Differentiation Of Marrow-derived Mesenchymal Stem Cells Research Articles

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.

Mechano-responsive hydrogel for direct stem cell manufacturing to therapy.

Bone marrow-derived mesenchymal stem cell (MSC) is one of the most actively studied cell types due to its regenerative potential and immunomodulatory properties. Conventional cell expansion methods using 2D tissue culture plates and 2.5D microcarriers in bioreactors can generate large cell numbers, but they compromise stem cell potency and lack mechanical preconditioning to prepare MSC for physiological loading expected in vivo. To overcome these challenges, in this work, we describe a 3D dynamic hydrogel using magneto-stimulation for direct MSC manufacturing to therapy. With our technology, we found that dynamic mechanical stimulation (DMS) enhanced matrix-integrin β1 interactions which induced MSCs spreading and proliferation. In addition, DMS could modulate MSC biofunctions including directing MSC differentiation into specific lineages and boosting paracrine activities (e.g., growth factor secretion) through YAP nuclear localization and FAK-ERK pathway. With our magnetic hydrogel, complex procedures from MSC manufacturing to final clinical use, can be integrated into one single platform, and we believe this 'all-in-one' technology could offer a paradigm shift to existing standards in MSC therapy.

Comparative analysis of proliferative and multilineage differentiation potential of human periodontal ligament stem cells from maxillary and mandibular molars.

Clinical experience indicates that wounds in alveolar bone and periodontal tissue heal faster and more efficiently in the maxilla compared with the mandible. Since stem cells are known to have a decisive influence on wound healing and tissue regeneration, the aim of this study was to determine whether differences in proliferation and differentiation of periodontal ligament stem cells (PDLSC) from upper (u-PDLSC) and lower jaw (l-PDLSC) contribute to the enhanced wound healing in the maxilla. u-PDLSC and l-PDLSC from the same donor were harvested from the periodontal ligament of extracted human maxillary and mandibular third molars. Cell differentiation potential was assessed by analyzing stem cell markers, proliferation rate, and multilineage differentiation among each other and bone marrow-derived mesenchymal stem cells (MSC). Successful differentiation of PDLSC and MSC toward osteoblasts, adipocytes, and chondrocytes was analyzed via reverse transcriptase-quantitative polymerase chain reaction and histochemical staining (Alizarin Red, Oil Red O, Toluidine Blue). u-PDLSC and l-PDLSC expressed the MSC-markers CD73+ , CD90+ , and CD105+ and lacked expression of CD34- and CD45- . Proliferation was significantly higher in u-PDLSC than in l-PDLSC, regardless of the culture conditions. Osteogenic (ALP, RunX2, and osteocalcin) and chondrogenic (SOX9 and ACAN) related gene expression as well as staining intensities were significantly higher in u-PDLSC than in l-PDLSC. No difference in adipogenic differentiation was observed. u-PDLSC showed a significantly higher proliferative and differentiation potential than l-PDLSC, offering a possible cell-based explanation for the differences in periodontal wound healing efficacy between maxilla and mandible.

Phosphate promotes osteogenic differentiation through non-canonical Wnt signaling pathway in human mesenchymal stem cells.

Phosphate is indispensable in osteogenesis and mineralization. However, mechanisms by which phosphate enhances osteogenic differentiation are not fully understood. In this study, we studied the effect of phosphate on osteogenic differentiation as well as signaling pathways induced by phosphate in the process. Induced human bone marrow-derived mesenchymal stem cells differentiation into osteoblasts by the change of media containing β-glycerophosphate (GP), 1mM inorganic phosphate, or 3mM inorganic phosphate (Pi). The differentiation of osteoblasts was verified by the expression of osteoblast differentiation markers and calcium deposition. RNA sequencing was performed to assess transcriptome in the early stage of osteogenic differentiation. Osteogenic differentiation and mineralization were promoted in the 3mM Pi group compared to those in the GP and 1mM Pi groups on day 7 of culture. RNA sequencing revealed that the gene expressions involved in osteogenesis and the components in the Wnt signaling pathway was increased in 3mM Pi group compared with those in the GP on day 7. Analysis with qPCR and Western blot suggested upregulation of components in the non-canonical Wnt signaling pathway, including WNT5b and phosphorylated-c-Jun in the 3mM Pi group on day 7. WNT11 mRNA expression was increased in the 2 induction groups on day 7. Inhibition of WNT5b by siRNA experiment attenuated the components in non-canonical Wnt signaling expression, including WNT5b, WNT11 and ROR2 mRNA expression and phosphorylated-c-Jun protein expression. In addition, osteogenic differentiation and mineralization were partly decreased in 3mM Pi group on day 7 by the inhibition of WNT5b. Pi promoted osteogenic differentiation through the up-regulation of the non-canonical Wnt signaling pathway, including WNT5b, WNT11, p-c-Jun/c-Jun, in the early stage of differentiation. These findings provide a new perspective into the association of Pi and the non-canonical Wnt signaling pathway during osteogenic differentiation.

Silymarin Effects on Ovine Fetal Bone Marrow-Derived Mesenchymal Stem Cells Differentiation into Osteogenic Lineage

هدف: سلول درمانی با استفاده از سلول­های بنیادی مزانشیمی (MSCs) می تواند ابزاری امیدوار کننده به عنوان طب بازساختی باشد. یکی از غنی ترین منابع سلول های بنیادی مزانشیمی مغز استخوان جنین است. سیلیمارین دارای فعالیت های آنتی اکسیدانی و ضد التهابی قوی با تأثیر مثبت بر تکثیر برخی از سلول ها و همچنین خاصیت ضد پوکی استخوان است. این مطالعه با هدف نشان دادن اثر سیلیمارین بر تمایز سلول های بنیادی مزانشیمی مشتق از مغز استخوان جنین گوسفند به رده استئوژنیک انجام شد. مواد و روش­ها: سلول­های بنیادی مزانشیمی از مغز استخوان جنین گوسفند جدا شدند. آزمون MTT جهت بررسی سمیت سلولی سیلیمارین روی سلول­ها درغلظت ­های مختلف در زمان­های 24 و 72 ساعت انجام شد. سپس، سلول‌ها در یکی از 8 گروه 1: شاهد منفی؛ 2: تیمار شده با 10 میکرومول بر لیتر سیلیمارین در محیط معمول، 3: تیمار شده با 20 میکرومول بر لیتر سیلیمارین در محیط معمول، 4: تیمار شده با 100 میکرومول بر لیتر استرادیول در محیط معمول، 5: شاهد مثبت، 6: تیمار شده با 10 میکرومول بر لیتر سیلیمارین در محیط تمایزی، 7: تیمار شده با 20 میکرومول بر لیتر سیلیمارین در محیط تمایزی، 8: تیمار شده با 100 میکرومول بر لیتر در محیط تمایزی، به‌مدت 21 روز کشت شدند.جهت تعیین تمایز استئوژنیک سلول­ها، با استفاده از رنگ آمیزی آلیزارین رد رسوب یون هیدروکسی آپاتیت بررسی شد و همچنین میزان ترشح آنزیم ALP در گروه های مورد مطالعه اندازه گیری شد. نتایج: مقایسه میانگین جذب نوری سلول­ها در غلظت­های مختلف بین زمان­های 24 و 72 ساعت پس از تیمار نشان داد که میانگین جذب نوری سلول­ها در غلظت صفر سیلیمارین، 72 ساعت پس از تیمار نسبت به زمان 24 ساعت پس از تیمار کاهش نشان داد(05/0P<) اما در سایر غلظت­ها تفاوت معنی دار مشاهده نشد(05/0P>). بررسی میزان ترشح آنزیم ALP ، 21 روز پس از تیمار در گروه­های مورد مطالعه نشان داد که در مجموع بیشترین میزان ترشح آنزیم در گروه 8 بود (05/0P≤). کمترین میزان ترشح آنزیم در گروه 1 (کنترل منفی) و پس از آن به ترتیب در گروه­ 2 و گروه 3 مشاهد شد(05/0P<). بین گروه­های 4، 5 و 6 تفاوت معنی دار مشاهده نشد(P>0.05). بر اساس نتایج حاصل از رنگ آمیزی آلیزارین رد، رسوب املاح کلسیم در تمام گروه­های مربوط به محیط کشت تمایزی مشاهده شد که به ترتیب در گروه­های 8، 7، 6 و 5 افزایش یافت. در گروه­های کشت شده در محیط معمول، در گروه 1 رسوبی مشاهده نشد و میزان رسوب به ترتیب در گروه­های 2، 3 و 4 افزایش نشان داد. در مجموع میزان رسوب در گروه­های محیط تمایزی بیشتر از محیط معمول بود. نتیجه گیری: سیلیمارین در غلظت­های مورد بررسی روی سلولهای بنیادی مزانشیمی مشتق از مغز استخوان جنین گوسفند، اثر سمیت ندارد و همچنین در غلظت­های مورد بررسی باعث افزایش تمایز سلول­ها به رده ی استخوانساز می شود که این افزایش وابسته به غلظت است. از این رو، به نظر می رسد با مطالعات بیشتر و شناسایی مسیرهای مولکولی اثر گذاری سیلیمارین، می­توان از آن در سلول درمانی جهت ترمیم ضایعات استخوانی استفاده کرد.

Silymarin Effects on Ovine Fetal Bone Marrow-Derived Mesenchymal Stem Cells Differentiation into Osteogenic Lineage

Silymarin Effects on Ovine Fetal Bone Marrow-Derived Mesenchymal Stem Cells Differentiation into Osteogenic Lineage

Anti-adipogenic peptides from ark shell protein hydrolysate: Purification, identification and anti-adipogenic effect

Ark shell protein hydrolysates-derived peptides showing anti-adipogenic effect were purified and identified using consecutive chromatographic techniques and tandem mass spectrometry. Two anti-adipogenic peptides LLRLTDL (843.02 Da) and GYALPCDCL (954.13 Da) were found to be highly effective for lipid accumulation inhibition in mouse bone marrow-derived mesenchymal stem cells differentiation into adipocyte. The inhibitory activity of LLRLTDL and GYALPCDCL for lipid accumulation was 48.53 % and 46.22 % at 100 μM, respectively. During adipocyte differentiation, two anti-adipogenic peptides LLRLTDL and GYALPCDCL significantly inhibited intracellular reactive oxygen species (ROS) generation by 47.39 % and 49.77 % at 100 μM. In addition, LLRLTDL and GYALPCDCL treatment increased cellular antioxidant enzyme activities, including superoxide dismutase, catalase, and glutathione peroxidase. Moreover, LLRLTDL and GYALPCDCL suppressed proinflammatory cytokine productions, including tumor necrosis factor-α, interleukin-1β and -6. These results suggest that LLRLTDL and GYALPCDCL could modulate adipocyte differentiation and be useful as anti-obesity ingredients or functional foods.

The inhibitory effects of vancomycin on rat bone marrow-derived mesenchymal stem cell differentiation.

The use of intrawound vancomycin powder in spine surgery has been shown to decrease the rate of surgical site infections; however, the optimal dose is unknown. High-dose vancomycin inhibits osteoblast proliferation in vitro and may decrease the rate of solid arthrodesis. Bone marrow-derived mesenchymal stem cells (BMSCs) are multipotent cells that are a source of osteogenesis in spine fusions. The purpose of this study was to determine the effects of vancomycin on rat BMSC viability and differentiation in vitro. BMSCs were isolated from the femurs of immature female rats, cultured, and then split into two equal groups; half were treated to stimulate osteoblastic differentiation and half were not. Osteogenesis was stimulated by the addition of 50 µg/mL l-ascorbic acid, 10 mM β-glycerol phosphate, and 0.1 µM dexamethasone. Vancomycin was added to cell culture medium at concentrations of 0, 0.04, 0.4, or 4 mg/mL. Early differentiation was determined by alkaline phosphatase activity (4 days posttreatment) and late differentiation by alizarin red staining for mineralization (9 days posttreatment). Cell viability was determined at both the early and late time points by measurement of formazan colorimetric product. Viability within the first 4 days decreased with high-dose vancomycin treatment, with cells receiving 4 mg/mL vancomycin having 40%-60% viability compared to the control. A gradual decrease in alizarin red staining and nodule formation was observed with increasing vancomycin doses. In the presence of the osteogenic factors, vancomycin did not have deleterious effects on alkaline phosphatase activity, whereas a trend toward reduced activity was seen in the absence of osteogenic factors when compared to osteogenically treated cells. Vancomycin reduced BMSC viability and impaired late osteogenic differentiation with high-dose treatment. Therefore, the inhibitory effects of high-dose vancomycin on spinal fusion may result from both reduced BMSC viability and some impairment of osteogenic differentiation.

Sol-Gel Synthesis, in vitro Behavior, and Human Bone Marrow-Derived Mesenchymal Stem Cell Differentiation and Proliferation of Bioactive Glass 58S

Background:Bioactive glasses 58S, are silicate-based materials containing calcium and phosphate, which dissolved in body fluid and bond to the bone tissue. This type of bioactive glass is highly biocompatible and has a wide range of clinical applications. Methods:The 58S glass powders were synthesized via sol-gel methods, using tetraethyl orthosilicate, triethyl phosphate, and calcium nitrate, as precursors. Upon the analyses of phase and chemical structures of bioactive glass in different gelation times (12, 48, and 100 h), the appropriate heat treatment (at 525, 575, and 625 °C) was performed to eliminate nitrate compounds and stabilize the glass powder samples. The in vitro assay in SBF solution revealed the bioactivity of the synthesized 58S glass through the morphological (SEM), chemical structure (FTIR), release of calcium, phosphorous and silicon elements, pH variations, and weight loss measurements. The behavior of MSCs in the presence of bioactive glass powders was studied by MTT cytotoxicity, cell staining, ALP activity and biomineralization tests, as well as by the evaluation of ALP, osteocalcin, osteonectin, collagen I, and RUNX2 gene expression. Results: The results confirmed a gelation time of 100 h and a calcination temperature of 575 °C at optimal conditions for the synthesis of nitrate-free bioactive glass powders. Conclusion:The glass spherical nanoparticles in the range of 20-30 nm possess the improved bioactivity and osteogenic properties as demanded for bone tissue engineering.

Sol-Gel Synthesis, in vitro Behavior, and Human Bone Marrow-Derived Mesenchymal Stem Cell Differentiation and Proliferation of Bioactive Glass 58S.

Bioactive glasses 58S, are silicate-based materials containing calcium and phosphate, which dissolved in body fluid and bond to the bone tissue. This type of bioactive glass is highly biocompatible and has a wide range of clinical applications. The 58S glass powders were synthesized via sol-gel methods, using tetraethyl orthosilicate, triethyl phosphate, and calcium nitrate, as precursors. Upon the analyses of phase and chemical structures of bioactive glass in different gelation times (12, 48, and 100 h), the appropriate heat treatment (at 525, 575, and 625 °C) was performed to eliminate nitrate compounds and stabilize the glass powder samples. The in vitro assay in SBF solution revealed the bioactivity of the synthesized 58S glass through the morphological (SEM), chemical structure (FTIR), release of calcium, phosphorous and silicon elements, pH variations, and weight loss measurements. The behavior of MSCs in the presence of bioactive glass powders was studied by MTT cytotoxicity, cell staining, ALP activity and biomineralization tests, as well as by the evaluation of ALP, osteocalcin, osteonectin, collagen I, and RUNX2 gene expression. The results confirmed a gelation time of 100 h and a calcination temperature of 575 °C at optimal conditions for the synthesis of nitrate-free bioactive glass powders. The glass spherical nanoparticles in the range of 20-30 nm possess the improved bioactivity and osteogenic properties as demanded for bone tissue engineering.

Biomaterials for Regenerative Medicine

Biomaterials for Regenerative Medicine

Osteogenic and Adipogenic Differentiation of Mesenchymal Stem Cells in Gelatin Solutions of Different Viscosities.

Accumulating evidence indicates that stem cell fate can be regulated by mechanical properties of the extracellular matrix. Most studies have focused onthe influence of matrix elasticity and viscoelasticity on stem cell differentiation. However, how matrix viscosity affects stem cell differentiation has been overlooked. In this study, a biphasic gelatin solution/hydrogel system is used for 3D culture of human bone marrow-derived mesenchymal stem cells (MSCs) to investigate the influence of gelatin solution viscosity on simultaneous osteogenic and adipogenic differentiation at the same culture condition. Gelatin solution promotes cell proliferation, while its promotive effect decreases with the increase of viscosity. The influence of viscosity on osteogenic and adipogenic differentiation of MSCs shows opposite trends. A high-viscosity gelatin solution results in an increase of alkaline phosphatase (ALP) activity, calcium deposition, and expression of osteogenesis-related genes. On the other hand, in a low-viscosity gelatin solution, a lot of lipid vacuoles are formed and adipogenesis-related genes are highly expressed. The results indicate high viscosity is beneficial for osteogenic differentiation, while low viscosity is beneficial for adipogenic differentiation. These findings suggest the importance of matrix viscosity on stem cell differentiation in 3D microenvironments.

ECM scaffolds mimicking extracellular matrices of endochondral ossification for the regulation of mesenchymal stem cell differentiation

Endochondral ossification (ECO) is an important process of bone tissue development. During ECO, extracellular matrices (ECMs) are essential factors to control cell functions and induce bone regeneration. However, the exact role of ECO ECMs on stem cell differentiation remains elusive. In this study, ECM scaffolds were prepared to mimic the ECO-related ECM microenvironments and their effects on stem cell differentiation were compared. Four types of ECM scaffolds mimicking the ECMs of stem cells (SC), chondrogenic (CH), hypertrophic (HY) and osteogenic (OS) stages were prepared by controlling differentiation of human bone marrow-derived mesenchymal stem cells (MSCs) at different stages. Composition of the ECM scaffolds was dependent on the differentiation stage of MSCs. They showed different influence on osteogenic differentiation of MSCs. HY ECM scaffold had the most promotive effect on osteogenic differentiation of MSCs. CH ECM and OS ECM scaffolds showed moderate effect, while SC ECM scaffold had the lowest effect on osteogenic differentiation of MSCs. Their effects on chondrogenic or adipogenic differentiation were not significantly different. The results suggested that the engineered HY ECM scaffold had superior effect for osteogenic differentiation of MSCs. Statement of significance ECM scaffolds mimicking endochondral ossification-related ECM microenvironments are pivotal for elucidation of their roles in regulation of stem cell functions and bone tissue regeneration. This study offers a method to prepare ECM scaffolds that mimic the ECMs from cells at hypertrophic, osteogenic, chondrogenic and stem cell stages. Their composition and impacts on osteogenic differentiation of MSCs were compared. The hypertrophic ECM scaffold had the highest promotive effect on osteogenic differentiation of MSCs. The results advance our understanding about the role of ECO ECMs in regulation of stem cell functions and provide perspective for bone defect repair strategies.

Effects of WNT5A signaling-mediated inflammation of macrophages upon cartilage homeostasis

Purpose: To construct and comparehumanbone marrow-derived mesenchymal stem cells (MSCs) two dimension (2D)and bone marrow aspirates in vitrtranswell separated co-cultural models with recombinant adeno-associated virus (rAAV)-Wnt5a-modified activated macrophages and investigate the effect of Wnt5a signaling upon cartilage homeostasis through the regulation of macrophage pro-inflammatory responses. Methods: Macrophages, MSCs,and bone marrow aspirates specimens were extracted from 6 patients with severe knee deformities undergoing total knee arthroplasty (2 males, 4 females, 58-71 years old). The synovial tissues of knee joints were exposed to type II collagenase and obtained single cell suspensions, and the purity of macrophages was determined by Ficoll gradient centrifugation and anti-CD14 antibody flow cytometry. The macrophages were transduced with rAAV-lacZ or Wnt5a respectively for 48 hours prior to the stimulation of IFN-γ combined TNF-α for 24 hours, and subsequently transferred to the upper compartment of the 96-well transwell plate, with hMSCs 2D monolayer, and bone marrow aspirates at the lower compartment, respectively (Figure 1 & 2). Cell morphology and proliferation (cellularity), viral transfection efficiency and secretion of proinflammatory cytokine were estimated by Haemotoxylin and Eosin (H&E) staining, toluidine blue staining, X-gal staining, anti-Wnt5a, anti-II and X collagen immunohistochemical staining and enzyme-linked immunosorbent assay (ELISA) assay, respectively. Results: The isolated synovial macrophages were positive against anti-CD14 primary antibody, with a percentage of 90.31%, indicating that the cells were macrophages. The level of monocyte chemotactic protein-1 in supernatants was significantly increased after stimulation with IFN-γ and TNF-α, indicating that the macrophages were activated and at proinflammatory condition (Figure 3). After transduction with rAAV-lacZ for 3 days, X-gal staining indicated that lacZ gene transfer could efficiently transducethe activated macrophages, with the efficiency over 97.50% for at least 21 days. rAAV-Wnt5a-modified macrophages enhanced the intrinsic expression of Wnt5a, stimulated the extrinsic expression of Wnt5a in the above two models and inhibited the secretion of MCP-1. The expression of MCP-1 in Wnt5a group was 14.76 and 61.51 pg/ml, respectively (F = 6.78 and F = 4.67, p<0.05 or 0.01). In addition, rAAV-Wnt5a gene transfer could promote cell proliferation (F = 42.15 and F = 99.38, p<0.05 or 0.01) and chondrogenic differentiation and cartilage matrix synthesis,with a statistical significance in toluidine blue (F = 5.05, p<0.05 or 0.01) and immunohistochemical staining against type II and type X collagen (F = 20.65 and 26.47, p<0.05 or 0.01). In the 2D single-layer transwell co-culture model, the cell density of the Wnt5a group was 37.75 / μm2, with 2.40, 2.19 and 2.16 times comparing the remaining 3 groups respectively (Figure 4), while the cell proliferation capacity of the bone marrow aspirates transwell co-culture model in Wnt5a group was significantly enhanced to 99.28/ μm2, with 1.59, 1.73 and 1.91 times comparing the remaining 3 groups respectively. Moreover, Wnt5a transfection also promotes the expression of type II and Type X collagen in the bone marrow aspirates transwell co-culture model, with semi-quantitative analysis of 93.50 and 61.50, respectively, with 1.45, 1.56, 1.41 and 3.24, 3.08, 2.80 times comparing to lacZ and lacZ + KN93 and Wnt5a + KN93 group respectively (Figure 5). Conclusions: Under the condition of macrophage and MSCs 2D monolayer or aspirates transwell co-culture, rAAV-mediated overexpression of Wnt5a can promote the maintenance of cartilage homeostasis and chondrogenic differentiation of MSCs via macrophage inflammatory response,macrophages may affect cartilage homeostasis and MSCs chondrogenesis through Wnt5a signaling pathway.Fig. 2View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Differentiation-inducing factor-1 accelerates bone marrow-derived mesenchymal stem cells differentiation into adipocyte

Differentiation-inducing factor-1 accelerates bone marrow-derived mesenchymal stem cells differentiation into adipocyte

Effects of macrophage with Wnt5a over-expression on inflammation and chondrogenic differentiation in co-culture system

To construct and compare macrophage by gene modification of Wnt5a which co-cultured by human bone marrow-derived mesenchymal stem cells (MSCs) with two dimension (2D) and bone marrow aspirates in vitr transwell, in order to investigate the effect of Wnt5a signaling on cartilage homeostasis through regulation of macrophage pro-inflammatory responses. Macrophages, MSCs and bone marrow aspirates specimens were extracted from 6 patients with severe knee deformities undergoing total knee arthroplasty(2 males, 4 females, aged from 58 to 71 years old) from September 2015 to December 2018. The synovial tissues of knee joints were exposed to type II collagenase and obtained single cell suspensions, and the purity of macrophages was determined by Ficoll gradient centrifugation and anti-CD14 antibody flow cytometry. The macrophages were transduced with IFN-γ combined with TNF-α for 48 h, and rAAV-lacZ or Wnt5a transfected for 24 h, then co-cultured model by human bone marrow-derived mesenchymal stem cells (MSCs) with two dimension (2D) and bone marrow aspirates in vitro transwell. HE staining, toluidine blue staining, X-gal staining and anti-Wnt5a, anti-II, X collagen immunohistochemical staining and enzyme-linked immunosorbent assay were applied to detect cell morphology and proliferation (cellularity), viral transfection efficiency respectively. Results of anti-CD68 immunohistochemistry showed macrophage of patients with osteoarthritis increased obviously. Anti-CD 14 flow cytometry confirmed that percentage of isolated synovial macrophages was 90.31%. The level of monocyte chemotactic protein-1 in supernatants was significantly increased after stimulation with IFN-γ and TNF-α, indicating that the macrophages were activated and at proinflammatory condition. After transduction with rAAV-lacZ for 3 days, X-gal staining indicated that lacZ gene transfer could efficiently transduce the activated macrophages with the efficiency over 97.50% for at least 21 days. After transfection macrophages by rAAV-Wnt5a stimulated expression of Wnt5a, and enhanced expression of Wnt5a between two models and inhibited the secretion of MCP-1. The expression of MCP-1 in Wnt5a group was 14.76 and 61.51 pg/ml respectively. In addition, rAAV-Wnt5a gene transfer could promote cell proliferation, chondrogenic differentiation and cartilage matrix synthesis. Under the condition of macrophage and MSCs 2D monolayer or aspirates transwe ll co-culture, rAAV-mediated over-expression of Wnt5a could promote maintenance of cartilage homeostasis and chondrogenic differentiation of MSCs via macrophage inflammatory response, macrophages may affect cartilage homeostasis and MSCs chondrogenesis through Wnt5a signaling pathway.

Synthesis of Novel Farnesoid X Receptor Agonists and Validation of Their Efficacy in Activating Differentiation of Mouse Bone Marrow-Derived Mesenchymal Stem Cells into Osteoblasts.

The modulators of farnesoid X receptor (FXR), a bile acid receptor, regulate various biological processes including bile acid metabolism, and are associated with the control of fatty liver and osteoporosis. Thus, the control of FXR activity and development of FXR modulators are critical not only for research, but also for clinical application. In this study, we synthesized novel FXR agonists 1–4 possessing isoxazole and N-substituted benzimidazole moieties, and compared their effects on osteoblast differentiation with the known FXR agonists, chenodeoxycholic acid and a synthetic compound, GW4064. Two (3 and 4) of the four novel FXR agonists 1–4 showed high specificities for FXR. Computer-assisted modeling suggested that the binding of the FXR agonist 3 with ligand binding domain of FXR was similar to GW4064. FXR was expressed in mouse bone marrow-derived mesenchymal stem cell (MSC)-like ST2 cells (ST-2 MSCs). The FXR agonists activated the BMP-2-induced differentiation of ST-2 MSCs into osteoblasts and enhanced the expression of RUNX2. Moreover, the potency of the FXR agonist 3 was comparable to GW4064 in promoting osteoblast differentiation of ST-2 MSCs. These results indicate that FXR activation enhanced the BMP-2-induced differentiation of MSCs into osteoblasts through activating RUNX2 expression. FXR could be a potential therapeutic target for the treatment of bone diseases such as osteoporosis.

Effects of various antimicrobial agents on multi-directional differentiation potential of bone marrow-derived mesenchymal stem cells.

Antimicrobial drugs of several classes play an important role in the treatment of bone and joint infections. In addition to fighting pathogenic microorganisms, the effects of drugs on local tissues and cells are also related to the course and prognosis of bone and joint infections. The multi-directional differentiation potential of bone marrow-derived mesenchymal stem cells (MSCs) is essential for tissue repair after local injury, which is directly related to the recovery of bone, cartilage, and medullary adipose tissue. Our previous studies and the literature indicate that certain antimicrobial agents can regulate the differentiation potential of bone marrow-derived MSCs. Here, in order to systematically analyze the effects of various antimicrobial drugs on local tissue regeneration, we comprehensively review the studies on the effects of these drugs on MSC differentiation, and classify them according to the three differentiation directions (osteogenesis, chondrogenesis, and adipogenesis). Our review demonstrates the specific effects of different antimicrobial agents on bone marrow-derived MSCs and the range of concentrations at which they work, and provides a basis for drug selection at different sites of infection.

Effect of GSK126 on the chondrogenic differentiation of mesenchymal stem cells

Objective To investigate the effect of enhancer of Zeste homolog (EZH2) inhibitor GSK126 on the chondrogenic differentiation of the bone marrow-derived mesenchymal stem cells (BMSCs). Methods BMSCs were isolated by ficoll density-gradient centrifugation. The cells at passage 3 were used in this study. The optimal concentration of GSK126 was confirmed by methyl thiazol tetrazolium (MTT). On reaching confluence, cells were digested, suspended and then centrifuged at a density of 5×106/ml to form pellets. The pellets were cultured for 3 weeks with CM supplemented with GSK126 or not. The expression of type Ⅱ collagen and glycosaminoglycan (GAG) was detected by immunohistochemistry and toluidine blue staining, respectively. Real-time quantitative polymerase chain reaction (Real-time PCR) was performed to detect the content of type Ⅱ collagen, sex determining region Y box 9 (SOX9) and aggrecan mRNA, and the amount of H3K27me3 protein was assayed by Western blotting. Results 5 μmol/L GSK126 was regarded as the optimal concentration since the cytotoxic effect has not been observed. The cell volumn in the experiment group was larger than that in the control group, with lower cell density. The histological staining showed that type Ⅱ collagen and GAG were synthesized and secreted in all the pellets with stronger deposit in the experiment group. The absorbance (A) of experiment group and control group was 0.368 5±0.040 5 and 0.132 2±0.021 1 respectively. Real-time PCR showed that the mRNA expression of type Ⅱ collagen and aggrecan in the experiment group was significantly higher than that in the control group. High level of H3K27me3 protein was noted in the control group, with the A value 0.6742±0.056 2. Treatment with 10 μmol/L GSK126 obviously decreased the amount of H3K27me3, with the A value of 0.326 5±0.030 2. Conclusion GSK126 obviously promoted the chondrogenic differentiation of BMSCs by demethylation of H3K27me3. Key words: GSK126; H3K27me3; Chondrogenic differentiation; Bone mesenchymal stem cells

Transcription factor Krüppel-like factor 4 inhibits the differentiation of bone marrow-derived mesenchymal stem cells to nucleus pulposus-like cells

Transcription factor Krüppel-like factor 4 inhibits the differentiation of bone marrow-derived mesenchymal stem cells to nucleus pulposus-like cells