derived paracrine action has been proved to mediate cardioprotection. Based on our previous study showing that MSC conditioned media (MSC CM) and MSC-exosomes (MSC Exo) confer improved donor heart preservation during cold ischemic storage, we aimed to test the effect of MSC CM and MSC Exo on ex vivo recovery of ischemia-damaged myocardium and on promoting the implanted DCD heart function using an in vivo murine heterotopic heart transplantation model. Methods: Donor hearts from adult male C57BL/6 mice with 0, 30’ or 50’ in vivo warm ischemia (WI) were divided into (n=5-7/group): 1) no WI; 2) 30’-WI; 3) 50’-WI; 4) no WI+4h cold storage at 0-4°C (no WI+CS); 5) 30’-WI+CS; 6) 50’-WI+CS; 7) 50’-WI+CS+vehicle; 8) 50’-WI+CS+MSC CM; and 9) 50’-WI+CS+MSC Exo. The CM and Exo were obtained from cultivation of human bone marrow-MSCs in serum-free media for 72h and was added into preservation solution. Donor hearts were implanted into C57BL/6 male recipients using cervical heterotopic heart transplantation. At 24h post-surgery, left ventricular (LV) function was detected in transplanted hearts. Abdominal artery pressure was measured in recipients to validate technical reproducibility of the HTx model. Mitochondrial structure was detected by electron microscopy on the fixed human LV (hDCD&hDBD). Results: WI significantly impaired LV function in transplanted DCD hearts vs. no-WI (Fig. A, B). Intriguingly, markedly improved graft function was observed in MSC CM- or Exo-treated DCD hearts vs. vehicle (Fig. C). Short and disrupted cristae in mitochondria were noticed in hDCD vs. hDBD (Fig. D). Conclusions: Our results represent the initial evidence that MSC secretome (CM&Exo) mediates ex vivo cardiac repair in DCD hearts and validate an approach on MSC derived therapy to rescue marginal DCD donor hearts and improve post-transplant graft function.

Bone marrow stromal cells enhance differentiation of acute myeloid leukemia induced by pyrimidine synthesis inhibitors.

Acute myeloid leukemia (AML) is a heterogeneous group of hematological malignancies characterized by differentiation arrest, high relapse rates, and poor survival. The bone marrow (BM) microenvironment is recognized as a critical mediator of drug resistance and a primary site responsible for AML relapse. Our previous study reported that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) induces AML cell differentiation by inhibiting pyrimidine synthesis and activating Checkpoint kinase 1. Although the protective effect of BM stroma on leukemia cells in response to cytotoxic drugs is well-documented, its effect on AML differentiation remains less explored. In this study, we investigated the impact of stromal cell lines and primary mesenchymal stromal cells (MSCs) on AML cell line differentiation triggered by AICAr and brequinar, a known dihydroorotate dehydrogenase (DHODH) inhibitor. Our findings indicate that the mouse MS-5 stromal cell line, known for its cytoprotective effects, does not inhibit AML cell differentiation induced by pyrimidine synthesis inhibitors. Interestingly, AICAr caused morphological changes and growth arrest in MS-5 stromal cells via an AMP-activated protein kinase (AMPK)-dependent pathway. Human stromal cell lines HS-5 and HS-27, as well as primary MSCs isolated from patient bone marrow, were superior in promoting AML differentiation compared with mouse cells in response to AICAr and brequinar, with the inhibitors not significantly affecting the stromal cells themselves. In conclusion, our study highlights the supportive role of human BM MSCs in enhancing the differentiation effects of pyrimidine synthesis inhibitors on AML cells, suggesting that AML treatment strategies focusing on differentiation rather than cell killing may be successful in clinical settings.NEW & NOTEWORTHY This study is the first to demonstrate that human stromal cell lines and primary mesenchymal stromal cells from patients enhance the in vitro differentiation of acute myeloid leukemia (AML) cells induced by pyrimidine synthesis inhibitors, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr), and brequinar. Furthermore, this is the first report to show that AICAr affects mouse bone marrow stromal cells by activating AMP-activated protein kinase (AMPK) and that human stromal cells are superior to mouse cells for testing the effects of drugs on AML differentiation.

Evaluation of biocompatibility and osteoconductivity of a hybrid cell-tissue graft for bone regenerative medicine

Aim – to evaluate in vitro the biocompatibility and osteoconductivity of a hybrid graft based on a bioorganic matrix, human bone marrow mesenchymal stromal cells (BM-MSC) and osteogenic growth factors. Material and methods. Bioorganic matrices were studied for biocompatibility with human BM-MSC culture used in traumatology and orthopedics. For promoted osteogenic differentiation of BM-MSCs, allogeneic plasma enriched with soluble platelet factors was used. The osteogenic potential of BM-MSCs by the synthesis of mRNAs of early (transcription factor 2 (Run X2), alkaline phosphatase (ALP)) and late genes (osteopontin (OSP)) osteogenesis was analyzed. The properties of cell adhesion and proliferation of MSCs in the conditions of a three-dimensional hybrid graft by the MTT test and fluorescence microscopy were assessed. Results. The biocompatibility of the studied bioorganic matrices with human BM-MSCs was established. The collagen matrix promoted rapid cell adhesion and proliferation between the scaffold fibrils. It has also been established that allogeneic platelet-rich plasma affects the osteogenic differentiation of human BM-MSCs in vitro, increasing the expression of marker genes RunX2, ALP, OSP. When modeling a hybrid graft in vitro, the formation of a tight contact between the alloimplant and collagen biopolymer using MSCs was shown. Conclusion. The biological properties of the developed hybrid cell-tissue graft characterize its biocompatibility and osteoconductivity of its constituent components, which makes it promising for use in regenerative medicine, especially in reconstructive surgery of bone defects.

Exosomes from human bone marrow MSCs alleviate PD-1/PD-L1 inhibitor-induced myocardial injury in melanoma mice by regulating macrophage polarization and pyroptosis

Myocarditis, which can be triggered by immune checkpoint inhibitor (ICI) treatment, represents a critical and severe adverse effect observed in cancer therapy. Thus, elucidating the underlying mechanism and developing effective strategies to mitigate its harmful impact is of utmost importance. The objective of this study is to investigate the potential role and regulatory mechanism of exosomes derived from human bone marrow mesenchymal stem cells (hBMSC-Exos) in providing protection against myocardial injury induced by ICIs. We observed that the administration of programmed death 1/programmed death-ligand 1 (PD-1/PD-L1) inhibitor BMS-1 in tumor-bearing mice led to evident cardiac dysfunction and myocardial injury, which were closely associated with M1 macrophage polarization and cardiac pyroptosis. Remarkably, these adverse effects were significantly alleviated through tail-vein injection of hBMSC-Exos. Moreover, either BMS-1 or hBMSC-Exos alone demonstrated the ability to reduce tumor size, while the combination of hBMSC-Exos with BMS-1 treatment not only effectively improved the probability of tumor inhibition but also alleviated cardiac anomalies induced by BMS-1.

Mesenchymal stromal cells can block palmitate training of macrophages via cyclooxygenase-2 and interleukin-1 receptor antagonist

Innate training of macrophages can be beneficial for the clearance of pathogens. However, for certain chronic conditions, innate training can have detrimental effects due to an excessive production of pro-inflammatory cytokines. Obesity is a condition that is associated with a range of increased pro-inflammatory training stimuli including the free fatty acid palmitate. Mesenchymal stromal cells (MSCs) are powerful immunomodulators and known to suppress inflammatory macrophages via a range of soluble factors. We show that palmitate training of murine bone-marrow-derived macrophages and human monocyte-derived macrophages (MDMs) results in an increased production of TNFα and IL-6 upon stimulation with lipopolysaccharide and is associated with epigenetic remodeling. Palmitate training led to metabolic changes, however, MSCs did not alter the metabolic profile of human MDMs. Using a transwell system, we demonstrated that human bone marrow MSCs block palmitate training in both murine and human macrophages suggesting the involvement of secreted factors. MSC disruption of the training process occurs through more than one pathway. Suppression of palmitate-enhanced TNFα production is associated with cyclooxygenase-2 activity in MSCs, while secretion of interleukin-1 receptor antagonist by MSCs is required to suppress palmitate-enhanced IL-6 production in MDMs.

Transcriptomic analysis of BM-MSCs identified EGR1 as a transcription factor to fully exploit their therapeutic potential

Bone marrow-mesenchymal stromal cells (BM-MSCs) are key components of the BM niche, where they regulate hematopoietic stem progenitor cell (HSPC) homeostasis by direct contact and secreting soluble factors. BM-MSCs also protect the BM niche from excessive inflammation by releasing anti-inflammatory factors and modulating immune cell activity. Thanks to these properties, BM-MSCs were successfully employed in pre-clinical HSPC transplantation models, increasing the rate of HSPC engraftment, accelerating the hematological reconstitution, and reducing the risk of graft failure. However, their clinical use requires extensive in vitro expansion, potentially altering their biological and functional properties. In this work, we analyzed the transcriptomic profile of human BM-MSCs sorted as CD45−, CD105+, CD73+, and CD90+ cells from the BM aspirates of heathy-donors and corresponding ex-vivo expanded BM-MSCs. We found the expression of immune and inflammatory genes downregulated upon cell culture and selected the transcription factor EGR1 to restore the MSC properties. We overexpressed EGR1 in BM-MSCs and performed in vitro tests to study the functional properties of EGR1-overexpressing BM-MSCs. We concluded that EGR1 increased the MSC response to inflammatory stimuli and immune cell control and potentiated the MSC hematopoietic supportive activity in co-culture assay, suggesting that the EGR1-based reprogramming may improve the BM-MSC clinical use.

Human liver derived mesenchymal stromal cells ameliorate murine ischemia-induced inflammation through macrophage polarization.

The immunomodulatory properties of mesenchymal stromal cells (MSC) have been well-characterized in in-vitro and in-vivo models. We have previously shown that liver MSC (L-MSC) are superior inhibitors of T-cell activation/proliferation, NK cell cytolytic function, and macrophage activation compared to adipose (A-MSC) and bone marrow MSC (BM-MSC) in-vitro. To test these observations in-vivo, we infused these types of MSC into mice with unilateral renal artery stenosis (RAS), an established model of kidney inflammation. Unilateral RAS was induced via laparotomy in 11-week-old, male 129-S1 mice under general anesthesia. Control mice had sham operations. Human L-MSC, AMSC, and BM-MSC (5x105 cells each) or PBS vehicle were injected intra-arterially 2 weeks after surgery. Kidney morphology was studied 2 weeks after infusion using micro-MRI imaging. Renal inflammation, apoptosis, fibrosis, and MSC retention were studied ex-vivo utilizing western blot, immunofluorescence, and immunohistological analyses. The stenotic kidney volume was smaller in all RAS mice, confirming significant injury, and was improved by infusion of all MSC types. All MSC-infused groups had lower levels of plasma renin and proteinuria compared to untreated RAS. Serum creatinine improved in micetreated with BM- and L-MSC. All types of MSC located to and were retained within the stenotic kidneys, but L-MSC retention was significantly higher than A- and BM-MSC. While all groups of MSC-treated mice displayed reduced overall inflammation and macrophage counts, L-MSC showed superior potency in-vivo at localizing to the site of inflammation and inducing M2 (reparative) macrophage polarization to reduce inflammatory changes. These in-vivo findings extend our in-vitro studies and suggest that L-MSC possess unique anti-inflammatory properties that may play a role in liver-induced tolerance and lend further support to their use as therapeutic agents for diseases with underlying inflammatory pathophysiology.

NSD2-mediated H3K36me2 exacerbates osteoporosis via activation of hoxa2 in bone marrow mesenchymal stem cells

BackgroundOsteoporosis (OP) is a prevalent disease associated with age, and one of the primary pathologies is the defect of osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). This study aimed to elucidate whether Nuclear Receptor Binding SET Domain Protein 2 (NSD2) transcriptionally regulates osteogenic differentiation of BMSCs in osteoporosis. MethodsIdentification of human BMSCs (hBMSCs) in vitro was measured by flow cytometry. Osteogenesis of hBMSCs in vitro was measured by Alizarin Red and Alkaline Phosphatase staining. The protein levels of H3K36me1/2/3, NSD2, and Hoxa2 were measured by western blotting. The mRNA levels of NSD2, Runx2, and BSP were measured by qPCR. The role of NSD2 in the osteogenic differentiation of BMSCs was further identified by silencing NSD2 via shRNA or overexpression of NSD2 via lentivirus transfection. The interactions of NSD2, H3K36me2 and Hoxa2 were identified via chromatin immunoprecipitation (ChIP). Luciferase reporting analysis was employed to confirm that NSD2 regulated the transcriptional activity of Hoxa2. Ovariectomized (OVX) was performed on mice to construct osteoporosis (OP) model. Subsequently, the bone mass was assessed by micro computed tomography (micro-CT) scan. ResultsDuring the osteogenesis of OP-derived hBMSCs, the levels of NSD2 and H3K36me2 significantly increased in 14 days of osteogenic induction. Inhibition of NSD2 via shRNA increased the RUNX2 and BSP expression of hBMSCs, while overexpression of NSD2 decreased RUNX2 and BSP expression of hBMSCs. ChIP analysis indicated NSD2-mediated H3K36me2 reduced the osteogenic differentiation of hBMSCs by regulating the osteogenic inhibitor Hoxa2. Accordingly, inhibition of NSD2 in vivo via tail vein injection of LV-shNSD2 lentivirus greatly alleviated OVX-induced osteoporosis in mice. ConclusionWe demonstrated that NSD2 inhibited the osteogenic differentiation in hBMSCs by transcriptionally downregulating Hoxa2 via H3K36me2 dimethylation. Inhibition of NSD2 effectively attenuated bone loss in murine osteoporosis and NSD2 is a promising target for clinical treatment of osteoporosis.

Vascular endothelial growth factor secretion and immunosuppression are distinct potency mechanisms of human bone marrow mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are investigated as cellular therapeutics for inflammatory bowel diseases and associated perianal fistula, although consistent efficacy remains a concern. Determining host factors that modulate MSCs' potency including their secretion of angiogenic and wound-healing factors, immunosuppression, and anti-inflammatory properties are important determinants of their functionality. We investigated the mechanisms that regulate the secretion of angiogenic and wound-healing factors and immune suppression of human bone marrow MSCs. Secretory analysis of MSCs focusing on 18 angiogenic and wound-healing secretory molecules identified the most abundancy of vascular endothelial growth factor A (VEGF-A). MSC viability and secretion of other angiogenic factors are not dependent on VEGF-A secretion which exclude the autocrine role of VEGF-A on MSC's fitness. However, the combination of inflammatory cytokines IFNγ and TNFα reduces MSC's VEGF-A secretion. To identify the effect of intestinal microvasculature on MSCs' potency, coculture analysis was performed between human large intestine microvascular endothelial cells (HLMVECs) and human bone marrow-derived MSCs. HLMVECs do not attenuate MSCs' viability despite blocking their VEGF-A secretion. In addition, HLMVECs neither attenuate MSC's IFNγ mediated upregulation of immunosuppressive enzyme indoleamine 2,3-dioxygenase nor abrogate suppression of T-cell proliferation despite the attenuation of VEGF-A secretion. We found that HLMVECs express copious amounts of endothelial nitric oxide synthase and mechanistic analysis showed that pharmacological blocking reverses HLMVEC-mediated attenuation of MSC's VEGF-A secretion. Together these results suggest that secretion of VEGF-A and immunosuppression are separable functions of MSCs which are regulated by distinct mechanisms in the host.

Human mesenchymal stromal cells ameliorate cisplatin-induced acute and chronic kidney injury via TSG-6.

Cisplatin is widely used in tumor chemotherapy, but nephrotoxicity is an unavoidable side effect of cisplatin. Several studies have demonstrated that mesenchymal stromal cells (MSCs) ameliorate cisplatin-induced kidney injury, but the underlying mechanisms are unknown. In this study, the cisplatin-induced kidney injury mouse model was established by subjecting a single intraperitoneal injection with cisplatin. One hour before cisplatin injection, the mice received human bone marrow MSCs (hBM-MSCs) with or without siRNA-transfection, recombinant human tumor necrosis factor-α-stimulated gene/protein 6 (rhTSG-6), or PBS through the tail vein. In addition, cisplatin-stimulated HK-2 cells were treated with hBM-MSCs or rhTSG-6. Human BM-MSCs treatment remarkably ameliorated cisplatin-induced acute and chronic kidney injury, as evidenced by significant reductions in serum creatinine (Scr), blood urea nitrogen, tubular injury, collagen deposition, α-smooth muscle actin accumulation, as well as inflammatory responses, and by remarkable increased anti-inflammatory factor expression and Treg cells infiltration in renal tissues. Furthermore, we found that only a few hBM-MSCs engrafted into damaged kidney and that the level of human TSG-6 in the serum of mice increased significantly following hBM-MSCs administration. Moreover, hBM-MSCs significantly increased the viability of damaged HK-2 cells and decreased the levels of inflammatory cytokines in the culture supernatant. However, the knockdown of the TSG-6 gene in hBM-MSCs significantly attenuated their beneficial effects in vivo and in vitro. On the contrary, treated with rhTSG-6 achieved similar beneficial effects of hBM-MSCs. Our results indicate that systemic administration of hBM-MSCs alleviates cisplatin-induced acute and chronic kidney injury in part by paracrine TSG-6 secretion.

MEG3 shuttled by exosomes released from human bone marrow mesenchymal stem cells promotes TP53 stability to regulate MCM5 transcription in keloid fibroblasts.

Despite the interest in mesenchymal stem cells (MSC), their potential to treat abnormal scarring, especially keloids, is yet to be described. The present study aimed to investigate the therapeutic potential of exosomes derived from human bone marrow MSCs (hBMSC-Exos) in alleviating keloid formation. Exosomes were isolated from hBMSC, and keloid fibroblasts (KFs) were treated with hBMSC-Exos. Cell counting kit-8, wound healing, transwell invasion, immunofluorescence, and western blot assays were conducted to study the malignant phenotype of KFs. Mice were induced with keloids and treated with hBMSC-Exos. The effect of hBMSC-Exos on keloid formation in vivo was evaluated by hematoxylin and eosin staining, Masson staining, immunohistochemistry, and western blotting. The GSE182192 dataset was screened for differentially expressed long non-coding RNA during keloid formation. Next, maternally expressed gene 3 (MEG3) was knocked down in hBMSC to obtain hBMSC-Exossh-MEG3. The molecular mechanism of MEG3 was investigated by bioinformatic screening, and the relationship between MEG3 and TP53 or MCM5 was verified. hBMSC-Exos inhibited the malignant proliferation, migration, and invasion of KFs at same time as promoting their apoptosis, Moreover, hBMSC-Exos reduced the expression of fibrosis- and collagen-related proteins in the cells and the formation of keloids caused by KFs. The reduction in MEG3 enrichment in hBMSC-Exos weakened the inhibitory effect of hBMSC-Exos on KF activity. hBMSC-Exos delivered MEG3 to promote MCM5 transcription by TP53 in KFs. Overexpression of MCM5 in KFs reversed the effects of hBMSC-Exossh-MEG3, leading to reduced KF activity. hBMSC-Exos delivered MEG3 to promote the protein stability of TP53, thereby activating MCM5 and promoting KF activity.

Multifunctional platform for future applications in cell and tissue engineering based on silicate phosphate hydroxyapatite co-doped with Li+, Eu3+ and Gd3+ ions

Although hydroxyapatite has established itself as a promising biomaterial for the simultaneous diagnosis and treatment of bone defects, several physicochemical properties can be further refined to achieve a specialized theranostic platform for cell and tissue engineering. In this context, we synthesized silicate-substituted hydroxyapatites co-doped with Li+, Eu3+, and Gd3+ ions (abbr. as Si-HAp-LEG) using a microwave-assisted hydrothermal method. The aim was to create a combination of dopants to stimulate bone regeneration (Li+ ion) while enabling bioimaging (Eu3+ and Gd3+ ions). Structural and morphological assessments included, e.g., XRPD (X-ray Powder Diffraction), HR-TEM (High-resolution Transmission Electron Microscopy) imaging, and BET (Brauner-Emmet-Teller) analysis. Unit-cell parameters were estimated via Rietveld refinement. The luminescence properties (emission, excitation spectra, and emission kinetics) were recorded depending on the varying Li+-ion concentration. The cytocompatibility of biomaterials was tested using hBMSCs (multipotent human bone-marrow mesenchymal stromal cells). In vitro assays evaluated biomaterials’ effects on morphology, metabolic activity (Alamar blue assay), and transcriptomic activity (RT-qPCR). The results indicate that co-doping with Li+, Eu3+, and Gd3+ ions introduces distortions while preserving the crystal structure of hydroxyapatite. Additionally, the cells maintained proper morphology and ultrastructure, internalizing biomaterials without significant cytotoxicity. The biomaterials also triggered the expression of key transcriptional factors. Si-HAp-LEG biomaterials, particularly LEG-222 and LEG-221, exhibit desired physicochemical and biological features, rendering them highly bioactive and promising for cell and tissue engineering applications.