Differentiation Of MSCs Research Articles

ROS Scavenging and Osteogenic Differentiation Potential of L-Methionine-Substituted Poly(Organophosphazene) Electrospun Fibers

This study investigated the application of poly[bis (ethylmethionato) phosphazene] (PαAPz-M) electrospun fibers in tissue engineering, focusing on their reactive oxygen species (ROS) scavenging capabilities and material-directed cell behavior, including the influence of their degradation products on cell viability and differentiation, and the scaffold topography’s influence on cell alignment. The ROS scavenging ability of PαAPz-M was assessed by DPPH assay, and then PαAPz-M’s protection against exogenous ROS was studied. The results showed enhanced cell viability on PαAPz-M fiber mats under oxidative stress conditions. This study also investigated the effects of the degradation products of PαAPz-M on cell viability and osteogenic differentiation. It was observed that the late-stage degradation product, phosphoric acid, can significantly influence the osteogenic differentiation of MSCs. In contrast, methionine, which is the early-stage degradation product, showed a minimal influence. Additionally, the study fabricated fiber mats that can lead to enhanced cell alignment while maintaining high porosity. Collectively, this study expanded the applications of PαAPz-M fiber mat protection against oxidative stress and guiding osteogenic differentiation and cell alignment.

Exploration of immunomodulatory mechanism of caprine Wharton’s jelly derived mesenchymal stem cells

The present study was aimed to explore the possible mechanisms by which caprine Wharton’s jelly-derived MSCs (WJ-MSCs) perform their immunomodulatory function. WJ-MSCs were isolated through explants culture and characterized as per ISCT criteria using culture behavior, expression of surface markers by PCR, FACS and immunocytochemical localization (ICC), trilineage differentiation potential etc. Secretory behavior for important biomolecules (IDO, TGFβ1, VEGF, IL6) was evaluated by ICC and western blot assay. Cell-to-cell communication was studied by culturing cells in cell–cell contact and trans-well system. The MSCs when co-cultured with activated Tc and Th cells, down-regulation of T cell cytokine as well as upregulation of immunomodulatory factors (VEGF A, IL10, IL6, IDO, iNOS, PTGS2, HGF, TGFβ, CXCL10, CXCL11) was noticed in both cell–cell contact and trans-well culture system which was significantly higher in cell–cell contact system. Trilineage differentiation of MSCs showed significant upregulation of MHC I (CAHI) and MHC II (CLA DRB3) molecules suggesting better clinical applications of MSCs without differentiation to avoid immune rejection. It can be concluded that WJ-MSCs perform their immunomodulation through the secretion of a battery of biomolecules and work in both cell–cell contact manner and through their secretome.

Modeling the profibrotic microenvironment in vitro: Model validation

Establishing the molecular and cellular mechanisms of fibrosis requires the development of validated and reproducible models. The complexity of in vivo models challenges the monitoring of an individual cell fate, in some cases making it impossible. However, the set of factors affecting cells in vitro culture systems differ significantly from in vivo conditions, insufficiently reproducing living systems. Thus, to model profibrotic conditions in vitro, usually the key profibrotic factor, transforming growth factor beta (TGFβ-1) is used as a single factor. TGFβ-1 stimulates the differentiation of fibroblasts into myofibroblasts, the main effector cells promoting the development and progression of fibrosis. However, except for soluble factors, the rigidity and composition of the extracellular matrix (ECM) play a critical role in the differentiation process. To develop the model of more complex profibrotic microenvironment in vitro, we used a combination of factors: decellularized ECM synthesized by human dermal fibroblasts in the presence of ascorbic acid if cultured as cell sheets and recombinant TGFβ-1 as a supplement. When culturing human mesenchymal stromal cells derived from adipose tissue (MSCs) under described conditions, we observed differentiation of MSCs into myofibroblasts due to increased number of cells with stress fibrils with alpha-smooth muscle actin (αSMA), and increased expression of myofibroblast marker genes such as collagen I, EDA-fibronectin and αSMA. Importantly, secretome of MSCs changed in these profibrotic microenvironment: the secretion of the profibrotic proteins SPARC and fibulin-2 increased, while the secretion of the antifibrotic hepatocyte growth factor (HGF) decreased. Analysis of transciptomic pattern of regulatory microRNAs in MSCs revealed 49 miRNAs with increased expression and 3 miRNAs with decreased expression under profibrotic stimuli. Bioinformatics analysis confirmed that at least 184 gene targets of the differently expressed miRNAs genes were associated with fibrosis. To further validate the developed model of profibrotic microenvironment, we cultured human dermal fibroblasts in these conditions and observed increased expression of fibroblast activation protein (FAPa) after 12 h of cultivation as well as increased level of αSMA and higher number of αSMA + stress fibrils after 72 h. The data obtained allow us to conclude that the conditions formed by the combination of profibrotic ECM and TGFβ-1 provide a complex profibrotic microenvironment in vitro. Thus, this model can be applicable in studying the mechanism of fibrosis development, as well as for the development of antifibrotic therapy.

Promotion of osteochondral repair through immune microenvironment regulation and activation of endogenous chondrogenesis via the release of apoptotic vesicles from donor MSCs

Utilizing transplanted human umbilical cord mesenchymal stem cells (HUMSCs) for cartilage defects yielded advanced tissue regeneration, but the underlying mechanism remain elucidated. Early after HUMSCs delivery to the defects, we observed substantial apoptosis. The released apoptotic vesicles (apoVs) of HUMSCs promoted cartilage regeneration by alleviating the chondro-immune microenvironment. ApoVs triggered M2 polarization in macrophages while simultaneously facilitating the chondrogenic differentiation of endogenous MSCs. Mechanistically, in macrophages, miR-100-5p delivered by apoVs activated the MAPK/ERK signaling pathway to promote M2 polarization. In MSCs, let-7i-5p delivered by apoVs promoted chondrogenic differentiation by targeting the eEF2K/p38 MAPK axis. Consequently, a cell-free cartilage regeneration strategy using apoVs combined with a decellularized cartilage extracellular matrix (DCM) scaffold effectively promoted the regeneration of osteochondral defects. Overall, new mechanisms of cartilage regeneration by transplanted MSCs were unconcealed in this study. Moreover, we provided a novel experimental basis for cell-free tissue engineering-based cartilage regeneration utilizing apoVs.

The Role of Long Non-Coding RNF144A-AS1 in Cancer Progression.

RNAs transcribing more than 200 nucleotides without encoding proteins are termed long non-coding RNAs (LncRNAs). LncRNAs can be used as decoy molecules, signal molecules, scaffolds, and guide molecules. Long non-coding RNAs can interact with DNA, chromatin-modifying complexes, and transcriptional regulatory proteins, regulating gene expression in the cell nucleus. It is distributed in cytoplasm; they also participate in mRNA degradation and translational regulation via miRNAs, other transcription products, and proteins. They play a significant role in the development of various diseases, including tumors. Cancer seriously threatens human life and health. Regretfully, a great deal of newly diagnosed cancer patients found to have metastasized. RNF144A-AS1, also referred to as GRASLND, was initially recognized for its regulation of chondrogenic differentiation in MSCs. Focusing on RNF144A-AS1, this review summarizes and discusses the latest progress of RNF144A-AS1 in bladder cancer, glioblastoma, papillary renal cell carcinoma, gastric cancer, osteosarcoma, head and neck squamous cell carcinoma, and ovarian cancer. RNF144A-AS1 has good potential in tumor treatment and diagnosis.

SDF-1α peptide-tethered SIS membrane enables biomimetic tissue regeneration via multifactorial synergetic regulation

Tissue injuries occur frequently in daily life, but remain a challenge to treat in clinical medicine. Significant progress has been made in designing materials capable of directing endogenous cells to promote tissue regeneration. However, few studies have described a material that simultaneously promotes endogenous cell recruitment, immunomodulation, angiogenesis, soft tissue healing and bone tissue regeneration. Small intestinal submucosa (SIS) membrane possesses good biocompatibility and a precise spatial structure. The stromal-derived factor-1 (SDF-1) plays an important role in recruiting resident or circulating stem cells. In this work, the derived SDF-1α peptide was modified with a collagen-binding peptide to specifically tether to the SIS membrane. During the initial phase of tissue repair, pSIS@SDF-1α promoted cell recruitment and M2 polarization of macrophages. Subsequently, pSIS@SDF-1α promoted angiogenesis as well as osteogenic differentiation of bone marrow MSCs. The results of in vivo experiments demonstrated that pSIS@SDF-1α promoted soft tissue wound repair and regenerative repair of defective bone tissue. In conclusion, these findings may have important implications for in situ tissue regeneration by orchestrating endogenous cell recruitment and host responses.

Initial therapeutic evidence of a borosilicate bioactive glass (BSG) and Fe3O4 magnetic nanoparticle scaffold on implant-associated Staphylococcal aureus bone infection

Implant-associated Staphylococcus aureus (S. aureus) osteomyelitis is a severe challenge in orthopedics. While antibiotic-loaded bone cement is a standardized therapeutic approach for S. aureus osteomyelitis, it falls short in eradicating Staphylococcus abscess communities (SACs) and bacteria within osteocyte-lacuna canalicular network (OLCN) and repairing bone defects. To address limitations, we developed a borosilicate bioactive glass (BSG) combined with ferroferric oxide (Fe3O4) magnetic scaffold to enhance antibacterial efficacy and bone repair capabilities. We conducted comprehensive assessments of the osteoinductive, immunomodulatory, antibacterial properties, and thermal response of this scaffold, with or without an alternating magnetic field (AMF). Utilizing a well-established implant-related S. aureus tibial infection rabbit model, we evaluated its antibacterial performance in vivo. RNA transcriptome sequencing demonstrated that BSG + 5%Fe3O4 enhanced the immune response to bacteria and promoted osteogenic differentiation and mineralization of MSCs. Notably, BSG + 5%Fe3O4 upregulated gene expression of NOD-like receptor and TNF pathway in MSCs, alongside increased the expression of osteogenic factors (RUNX2, ALP and OCN) in vitro. Flow cytometry on macrophage exhibited a polarization effect towards M2, accompanied by upregulation of anti-inflammatory genes (TGF-β1 and IL-1Ra) and downregulation of pro-inflammatory genes (IL-6 and IL-1β) among macrophages. In vivo CT imaging revealed the absence of osteolysis and periosteal response in rabbits treated with BSG + 5%Fe3O4 + AMF at 42 days. Histological analysis indicated complete controls of SACs and bacteria within OLCN by day 42, along with new bone formation, signifying effective control of S. aureus osteomyelitis. Further investigations will focus on the in vivo biosafety and biological mechanism of this scaffold within infectious microenvironment.

Decellularized extracellular matrix enriched with GDNF enhances neurogenesis and remyelination for improved motor recovery after spinal cord injury

Motor functional improvement represents a paramount treatment objective in the post-spinal cord injury (SCI) recovery process. However, neuronal cell death and axonal degeneration following SCI disrupt neural signaling, impeding the motor functional recovery. In this study, we developed a multifunctional decellularized spinal cord-derived extracellular matrix (dSECM), crosslinked with glial cell-derived neurotrophic factor (GDNF), to promote differentiation of stem cells into neural-like cells and facilitate axonogenesis and remyelination. After decellularization, the immunogenic cellular components were effectively removed in dSECM, while the crucial protein components were retained which supports stem cells proliferation and differentiation. Furthermore, sustained release of GDNF from the dSECM facilitated axonogenesis and remyelination by activating the PI3K/Akt and MEK/Erk pathways. Our findings demonstrate that the dSECM-GDNF platform promotes neurogenesis, axonogenesis, and remyelination to enhance neural signaling, thereby yielding promising therapeutic effects for motor functional improvement after SCI. Statement of significanceThe dSECM promotes the proliferation and differentiation of MSCs or NSCs by retaining proteins associated with positive regulation of neurogenesis and neuronal differentiation, while eliminating proteins related to negative regulation of neurogenesis.After crosslinking, GDNF can be gradually released from the platform, thereby promoting neural differentiation, axonogenesis, and remyelination to enhance neural signaling through activation of the PI3K/Akt and MEK/Erk pathways.In vivo experiments demonstrated that dSECM-GDNF/MSC@GelMA hydrogel exhibited the ability to facilitate neuronal regeneration at 4 weeks post-surgery, while promoting axonogenesis and remyelination at 8 weeks post-surgery, ultimately leading to enhanced motor functional recovery.This study elucidates the ability of neural regeneration strategy to promote motor functional recovery and provides a promising approach for designing multifunctional tissue for SCI treatment.

Novel polycaprolactone-based biomimetic grafts enriched with graphene oxide and cerium oxide: Exploring improved osteogenic potential

In bone tissue engineering, the development of advanced biomaterials is crucial to address the challenges associated with the treatment and regeneration of bone defects. This study presents a novel polymeric nanocomposite consisting of Polycaprolactone (PCL) integrated with graphene oxide (GO) and cerium oxide (CeO2) nanoparticles, aiming to comprehensively explore its multifaceted potential for bone tissue engineering applications. The physicochemical properties of the composite films were analyzed. Biodegradation studies showed superior degradability to pure PCL due to added hydrophilic features and increased nanofiller-induced surface porosity. The remarkable improvement in mechanical stability is attributed to the uniform dispersion of nanofillers. Assessments of surface wettability, demonstrate a noteworthy shift towards heightened hydrophilicity, rendering a more conducive environment for biomolecules. The hybrid polymer nanocomposite exhibits remarkable hemocompatibility, antibacterial properties, antioxidant activity, and excels in facilitating apatite deposition and promoting osteogenic differentiation. Biomineralization investigations and ARS assay underscore the nanocomposite's exceptional ability to promote apatite deposition, a pivotal mineral in bone formation, owing to the altered surface properties introduced by the nanofillers. The ALP findings imply that the PCL-GO-CeO2 nanocomposite actively supports and accelerates the process of osteogenic differentiation in MSCs, indicating its potential utility in bone tissue engineering. Additionally, cell culture studies conducted on HOS and rBMSC cell lines validate the nanocomposite's excellent cell viability, attachment, and proliferation, affirming its biocompatibility and potential for bone tissue engineering. Collectively, these findings establish PCL-GO-CeO2 nanocomposite as a favourable option for enhancing bone regeneration.

Regulatory T cell-derived exosome mediated macrophages polarization for osteogenic differentiation in fracture repair

Refractory fracture presents an intractable challenge in trauma treatment. Selective polarization of macrophages as well as the recruitment of osteogenic precursor cells play key roles in osteogenic differentiation during fracture healing. Here we constructed regulatory T cell (Treg)-derived exosomes (Treg-Exo) for the treatment of fracture. The obtained exosomes displayed a spheroid shape with a hydrated particle size of approximately 130 nm. With further purification using CD39 and CD73 antibody-modified microfluidic chips, CD39 and CD73 specifically expressing exosomes were obtained. This kind of Treg-Exo utilized the ectonucleotidases of CD39 and CD73 to catalyze the high level of ATP in the fracture area into adenosine. The generated adenosine further promoted the selective polarization of macrophages. When interacting with mesenchymal stem cells (MSCs, osteogenic precursor cells), both Treg-Exo and Treg-Exo primed macrophages facilitated the proliferation and differentiation of MSCs. After administration in vivo, Treg-Exo effectively promoted fracture healing compared with conventional T cell-derived exosome. To further improve the delivery efficacy of exosomes and integrate multiple biological processes of fracture healing, an injectable hydrogel was fabricated to co-deliver Treg-Exo and stromal cell-derived factor 1 alpha (SDF-1α). With the dual effect of Treg-Exo for macrophage polarization and SDF-1α for MSC recruitment, the multifunctional hydrogel exerted a synergistic effect on fracture repair acceleration. This study provided a promising therapeutic candidate and synergistic strategy for the clinical treatment of fracture.

Long Non-Coding RNA-Cardiac-Inducing RNA 6 Mediates Repair of Infarcted Hearts by Inducing Mesenchymal Stem Cell Differentiation into Cardiogenic Cells through Cyclin-Dependent Kinase 1.

This study aims to investigate the induction effect of LncRNA-CIR6 on MSC differentiation into cardiogenic cells in vitro and in vivo. In addition to pretreatment with Ro-3306 (a CDK1 inhibitor), LncRNA-CIR6 was transfected into BMSCs and hUCMSCs using jetPRIME. LncRNA-CIR6 was further transfected into the hearts of C57BL/6 mice via 100 μL of AAV9-cTnT-LncRNA-CIR6-ZsGreen intravenous injection. After three weeks of transfection followed by AMI surgery, hUCMSCs (5 × 105/100 μL) were injected intravenously one week later. Cardiac function was evaluated using VEVO 2100 and electric mapping nine days after cell injection. Immunofluorescence, Evans blue-TTC, Masson staining, FACS, and Western blotting were employed to determine relevant indicators. LncRNA-CIR6 induced a significant percentage of differentiation in BMSCs (83.00 ± 0.58)% and hUCMSCs (95.43 ± 2.13)% into cardiogenic cells, as determined by the expression of cTnT using immunofluorescence and FACS. High cTNT expression was observed in MSCs after transfection with LncRNA-CIR6 by Western blotting. Compared with the MI group, cardiac contraction and conduction function in MI hearts treated with LncRNA-CIR6 or combined with MSCs injection groups were significantly increased, and the areas of MI and fibrosis were significantly lower. The transcriptional expression region of LncRNA-CIR6 was on Chr17 from 80209290 to 80209536. The functional region of LncRNA-CIR6 was located at nucleotides 0-50/190-255 in the sequence. CDK1, a protein found to be related to the proliferation and differentiation of cardiomyocytes, was located in the functional region of the LncRNA-CIR6 secondary structure (from 0 to 17). Ro-3306 impeded the differentiation of MSCs into cardiogenic cells, while MSCs transfected with LncRNA-CIR6 showed a high expression of CDK1. LncRNA-CIR6 mediates the repair of infarcted hearts by inducing MSC differentiation into cardiogenic cells through CDK1.

Polycaprolactone/graphene oxide/magnesium oxide as a novel composite scaffold for bone tissue engineering: Preparation and physical/biological assessment

Mechanically robust biocomposite scaffolds were fabricated by the electrospinning method for bone tissue engineering using a blend of polycaprolactone (PCL), and graphene oxide (GO) with magnesium oxide (MgO) nanoparticles. Physicochemical characteristics including morphology, tensile strength, swelling behavior, biodegradability, contact angle, cell viability, alkaline phosphatase (ALP) activity, mineralization ability, and osteogenic gene expression of the scaffolds were characterized. The addition of small amounts of GO and MgO nanoparticles significantly improved the morphological and mechanical properties of the PCL scaffold. The hydrophilicity, swelling ratio, and biodegradability of the developed composite scaffolds were improved. The PCL/GO/MgO scaffold demonstrated excellent biocompatibility and in-vitro biological performance with adipose-derived mesenchymal stem cells. Cell attachment, proliferation, ALP activity, mineral deposition, and osteogenesis-related gene expression were enhanced when compared to the pure PCL and PCL/GO scaffolds. Simultaneous incorporation of GO and MgO nanoparticles at a concentration of 2 wt% dramatically increased the differentiation of MSCs into osteoblasts. These findings may suggest that the hydrophilic properties and high protein adsorption of PCL/GO/MgO scaffold can stimulate cell proliferation, and nucleation to help improve bone mineralization.

Effects of crocin on the enhancement of in vitro neurogenesis: Involvement of Notch and CREB/BDNF signaling pathways.

Adult neurogenesis, the process of generating new neurons, continues throughout life. Unfortunately, this process is insufficient in pathological conditions and needs to be promoted. Crocin, the active component of saffron, affects neurogenesis in vivo and in vitro. We aimed to investigate the enhancing effects of crocin on the neurogenesis of adipose-derived mesenchymal stem cells in the presence of retinoic acid, as well as the molecular pathways involved. Differentiation capacities and stemness potential of harvested ADSCs were evaluated by differentiating into osteocytes and adipocytes, and expression of mesenchymal CD markers by flow cytometry. The optimum dose of crocin was assessed with an MTT assay. Crocin, retinoic acid, CREB/BDNF, and Notch inhibitors and their combination were added to the culture medium. Jag1, Hes1, Notch, and BDNF gene expression were analyzed by RT-PCR on days 7, 14, and 21, while CREB, DCX, SOX2, and NeuN expression were analyzed by immunofluorescence. Expression of mesenchymal CD markers as well as adipogenic and osteogenic differentiation confirmed the origin and properties of ADSCs. The optimal dose of crocin was 1 mM. Crocin significantly (P<0.05) increased, while inhibitors (DATP&Naphthol) significantly (P<0.05) decreased Jag1, Hes1, Notch, and BDNF expression. Immunofluorescent assessments showed that expression of DCX, BDNF, NeuN, and Sox2 proteins increased significantly (P<0.05) after crocin administration and decreased significantly (P<0.05) after inhibitor administration. Crocin can be used as an enhancer for neural differentiation of MSCs in vitro in the presence of retinoic acid. The mechanism is proposed through Notch and CREB/BDNF signaling pathways.

Titanium-based substrate modified with nanoenzyme for accelerating the repair of bone defect

Titanium (Ti) and titanium alloy are the most common metal materials in clinical orthopedic surgery. However, in the initial stage of surgery and implantation, the production of excessive reactive oxygen species (ROS) can induce oxidative stress (OS) microenvironment. OS will further inhibit the growth of new bone, resulting in surgical failure. In this study, based on the fact that nanoscale manganese dioxide (MnO2) can show H2O2-like enzyme activity, a MnO2 nanocoating was prepared on mciro-nano structured surface of Ti substrate via a two-step method of alkaline thermal and hydrothermal treatment. The results of scanning electron microscopy (SEM), X-ray diffractometer (XRD) and X-ray photoelectron spectroscopy (XPS) showed that the nano-MnO2 coating was successfully fabricated on the surface of Ti substrate. The results of measurement of H2O2, dissolved O2 and intracellular ROS in vitro showed that the treated Ti substrate could efficiently eliminate H2O2 and reduce ROS. Furthermore, the modified Ti substrate could promote the early adhesion, proliferation and osteogenic differentiation of MSCs, which was demonstrated by experimental results of cell morphology, cell viability, alkaline phosphatase, collagen, and mineralization deposition. The results of quantitative real-time polymerase chain reaction (qRT-PCR) of MSCs adhered the modified Ti substrate showed that the expression of genes related to osteogenic differentiation significantly increased. More importantly, the modified Ti implant could eliminate ROS at the injury site, reduce OS and promote the regeneration of bone tissue, which was demonstrated via hematoxylin/eosin, Masson's trichrome and immunohistochemical staining. In conclusion, the modified Ti implant presented here had the effect of reducing OS and promoting osseointegration. Relevant research ideas and results provide new methods for the research and development of functional implants, which have potential application value in the field of orthopedics.

Ellagic acid promotes osteoblasts differentiation via activating SMAD2/3 pathway and alleviates bone mass loss in OVX mice

Characterized by bone mass loss, osteoporosis is an orthopedic disease typically found in postmenopausal women and aging individuals. Consistent with its pathogenesis summarized as an imbalance in bone formation/resorption, current pharmacologically therapeutic strategies for osteoporosis mainly aim to promote bone formation or/and inhibit bone resorption. However, few effective drugs with mild clinical side effects have been developed, making it a well-concerned issue to seek appropriate drugs for osteoporosis. In this study, we investigated the effect of ellagic acid (EA) on osteogenesis in vitro and in vivo and searched for its molecular mechanism. Here, we showed that EA promoted osteogenic differentiation of MSCs, increased mRNA and protein expression levels of osteoblast marker genes Runt-related transcription factor2, Osterix, Alkaline phosphatase, Collagen type I alpha 1, Osteopontin and Osteocalcin. Furthermore, ovariectomized mice with orally administered EA (10 mg/kg, 50 mg/kg) had significantly higher bone mass than those in controls. And experiments such as fluorescence double-labeling and enzyme-linked immunosorbent assay also demonstrated that EA could promote osteogenesis in vivo. To probe the molecular mechanism of EA, we performed RNA sequencing analysis using EA-treated BMSCs. Significant up-regulation of SMAD2/3 transcription factors was identified by RNA-seq, and it was confirmed in vitro that EA promoted bone formation by activating the SMAD2/3 signaling pathway. Evidence from our present experiments indicates that EA may be a promising candidate for clinical treatment for osteoporosis in future.

Investigation the effects of bee venom and H-dental-derived mesenchymal stem cells on non-small cell lung cancer cells (A549).

Lung cancer, one of the most common oncological diseases worldwide, continues to be the leading cause of cancer-related deaths. The development of new approaches for lung cancer, which still has a low survival rate despite medical advances, is of great importance. In this study, bee venom (BV), conditioned medium of MSCs isolated from dental follicles (MSC-CM) and cisplatin were applied at different doses and their effects on A549 cell line were evaluated. Dental follicles were used as a source of MSCs source and differentiation kits, and characterization studies (flow cytometry) were performed. Cell viability was measured by the MTT method and apoptosis was measured by an Annexin V-FITC/PI kit on flow cytometer. IC50 dose values were determined according to the 24th hour and were determined as 15.8 µg/mL for BV, 10.78% for MSC-CM and 5.77 µg/mL for cisplatin. IC50 values found for BV and MSC-CM were also given in combination and the effects were observed. It was found that the applied substances caused BV to decrease in cell viability and induced apoptosis in cells. In addition to the induction of apoptosis in BV, MSC-CM, and combined use, all three applications led to an increase in Bax protein expression and a decrease in Bcl-2 protein expression. The molecular mechanism of anticancer activity through inhibition of Bax and Bcl-2 proteins and the NF-κB signaling pathway may be suggested. Isolated MSCs in our study showed anticancer activity and BV and MSC-CM showed synergistic antiproliferative and apoptotic effects.

Enhancer-mediated FOXO3 expression promotes MSC adipogenic differentiation by activating autophagy

BackgroundMesenchymal stem cells (MSCs) are pluripotent stem cells capable of differentiating into osteocytes, adipocytes and chondrocytes. However, in osteoporosis, the balance of differentiation is tipped toward adipogenesis and the key mechanism is controversial. Researches have shown that, as upstream regulatory elements of gene expression, enhancers ar involved in the expression of identity genes. In this study, we identified enhancers-mediated gene FOXO3 promoting MSC adipogenic differentiation by activating autophagy. MethodsWe integrated data of RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq) and ATAC-sequencing (ATAC-seq) to find the identity gene FOXO3. The expression of FOXO3 protein, adipogenic transcription factors and the substrate of autophagy were measured by western blotting. The Oil Red O (ORO) staining was used to visualize the adipogenesis of MSCs. Immunohistochemistry was used to visualize the FOXO3 expression in adipocytes in bone marrow. Immunofluorescence was used to detect the expression of PPARγ and LC3B. ResultsDuring adipogenesis, enhancers redistribute to genes associated with adipogenic differentiation, among which we identified the pivotal identity gene FOXO3. FOXO3 could promote the expression of the adipogenic transcription factors PPARγ, CEBPα, and CEBPβ during adipogenic differentiation, while PPARγ, CEBPα, and CEBPβ could in turn bind to FOXO3 and continue to promote FOXO3 expression to form a positive feedback loop. Consistently elevated FOXO3 expression promotes autophagy by activating the PI3K-AKT pathway which mediates adipogenic differentiation. ConclusionsPivotal identity gene FOXO3 promotes autophagy by activating PI3K-AKT pathway, which provokes adipogenic differentiation of MSCs. Enhancer-regulated adipogenic identity gene FOXO3 could be an attractive treatment for osteoporosis.

The role of integrin αvβ3 in biphasic calcium phosphate ceramics mediated M2 Macrophage polarization and the resultant osteoinduction

Calcium phosphate ceramics-based biomaterials were reported to have good biocompatibility and osteoinductivity and have been widely applied for bone defect repair and regeneration. However, the mechanism of their osteoinductivity is still unclear. In our study, we established an ectopic bone formation in vivo model and an in vitro macrophage cell co-culture system with calcium phosphate ceramics to investigate the effect of biphasic calcium phosphate on osteogenesis via regulating macrophage M1/M2 polarization. Our micro-CT data suggested that biphasic calcium phosphate had significant osteoinductivity, and the fluorescence co-localization detection found increased F4/80+/integrin αvβ3+ macrophages surrounding the biphasic calcium phosphate scaffolds. Besides, our study also revealed that biphasic calcium phosphate promoted M2 polarization of macrophages via upregulating integrin αvβ3 expression compared to tricalcium phosphate, and the increased M2 macrophages could subsequently augment the osteogenic differentiation of MSCs in a TGFβ mediated manner. In conclusion, we demonstrated that macrophages subjected to biphasic calcium phosphate could polarize toward M2 phenotype via triggering integrin αvβ3 and secrete TGFβ to increase the osteogenesis of MSCs, which subsequently enhances bone regeneration.

The influence of inflammation on the characteristics of adipose-derived mesenchymal stem cells (ADMSCs) and tissue repair capability in a hepatic injury mouse model

BackgroundMesenchymal stem cells (MSCs) are adult stem cells with self-renewal and multi-directional differentiation potential and possess the functions of immunomodulation, regulation of cell growth, and repair of damage. Over recent years, MSCs have been found to regulate the secretion of inflammatory factors and to exert regulatory effects on various lymphocytes in inflammatory states, and on the subsequent repair of tissue damage caused by inflammation. In the present study, we analyzed the effects of tissue inflammation on the characteristics of MSCs.MethodsHuman fat derived from the infrapatellar fat pad (IPFP) of knees with differing degrees of inflammation was extracted from specimens derived from total knee arthroplasties. HE and immunohistochemical staining was performed to directly observe the evidence and degree of inflammation in human infrapatellar fat pad tissue in order to classify MSCs cells, by their origin, into highly inflamed and lowly inflamed groups, and to study the effect of tissue inflammation on cell acquisition rates via cellular counting data. Flow cytometry assays were performed to investigate the effect of tissue inflammation on MSC surface marker expression. Trilineage differentiation, including osteogenesis, adipogenesis, and chondrogenesis, was performed to assess the effect of tissue inflammation on the ability of MSCs to undergo directed differentiation. The effect of tissue inflammation on the ability of MSCs to proliferate was investigated via clone formation studies. RNA-sequencing was performed to evaluate the transcriptomes of MSCs derived from different areas of inflammation. The effect of tissue inflammation on tissue repair capacity and safety of MSCs was investigated via a murine model of acute liver injury.ResultsThe results of cell count data indicate that a high degree of tissue inflammation significantly decreases the acquisition rate of MSCs, and the proportion of CD34+ and CD146+ cells. The results of our trilineage differentiation assay show that a higher degree of inflammation decreases osteogenic differentiation and enhances adipogenic and chondrogenic differentiation of MSCs. However, these differences were not statistically significant. Clone formation assays indicate that the degree of tissue inflammation at the MSC source does not significantly affect the proliferative capacity of MSCs. The transcriptomes of MSCs remain relatively stable in fat pad tissues derived from both highly and lowly inflamed samples. The results of acute liver injury investigations in mice indicate that MSCs of high and low inflammatory tissue origin have no significant difference in their tissue repair capability.ConclusionsHigh tissue inflammation at the source of MSCs reduces the acquisition rate of MSCs and the percentage of CD34+ and CD146+ cells acquisition. However, source tissue inflammation may not significantly affect trilineage differentiation potential and proliferative capacity of MSCs. Also, MSCs obtained from differing source degrees of inflammation retain stable and similar transcriptomic profile and are both safe and efficacious for tissue repair/regeneration without detectable differences.Graphical

Novel_circ_003686 regulates the osteogenic differentiation of MSCs in patients with myeloma bone disease through miR-142-5p/IGF1 axis

ObjectivesCirc_003686 is a novel_circRNA with abnormally low expression found in the samples of multiple myeloma bone disease (MBD) patients. The current research intended to investigate the effects of novel_circ_003686 in osteogenesis-induced differentiation of bone marrow mesenchymal stem cells (BMSCs) in MBD. MethodsBMSCs were extracted from MBD patients and normal participants, the pcDNA3.1 encoding the circ_003686 (ov-circ_003686), miR-142-5p-mimic/inhibitor and siRNA oligonucleotides targeting insulin like growth factor 1 (IGF1, si-IGF1) were applied to intervene circ_003686, miR-142-5p and IGF1 levels, respectively. Results: Results showed that ov-circ_003686 could mediate the osteogenesis-induced differentiation of MBD-BMSC, and luciferase assay and RIP experiments confirmed that circ_003686 could bind to miR-142-5p. MiR-142-5p-inhibitor helped osteogenesis-induced differentiation, while miR-142-5p-mimic inhibited osteogenesis-induced differentiation and reversed the promoting effect of ov-circ_003686, suggesting that circ_003686/miR-142-5p axis participated in osteogenesis-induced differentiation of MBD-BMSC. In addition, miR-142-5p binds to the target gene IGF1 and negatively adjust its expression. Si-IGF1 significantly inhibited the osteogenesis-induced differentiation and reversed the promotion effects of miR-142-5p-inhibitor and ov-circ_003686. Moreover, circ_003686/miR-142-5p/IGF1 axis meaningfully regulates protein expressions in the PI3K/AKT pathway. ConclusionIn conclusion, this research confirmed that circ_003686 regulated the osteogenesis-induced differentiation of MBD-BMSC by sponging miR-142-5p and mediating IGF1, and the PI3K/AKT pathway may also be involved.