New advances of NG2-expressing cell subset in marrow mesenchymal stem cells as novel therapeutic tools for liver fibrosis/cirrhosis.

BACKGROUND: Bone marrow-derived mesenchymal stem cell (BMMSC)-based therapy has become a major focus for treating liver fibrosis/cirrhosis. However, although these cell therapies promote the treatment of this disease, the heterogeneity of BMMSCs, which causes insufficient efficacy during clinical trials, has not been addressed. In this study, we describe a novel Percoll-Plate-Wait procedure (PPWP) for the isolation of an active cell subset from BMMSC cultures that was characterized by the expression of neuroglial antigen 2 (NG2/BMMSCs). METHODS: By using the key method of PPWP and other classical biological techniques we compared NG2/BMMSCs with parental BMMSCs in biological and functional characteristics within a well-defined diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis injury male C57BL/6 mouse model also in a culture system. Of note, the pathological alterations in the model is quite similar to humans'. RESULTS: The NG2/BMMSCs revealed more advantages compared to parentalBMMSCs. They exhibited greater proliferation potential than parental BMMSCs, as indicated by Ki-67 immunofluorescence (IF) staining. Moreover, higher expression of SSEA-3 (a marker specific for embryonic stem cells) was detected in NG2/BMMSCs than in parental BMMSCs, which suggested that the "stemness" of NG2/BMMSCs was greater than that of parental BMMSCs. In vivo studies revealed that an injection of NG2/BMMSCs into mice with ongoing DEN-induced liver fibrotic/cirrhotic injury enhanced repair and functional recovery to a greater extent than in mice treated with parental BMMSCs. These effects were associated with the ability of NG2/BMMSCs to differentiate into bile duct cells (BDCs). In particular, we discovered for the first time that NG2/BMMSCs exhibit unique characteristics that differ from those of parental BMMSCs in terms of producing liver sinusoidal endothelial cells (LSECs) to reconstruct injured blood vessels and sinusoidal structures in the diseased livers, which are important for initiating hepatocyte regeneration. This unique potential may also suggest that NG2/BMMSCs could be an novel off-liver progenitor of LSECs. Ex vivo studies revealed that the NG2/BMMSCs exhibited a similar trend to that of their in vivo in terms of functional differentiation responding to the DEN-diseased injured liver cues. Additionally, the obvious core role of NG2/BMMSCs in supporting the functions of BMMSCs in bile duct repair and BDC-mediated hepatocyte regeneration might also be a novel finding. CONCLUSIONS: Overall, the PPWP-isolated NG2/BMMSCs could be a novel effective cell subset with increased purity to serve as a new therapeutic tool for enhancing treatment efficacy of BMMSCs and special seed cell source (BDCs, LSECs) also for bioliver engineering.

Investigation on the molecular mechanism of SPA interference with osteogenic differentiation of bone marrow mesenchymal stem cells.

Binding of Staphylococcus aureus protein A (SPA) to osteoblasts induces apoptosis and inhibits bone formation. Bone marrow-derived mesenchymal stem cells (BMSCs) have the ability to differentiate into bone, fat and cartilage. Therefore, it was important to analyze the molecular mechanism of SPA on osteogenic differentiation. We introduced transcript sequence data to screen out differentially expressed genes (DEGs) related to SPA-interfered BMSC. Protein-protein interaction (PPI) network of DEGs was established to screen biomarkers associated with SPA-interfered BMSC. Receiver operating characteristic (ROC) curve was plotted to evaluate the ability of biomarkers to discriminate between two groups of samples. Finally, we performed GSEA and regulatory analysis based on biomarkers. We identified 321 DEGs. Subsequently, 6 biomarkers (Cenpf, Kntc1, Nek2, Asf1b, Troap and Kif14) were identified by hubba algorithm in PPI. ROC analysis showed that six biomarkers could clearly discriminate between normal differentiated and SPA-interfered BMSC. Moreover, we found that these biomarkers were mainly enriched in the pyrimidine metabolism pathway. We also constructed '71 circRNAs-14 miRNAs-5 mRNAs' and '10 lncRNAs-5 miRNAs-2 mRNAs' networks. Kntc1 and Asf1b genes were associated with rno-miR-3571. Nek2 and Asf1b genes were associated with rno-miR-497-5p. Finally, we found significantly lower expression of six biomarkers in the SPA-interfered group compared to the normal group by RT-qPCR. Overall, we obtained 6 biomarkers (Cenpf, Kntc1, Nek2, Asf1b, Troap, and Kif14) related to SPA-interfered BMSC, which provided a theoretical basis to explore the key factors of SPA affecting osteogenic differentiation.

microRNA-181a-5p promotes fibroblast differentiation of mesenchymal stem cells in rats with pelvic floor dysfunction.

The use of stem cells capable of multilineage differentiation in treating Pelvic Floor Dysfunction (PFD) holds great promise since they are susceptible to entering connective tissue of various cell types and repairing damaged tissues. This research investigated the effect of microRNA-181a-5p (miR-181a-5p) on Bone Marrow Mesenchymal Stem Cells (BMSCs) in rats with PFD. BMSCs were transfected and analyzed for their fibroblast differentiation ability. miR-181a-5p, MFN1, and fibroblast-related genes were quantitatively analyzed. Whether MFN1 is a target gene of miR-181a-5p was predicted and confirmed. The efficacy of BMSCs in vivo rats with PFD was evaluated by measuring Leak Point Pressure (LPP), Conscious Cystometry (CMG), hematoxylin and eosin staining, and Masson staining. The present results discovered that miR-181a-5p was up-regulated and MFN1 was down-regulated during the differentiation of BMSCs into fibroblasts. Fibroblast differentiation of BMSCs was promoted after miR-181a-5p was induced or MFN1 was suppressed, but it was suppressed after miR-181a-5p was silenced. miR-181a-5p improved LPP and conscious CMG outcomes in PDF rats by targeting MFN1 expression, thereby accelerating fibroblast differentiation of BMSCs. In brief, miR-181a-5p induces fibroblast differentiation of BMSCs in PDF rats by MFN1, potentially targeting PDF therapeutics.

Stem cell therapy in diabetic men with erectile dysfunction: a 24-month follow-up of safety and efficacy of two intracavernous autologous bone marrow derived mesenchymal stem cells injections, an open label phase 2 clinical trial.

BACKGROUND: Recently we reported results of phase 1 pilot clinical trial of 2 consecutive intracavernous (IC) injection of autologous bone marrow-derived mesenchymal stem cells (BM-MSCs) for the first time in the treatment of diabetic patients with erectile dysfunction (DM-ED). In phase 2 of this study our aim is to evaluate long term safety and efficacy of IC injections of BM-MSC on additional eight patients with DM-ED. RESULTS: Each patient received 2 consecutive IC injections of BM-MSC and evaluated at 1, 3, 6, 12, and 24-month time points. Primary outcome was the tolerability and safety of stem cells therapy (SCT), while the secondary outcome was improvement of erectile function (EF) as assessed using the International Index of Erectile Function-5 (IIEF-5), Erection Hardness Score (EHS) questionnaires, and Color Duplex Doppler Ultrasound (CDDU). IC injections of BM-MSCs was safe and well-tolerated. Minor local and short-term adverse events related to the bone marrow aspiration and IC injections were observed and treated conservatively. There were significant improvement in mean IIEF-5, EHS, all over the follow-up time points in comparison to the baseline. At 24-month follow up there were significant decline in the mean IIEF-5, and EHS compared to the baseline. The mean basal and 20-min peak systolic velocity was significantly higher at 3-month after the IC injections compared to baseline. CONCLUSIONS: This phase 2 clinical trial confirmed that IC injections of BM-MSC are safe and improve EF. The decline in EF over time suggests a need for assessing repeated injections. CLINICAL TRIAL REGISTRATION: NCT02945462.

RéSUMé: CONTEXTE: Récemment, nous avons rapporté les résultats d'un essai clinique pilote de phase 1, de 2 injections intracaverneuses (IC) consécutives de cellules souches mésenchymateuses autologues dérivées de la moelle osseuse (BM-MSC), pour la première fois dans le traitement de patients diabétiques atteints de dysfonction érectile (DM-ED). Dans la phase 2 de cette étude, notre objectif est d'évaluer l'innocuité et l'efficacité à long terme des injections IC de BM-MSC sur huit autres patients atteints de dysfonction érectile. RéSULTATS: Chaque patient a reçu 2 injections IC consécutives de BM-MSC, et a été évalué à des intervalles de temps de 1, 3, 6, 12 et 24 mois. Le critère de jugement principal était la tolérance et l'innocuité de la thérapie par cellules souches, tandis que le critère de jugement secondaire était l'amélioration de la fonction érectile (FE) évaluée à l'aide de l'indice international de la fonction érectile-5 (IIEF-5), de questionnaires sur le score de dureté de l'érection (EHS) et de l'échographie Doppler duplex couleur. Les injections IC de BM-MSC se sont avérées sûres et ont été bien tolérées. Des effets indésirables locaux et à court terme mineurs, liés à l'aspiration de la moelle osseuse et aux injections d'IC, ont été observés et traités de manière conservatrice. Il y a eu une amélioration significative des moyennes de l'IIEF-5 moyen, de l'EHS à tous les points de suivi par rapport à la l'état basal. A 24 mois de suivi, il y a eu une baisse significative de l'IIEF-5 moyen et de l'EHS par rapport à l'état basal. La moyenne se base et celle du pic maximal de la  vitesse systolique à 20 minutes étaient significativement plus élevées 3 mois après les injections de CI par rapport à l'état de base. CONCLUSIONS: Cet essai clinique de phase 2 a confirmé que les injections de BM-MSC par injections intracaverneuses sont sûres et améliorent la fonction érectile. La baisse de cette dernière au fil du temps suggère une nécessité d'évaluation des injections répétées.

Comparison of aquaporin profile of advanced passage mesenchymal stem cells with early passage mesenchymal stem cells and determination of its effect on adipogenic differentiation efficiency.

OBJECTIVE: Our study aimed to compare aquaporin profiles in advanced and early passage bone marrow-derived mesenchymal stem cells (BM-MSCs) and assess the impact of aquaporin changes after adipogenic differentiation. Aquaporins are crucial for stem cell survival and differentiation during their life cycle. We focused on the role of aquaporins in the cell structures of advanced and early passage stem cells. METHODS: In our study, BM-MSCs were used for our objectives. Characterization of the cells was evaluated via flow cytometry using stem cell surface markers. The characterized BM-MSCs were divided into control and differentiation groups at passages 3 (P3) and 8 (P8). AQP1, AQP3, AQP7, AQP9, and AQP10 expression levels on days 0, 1, 3, 7, 14, and 21 were evaluated using Real Time-PCR, ELISA, and immunofluorescence studies. RESULTS: The cells were characterized by flow cytometry and confirmed to exhibit BM-MSC characteristics. At P3 and P8, differentiation was initiated, and AQP protein expression was observed to initially increase and then decrease on subsequent days. The increase in AQP protein expression at P3 occurred earlier than that at P8. Gene expression analysis demonstrated a statistically significant increase in AQP gene expression on days when AQP protein expression decreased. Moreover, statistical differences were observed between late and early passage AQP profiles. CONCLUSION: Our study examined the composition of AQPs in BM-MSCs in association with cell passage, and found that AQPs play a role in the differentiation process. The connection between the AQP profile and aging might be related to differentiation capacity, which could have implications for slowing down cellular aging and developing new therapeutic approaches.

Vitamin D supplementation affects bone marrow-derived mesenchymal stem cells differentiation into insulin-producing cells.

Background this study was conducted to assess the effects of vitamin D on differentiation of bone marrow- derived mesenchymal stem cells (BM-MSCs) into insulin producing cells (IPCs). Method BM-MSCs were isolated from femur and tibia of rats and incubated in low (LG) or high glucose (HG) (5mM or 25mM), or high glucose DMEM media supplemented with vitamin D (0.2nM) (HGD) for 14 days. Cells viability was analysis by MTT assay. Differentiation of SCs was confirmed using measuring genes expression level of pdx1 and insulin, and insulin secretion, glucose stimulated insulin secretion, and insulin content by ELISA method. Results Cell viability was significantly higher in HGD than LG (p < 0.05) in day 3, also, in HG and HGD than LG (p < 0.001), and HGD vs. HG (p < 0.001) in day 7. Pdx1 and insulin level was markedly higher in HGD than LG (p < 0.05 and p < 0.01). pdx1 expression was markedly higher in HGD (p < 0.05) than LG, also insulin expression the HG (p < 0.05), and HGD (p < 0.01) groups compared to the LG group. Insulin release at 5mM glucose was notably higher in the HGD group compared to LG (p < 0.05), and at 25mM glucose, both HG and HGD showed significant increases vs. LG (p < 0.05 and p < 0.01, respectively). Insulin content was significantly higher in both 5mM and 25mM glucose for HG and HGD vs. LG (p < 0.01 and p < 0.001, respectively). In conclusion, treatment BM-MSCs with vitamin D could increase their differentiation into IPCs and it can be considered as a potential supplementary agent in enhancing differentiation SCs into insulin generating cells.

A Multidisciplinary Evaluation of Three-Dimensional Polycaprolactone Bioactive Glass Scaffolds for Bone Tissue Engineering Purposes.

In the development of bone graft substitutes, a fundamental step is the use of scaffolds with adequate composition and architecture capable of providing support in regenerative processes both on the tissue scale, where adequate resistance to mechanical stress is required, as well as at the cellular level where compliant chemical-physical and mechanical properties can promote cellular activity. In this study, based on a previous optimization study of this group, the potential of a three-dimensional construct based on polycaprolactone (PCL) and a novel biocompatible Mg- and Sr-containing glass named BGMS10 was explored. Fourier-transform infrared spectroscopy and scanning electron microscopy showed the inclusion of BGMS10 in the scaffold structure. Mesenchymal stem cells cultured on both PCL and PCL-BGMS10 showed similar tendencies in terms of osteogenic differentiation; however, no significant differences were found between the two scaffold types. This circumstance can be explained via X-ray microtomography and atomic force microscopy analyses, which correlated the spatial distribution of the BGMS10 within the bulk with the elastic properties and topography at the cell scale. In conclusion, our study highlights the importance of multidisciplinary approaches to understand the relationship between design parameters, material properties, and cellular response in polymer composites, which is crucial for the development and design of scaffolds for bone regeneration.

Corrigendum: BM-MSCs alleviate diabetic nephropathy in male rats by regulating ER stress, oxidative stress, inflammation, and apoptotic pathways.

[This corrects the article DOI: 10.3389/fphar.2023.1265230.].

Cadherin-6 is a novel mediator for the migration of mesenchymal stem cells to glioblastoma cells in response to stromal cell-derived factor-1.

Glioblastoma recruits various nontransformed cells from distant tissues. Although bone marrow-derived mesenchymal stem cells (MSCs) have been observed migrating to glioblastoma, the underlying mechanism driving MSC migration toward glioblastoma remains unclear. Tumor vascularity is critical in the context of recurrent glioblastoma and is closely linked to the expression of stromal cell-derived factor-1 (SDF-1). We demonstrated that cadherin-6 mediated MSC migration both toward SDF-1 and toward glioblastoma cells. Cadherin-6 knockdown resulted in the downregulation of MSCs capacity to migrate in response to SDF-1. Furthermore, MSCs with cadherin-6 knockdown exhibited impaired migration in response to conditioned media derived from glioblastoma cell lines (U87 and U373) expressing SDF-1, thus simulating the glioblastoma microenvironment. Moreover, MSCs enhanced the vasculogenic capacity of U87 cells without increasing the proliferation, cancer stem cell characteristics, or migration of U87. These results suggest that the current strategy of utilizing MSCs as carriers for antiglioblastoma drugs requires careful examination. Furthermore, cadherin-6 may represent a novel potential target for controlling the recruitment of MSCs toward glioblastoma.

Mechanism Exploration on the Immunoregulation of Allogeneic Heart Transplantation Rejection in Rats With Exosome miRNA and Proteins From Overexpressed IDO1 BMSCs.

Immunoregulation and indoleamine 2,3-dioxygenase 1 (IDO1) play pivotal roles in the rejection of allogeneic organ transplantation. This study aims to elucidate the immune-related functional mechanisms of exosomes (Exos) derived from bone marrow-derived mesenchymal stem cells (BMSCs) overexpressing IDO1 in the context of allogeneic heart transplantation (HTx) rejection. A rat model of allogeneic HTx was established. Exos were extracted after transfection with oe-IDO1 and oe-NC from rat BMSCs. Exos were administered via the caudal vein for treatment. The survival of rats was analyzed, and reverse transcription qualitative PCR (RT-qPCR) and immunohistochemistry (IHC) were employed to detect the expression of related genes. Histopathological examination was conducted using hematoxylin and eosin (HE) staining, and flow cytometry was utilized to analyze T-cell apoptosis. Proteomics and RNA-seq analyses were performed on Exos. The data were subjected to functional enrichment analysis using the R language. A protein interaction network was constructed using the STRING database, and miRWalk, TargetScan, and miRDB databases predicted the target genes, differentially expressed miRNAs, and transcription factors (TFs). Exos from BMSCs overexpressing IDO1 prolonged the survival time of rats undergoing allogeneic HTx. These Exos reduced inflammatory cell infiltration, mitigated myocardial damage, induced CD4 T-cell apoptosis, and alleviated transplantation rejection. The correlation between Exos from BMSCs overexpressing IDO1 and immune regulation was profound. Notably, 13 immune-related differential proteins (Anxa1, Anxa2, C3, Ctsb, Hp, Il1rap, Ntn1, Ptx3, Thbs1, Hspa1b, Vegfc, Dcn, and Ptpn11) and 10 significantly different miRNAs were identified. Finally, six key immune proteins related to IDO1 were identified through common enrichment pathways, including Thbs1, Dcn, Ptpn11, Hspa1b, Il1rap, and Vegfc. Thirteen TFs of IDO1-related key miRNAs were obtained, and a TF-miRNA-mRNA-proteins regulatory network was constructed. Exosome miRNA derived from BMSCs overexpressing IDO1 may influence T-cell activation and regulate HTx rejection by interacting with mRNA.

mir-150-5p inhibits the osteogenic differentiation of bone marrow-derived mesenchymal stem cells by targeting irisin to regulate the p38/MAPK signaling pathway.

PURPOSE: To study the effect of miR-150-5p on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs), and further explore the relationship between its regulatory mechanism and irisin. METHODS: We isolated mouse BMSCs, and induced osteogenic differentiation by osteogenic induction medium. Using qPCR to detect the expression of osteogenic differentiation-related genes, western blot to detect the expression of osteogenic differentiation-related proteins, and luciferase reporter system to verify that FNDC5 is the target of miR-150-5p. Irisin intraperitoneal injection to treat osteoporosis in mice constructed by subcutaneous injection of dexamethasone. RESULTS: Up-regulation of miR-150-5p inhibited the proliferation of BMSCs, and decreased the content of osteocalcin, ALP activity, calcium deposition, the expression of osteogenic differentiation genes (Runx2, OSX, OCN, OPN, ALP and BMP2) and protein (BMP2, OCN, and Runx2). And down-regulation of miR-150-5p plays the opposite role of up-regulation of miR-150-5p on osteogenic differentiation of BMSCs. Results of luciferase reporter gene assay showed that FNDC5 gene was the target gene of miR-150-5p, and miR-150-5p inhibited the expression of FNDC5 in mouse BMSCs. The expression of osteogenic differentiation genes and protein, the content of osteocalcin, ALP activity and calcium deposition in BMSCs co-overexpressed by miR-150-5p and FNDC5 was significantly higher than that of miR-150-5p overexpressed alone. In addition, the overexpression of FNDC5 reversed the blocked of p38/MAPK pathway by the overexpression of miR-150-5p in BMSCs. Irisin, a protein encoded by FNDC5 gene, improved symptoms in osteoporosis mice through intraperitoneal injection, while the inhibitor of p38/MAPK pathway weakened this function of irisin. CONCLUSION: miR-150-5p inhibits the osteogenic differentiation of BMSCs by targeting irisin to regulate the/p38/MAPK signaling pathway, and miR-150-5p/irisin/p38 pathway is a potential target for treating osteoporosis.

TGF-ß1-triggered BMI1 and SMAD2 cooperatively regulate miR-191 to modulate bone formation.

Transforming growth factor ß 1 (TGF-ß1), as the most abundant signaling molecule in bone matrix, is essential for bone homeostasis. However, the signaling transduction of TGF-ß1 in the bone-forming microenvironment remains unknown. Here, we showed that microRNA-191 (miR-191) was downregulated during osteogenesis and further decreased by osteo-favoring TGF-ß1 in bone marrow mesenchymal stem cells (BMSCs). MiR-191 was lower in bone tissues from children than in those from middle-aged individuals and it was negatively correlated with collagen type I alpha 1 chain (COL1A1). MiR-191 depletion significantly increased osteogenesis and bone formation in vivo. Hydrogels embedded with miR-191-low BMSCs displayed a powerful bone repair effect. Mechanistically, transcription factors BMI1 and SMAD2 coordinately controlled miR-191 level. In detail, BMI1 and pSMAD2 were both upregulated by TGF-ß1 under osteogenic condition. SMAD2 activated miR-191 transcription, while BMI1 competed with SMAD2 for binding to miR-191 promoter region, thus disturbing the activation of SMAD2 on miR-191 and reducing miR-191 level. Altogether, our findings reveal that miR-191 regulated by TGF-ß1-induced BMI1 and SMAD2 negatively modulated bone formation and regeneration, and inhibition of miR-191 might be therapeutically useful to enhance bone repair in clinic.

Poly-L-lactic acid/gelatin electrospun membrane-loaded bone marrow-derived mesenchymal stem cells attenuate erectile dysfunction caused by cavernous nerve injury.

Radical prostatectomy (RP) can cause neurogenic erectile dysfunction (ED), which negatively affects the quality of life of patients with prostate cancer. Currently, there is a dearth of effective therapeutic strategies. Although stem cell therapy is promising, direct cell transplantation to injured cavernous nerves is constrained by poor cell colonization. In this study, poly-L-lactic acid (PLLA)/gelatin electrospun membranes (PGEM) were fabricated to load bone marrow-derived mesenchymal stem cells (BM-MSCs) as a patch to be placed on injured nerves to alleviate ED. This study aimed to establish a promising and innovative approach to mitigate neurogenic ED post-RP and lay the foundation for modifying surgical procedures. Electrospinning and molecular biotechnology were performed in vitro and in vivo, respectively. It was observed that PGEM enhanced the performance of BM-MSCs and Schwann cells due to their excellent mechanical properties and biocompatibility. The transplanted PGEM and loaded BM-MSCs synergistically improved bilateral cavernous nerve injury-related ED and the corresponding histopathological changes. Nevertheless, transplantation of BM-MSCs alone has been verified to be ineffective. Overall, PGEM can serve as an ideal carrier to supply a more suitable survival environment for BM-MSCs and Schwann cells, thereby promoting the recovery of injured cavernous nerves and erectile function.

Validation of Human Bone Marrow-derived Mesenchymal Stem Cells and MCF-7 Breast Cancer Cells Co-culture Using a 3D Perfused Biomimetic Microfluidic Platform.

BACKGROUND/AIM: Microfluidic experimental models allow to study the mutual interrelation between tumor development and the microvasculature avoiding animal use and lacking interspecies differences. This study aimed to develop and characterize a 3D tissue culture model employing a two-compartment microfluidic chip-perfused platform to visualize and quantify human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and MCF-7 breast cancer cell-cell interactions in real time. MATERIALS AND METHODS: MCF-7 cells were implanted in the tumor chamber and hBM-MSCs were injected into microvascular channels. hBM-MSCs culture media was perfused into microvascular compartments. The microfluidic device was microscopically examined weekly for four weeks. RESULTS: VE- and E-cadherin immunofluorescence validated hBM-MSCs differentiation into endothelial cells and MCF-7 cell tumor formation. hBM-MSCs differentiation was highly heterogeneous along the microvascular channels, due to different perfusion flow. hBM-MSCs lining microvascular channels acquired VE-cadherin positive endothelial phenotype and continuously covered microchannels as an endothelium like layer. MCF-7 cells were constantly grown as spheroidal aggregates and later formed a compact area of E-cadherin-positive tumor cells inside tumor compartment. CONCLUSION: Our study provides valuable knowledge on the properties of hBM-MSCs as vasculogenesis-supporting cells when co-cultured with MCF-7 cells on a 3D perfused biomimetic microfluidic device. This newly established model may serve as an experimental platform for testing anti-tumor/anti-angiogenic drugs.

Vimentin protein is a factor for decreasing breast cancer cell proliferation co-culture with human bone marrow-derived mesenchymal stem cells pre-treated with thiazolidinedione solutions.

BACKGROUND: Our previous study investigated the levels of soluble growth factors in the conditioned media of bone marrow-derived mesenchymal stem cells (BMSCs) pre-treated with thiazolidinedione solutions. The present study aimed to investigate the complex intracellular proteins extracted from BMSCs pre-treated with pioglitazone and/or rosiglitazone using proteomics. METHODS: The proliferative effect of the identified protein on MCF-7 cells that interacted non-adhesively with BMSCs pre-treated with pioglitazone and/or rosiglitazone was evaluated using cell culture inserts and conditioned media. The mRNA expression of proliferation and lipid accumulation markers was also evaluated in the interacted MCF-7 cells by reverse transcription-quantitative PCR. Finally, the correlation between the identified protein and fibroblast growth factor 4 (FGF-4) protein in the conditioned media of the pre-treated BMSCs was evaluated by ELISA. RESULTS: The present study identified vimentin as the specific protein among the complex intracellular proteins that likely plays a role in MCF-7 cell proliferation when the breast cancer cells interacted non-adhesively with BMSCs pre-treated with a combination of pioglitazone and rosiglitazone. The inhibition of this protein promoted the proliferation of MCF-7 cells when the breast cancer cells interacted with pre-treated BMSCs. Gene expression analysis indicated that pre-treatment of BMSCs with a combination of pioglitazone and rosiglitazone decreased the mRNA expression of Ki67 and proliferating cell nuclear antigen in MCF-7 cells. The pre-treatment did not induce mRNA expression of PPARγ, which is a sign of lipid accumulation. The level of vimentin protein was also associated with the FGF-4 protein expression level in the conditioned media of the pre-treated BMSCs. Bioinformatics analysis revealed that vimentin regulated the expression of FGF-4 through its interaction with SRY-box 2 and POU class 5 homeobox 1. CONCLUSIONS: The present study identified a novel intracellular protein that may represent the promising target in pre-treated BMSCs to decrease the proliferation of breast cancer MCF-7 cells for human health and wellness.

The Impact of Centrifugal Force on Isolation of Bone Marrow Mononuclear Cells Using Density Gradient Centrifugation.

BACKGROUND: Bone marrow mononuclear cells (BMMNCs) have great potential in bone regenerative therapy. The main method used today to obtain BMMNCs is Ficoll density gradient centrifugation. However, the centrifugal force for this isolation method is still suboptimal. OBJECTIVES: To determine the optimal centrifugal force in Ficoll density gradient centrifugation of bone marrow (BM) to achieve high stem/progenitor cell content BMMNCs for regenerative therapy. METHODS: BM was aspirated from nine minipigs and divided into three groups according to different centrifugal forces (200 g, 300 g and 400 g). Immediately after BMMNCs were obtained from each group by Ficoll density gradient centrifugation, residual red blood cell (RBC) level, nucleated cell counting, viability and flow cytometric analyses of apoptosis and reactive oxygen species (ROS) generation were measured. The phenotypic CD90 and colony formation analyses of BMMNCs of each group were performed as well. Bone marrow-derived mesenchymal stem cells (BMSCs) were harvested at passage 2, then morphology, cell phenotype, proliferation, adipogenic, chondrogenic and osteogenic lineage differentiation potential of BMSCs from each group were compared. RESULTS: The 300 g centrifugal force was able to isolate BMMNCs from BM with the same efficiency as 400 g and provided significantly higher yields of CD90+ BMSCs and fibroblastic colony-forming units of BMSC (CFU-f(BMSC)), which is more crucial for the regenerative efficacy of BMMNCs. Meanwhile, 200 g hosted the most RBC contamination and minimum CFU-f (BMSC) yield, which will be disadvantageous for BMMNC-based cell therapy. As for in vitro cultured BMSCs which were isolated from BMMNCs by different centrifugal forces, no significant differences were found on morphology, cell proliferation rate, phenotypic marker, adipogenic, chondrogenic and osteogenic differentiation potential. CONCLUSIONS: 300 g may be the optimal centrifugal force when using Ficoll density gradient centrifugation to isolate BMMNCs for bone regenerative therapy. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Proliferation and Differentiation Potential of Bone Marrow-Derived Mesenchymal Stem Cells From Children With Polydactyly and Adults With Basal Joint Arthritis.

This study compared the proliferation and differentiation potential of bone marrow-derived mesenchymal stem cells (BMSCs) derived from infants with polydactyly and adults with basal joint arthritis. The proliferation rate of adult and infant BMSCs was determined by the cell number changes and doubling times. The γH2AX immunofluorescence staining, age-related gene expression, senescence-associated ß-galactosidase (SA-ß-gal) staining were analyzed to determine the senescence state of adult and infant BMSCs. The expression levels of superoxide dismutases (SODs) and genes associated with various types of differentiation were measured using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR). Differentiation levels were evaluated through histochemical and immunohistochemical staining. The results showed that infant BMSCs had a significantly higher increase in cell numbers and faster doubling times compared with adult BMSCs. Infant BMSCs at late stages exhibited reduced γH2AX expression and SA-ß-gal staining, indicating lower levels of senescence. The expression levels of senescence-related genes (p16, p21, and p53) in infant BMSCs were also lower than in adult BMSCs. In addition, infant BMSCs demonstrated higher antioxidative ability with elevated expression of SOD1, SOD2, and SOD3 compared with adult BMSCs. In terms of differentiation potential, infant BMSCs outperformed adult BMSCs in chondrogenesis, as indicated by higher expression levels of chondrogenic genes (SOX9, COL2, and COL10) and positive immunohistochemical staining. Moreover, differentiated cells derived from infant BMSCs exhibited significantly higher expression levels of osteogenic, tenogenic, hepatogenic, and neurogenic genes compared with those derived from adult BMSCs. Histochemical and immunofluorescence staining confirmed these findings. However, adult BMSCs showed lower adipogenic differentiation potential compared with infant BMSCs. Overall, infant BMSCs demonstrated superior characteristics, including higher proliferation rates, enhanced antioxidative activity, and greater differentiation potential into various lineages. They also exhibited reduced cellular senescence. These findings, within the context of cellular differentiation, suggest potential implications for the use of allogeneic BMSC transplantation, emphasizing the need for further in vivo investigation.

Ultrasound-Targeted Microbubble Destruction Increases BBB Permeability and Promotes Stem Cell-Induced Regeneration of Stroke by Downregulating MMP8.

The objective of this study was to evaluate the feasibility, safety, and effectiveness of intravenous stem cell delivery utilizing ultrasound-targeted microbubble destruction (UTMD) in a rat model of middle cerebral artery occlusion (MCAO), while investigating the underlying mechanisms. Acute cerebral infarction (ACI) was induced surgically in adult rats to create the MCAO rat model. Intravenous injection of SonoVue microbubbles and bone marrow-derived mesenchymal stem cells (BMSC) was performed concurrently, with or without ultrasound targeting the stroke. The animals were divided into four groups: sham-operated group, ACI-MCAO rats treated with phosphate-buffered saline (ACI+PBS), rats receiving intravenous delivery of BMSC expressing green fluorescent protein (GFP-BMSC; ACI+BMSC), and rats receiving intravenous GFP-BMSC with simultaneous UTMD exposure (ACI+BMSC+UTMD). The efficacy of the treatments was assessed by evaluating the animals' neurological function using the Longa score and examining histopathological changes such as cerebral infarct volume, cerebral edema, and cell apoptosis. A rat cytokine array was utilized to identify the potential cytokines that may be responsible for the therapeutic effect of UTMD-mediated BMSC treatment. Optimal UTMD parameters resulted in an increase in blood-brain barrier (BBB) permeability after 30 min, which returned to baseline 72 h later without causing any residual injury. UTMD application significantly increased the homing of intravenously delivered BMSC, resulting in a 2.2-fold increase in GFP-BMSC cell count on day 3 and a 2.6-fold increase on day 7 compared with intravenous delivery alone. This effect persisted for up to 6 weeks after injection. Intravenous BMSC delivery significantly reduced the volume of cerebral infarct and decreased cerebral edema, leading to a lower Longa score. Furthermore, this effect was further enhanced by UTMD. Acute cerebral infarction induced by MCAO led to elevated matrix metalloproteinase 8 (MMP8) levels in the cerebrospinal fluid, which were significantly reduced following UTMD-mediated BMSC treatment. Ultrasound-targeted microbubble destruction facilitates the migration and homing of BMSC into the brain, possibly by transiently increasing blood-brain barrier (BBB) permeability, thereby improving therapeutic outcomes in an ACI rat model. The observed effect may be partly attributed to modulation of MMP8 levels.Advances in knowledge: UTMD-mediated intravenously delivered BMSC transplantation led to a significant increase in cell homing and reduction of MMP8 levels, resulting in increased therapeutic effect in an acute ischemic cerebral infarction model.

An Advanced Bioconjugation Strategy for Covalent Tethering of TGFß3 with Silk Fibroin Matrices and its Implications in the Chondrogenesis Profile of Human BMSCs and Human Chondrocytes: A Paradigm Shift in Cartilage Tissue Engineering.

The transforming growth factor-ß class of cytokines plays a significant role in articular cartilage formation from mesenchymal condensation to chondrogenic differentiation. However, their exogenous addition to the chondrogenic media makes the protocol expensive. It reduces the bioavailability of the cytokine to the cells owing to their burst release. The present study demonstrates an advanced bioconjugation strategy to conjugate transforming growth factor-ß3 (TGFß3) with silk fibroin matrix covalently via a cyanuric chloride coupling reaction. The tethering and change in secondary conformation are confirmed using various spectroscopic analyses. To assess the functionality of the chemically modified silk matrix, human bone marrow-derived mesenchymal stem cells (hBMSCs) and chondrocytes are cultured for 28 days in a chondrogenic differentiation medium. Gene expression and histological analysis reveal enhanced expression of chondrogenic markers with intense Safranin-O and Alcian Blue staining in TGFß3 conjugated silk matrices than where TGFß3 is exogenously added to the media for both hBMSCs and chondrocytes. Therefore, this study successfully recapitulates the native niche of TGFß3 and the role of the silk as a growth factor stabilizer. When cultured over TGFß3 conjugated silk matrices, hBMSCs display increased proteoglycan secretion and maximum chondrogenic trait with attenuation of chondrocyte hypertrophy over human chondrocytes.

Morinda officinalis polysaccharide promotes the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells via microRNA-210-3p/scavenger receptor class A member 3.

Morinda officinalis polysaccharide (MOP) is the bioactive ingredient extracted from the root of Morinda officinalis, and Morinda officinalis is applied to treat osteoporosis (OP). The purpose of this study was to determine the role of MOP on human bone marrow mesenchymal stem cells (hBMSCs) and the underlying mechanism. HBMSCs were isolated from bone marrow samples of patients with OP and treated with MOP. Quantitative real-time polymerase chain reaction was adopted to quantify the expression of microRNA-210-3p (miR-210-3p) and scavenger receptor class A member 3 (SCARA3) mRNA. Cell Counting Kit-8 assay was employed to detect cell viability; Terminal-deoxynucleotidyl Transferase Mediated Nick End Labeling assay and flow cytometry were adopted to detect apoptosis; Alkaline Phosphatase (ALP) activity assay kit was applied to detect ALP activity; Western blot was executed to quantify the expression levels of SCARA3, osteogenic and adipogenic differentiation markers. Ovariectomized rats were treated with MOP. Bone mineral density (BMD), serum tartrate-resistant acid phosphatase 5b (TRACP 5b), and N-telopeptide of type I collagen (NTx) levels were assessed by BMD detector and Enzyme-linked immunosorbent assay kits. It was revealed that MOP could promote hBMSCs' viability and osteogenic differentiation and inhibit apoptosis and adipogenic differentiation. MOP could also upregulate SCARA3 expression through repressing miR-210-3p expression. Treatment with MOP increased the BMD and decreased the TRACP 5b and NTx levels in ovariectomized rats. MOP may boost the osteogenic differentiation and inhibit adipogenic differentiation of hBMSCs by miR-210-3p/SCARA3 axis.