Effects of recombinant human interferon β1a on the chondrogenic differentiation of human bone marrow mesenchymal stem cell / 生物工程学报

Recombinant human interferon beta (rhIFN-β) is a glycoprotein produced by genetically engineered cells and has anti-virus, anti-tumor and immunoregulation functions. Although studies have shown that other subtypes of IFN such as IFN-γ affects cell proliferation and differentiation to some extent, the effect of rhIFN-β on chondrogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) is less known. In this study we studied the effect of rhIFN-β on the chondrogenic differentiation of hMSCs by inducing hMSCs into cartilage pellet via adding IFN-β1a into regular TGF-β3 chondrogenic differentiation medium. We collected the induced pellets and then detected GAG content, assessed pellets size, observed agreecan using alcian blue staining, and analyzed the expression of Sox and CollangenⅡusing real-time PCR and Western blotting. Addition of 100 ng/mL IFN-β1a to regular TGF-β3 chondrogenic differentiation medium could improve the concentration of GAG, increase the size of pellets, promote the formation of aggrecan and up-regulate the expression of CollangenII and Sox9. IFN-β1a combined with TGF-β3 could promote chondrogenic differentiation of hMSCs.

Platelet Rich Plasma and Culture Configuration Affect the Matrix Forming Phenotype of Bone Marrow Stromal Cells

We aim to examine the influence of platelet rich plasma (PRP) and spatial cues in cartilage/bone matrix forming proteins, and to evaluate the mitotic and chemotactic potential of PRP on human mesenchymal stem cells (hMSCs). Directed cell migration towards PRP gradients was assessed in chemotactic chambers, and recorded by time-lapse microscopy. hMSCs cultured in three-dimensional (3D) scaffolds were visualized by scanning electron microscopy; Hoechst dye was used to confirm cell confluence in 3D-constructs and monolayers before experimental treatment. MSCs were treated with 10% PRP lysate or 10% PRP lysate supplemented with TGF-β-based differentiation medium. The expression of cartilage (COL2A1, Sox9, ACAN, COMP), and bone (COL1A1, VEGF, COL10A1, Runx2) fundamental genes was assessed by real time PCR in monolayers and 3D-constructs. PRP had mitotic (p <.001), and chemotactic effect on hMSCs, Ralyleigh test p = 1.02E - 10. Two and three-week exposure of MSCs to PRP secretome in 3Dconstructs or monolayers decreased Sox9 expression (p <0.001 and p = 0.050) and COL2A1, (p = 0.011 and p = 0.019). MSCs in monolayers exposed to PRP showed increased ACAN (p = 0.050) and COMP (p <0.001). Adding TGF-β-based differentiation medium to PRP increased COMP, and COL2A1 expression at gene and protein level, but merely in 3D-constructs, p <0.001. TGF-β addition to monolayers reduced Sox9 (p <0.001), aggrecan (p = 0.004), and VEGF (p = 0.004). Cells exposed to PRP showed no changes in hypertrophy associated genes in either monolayers or 3Dconstructs. Our study suggests hMSCs have high-degree of plasticity having the potential to change their matrix-forming phenotype when exposed to PRP and according to spatial configuration.

Expression regulation and functional analysis of RGS2 and RGS4 in adipogenic and osteogenic differentiation of human mesenchymal stem cells

BACKGROUND: Understanding the molecular basis underlying the formation of bone-forming osteocytes and lipid-storing adipocytes will help provide insights into the cause of disorders originating in stem/progenitor cells and develop therapeutic treatments for bone- or adipose-related diseases. In this study, the role of RGS2 and RGS4, two members of the regulators of G protein signaling (RGS) family, was investigated during adipogenenic and osteogenenic differentiation of human mesenchymal stem cells (hMSCs). RESULTS: Expression of RGS2 and RGS4 were found to be inversely regulated during adipogenesis induced by dexamethasone (DEX) and 3-isobutyl-methylxanthine, regardless if insulin was present, with RGS2 up-regulated and RGS4 down-regulated in response to adipogenic induction. RGS2 expression was also up-regulated during osteogenesis at a level similar to that induced by treatment of DEX alone, a shared component of adipogenic and osteogenic differentiation inducing media, but significantly lower than the level induced by adipogenic inducing media. RGS4 expression was down-regulated during the first 48 h of osteogenesis but up-regulated afterwards, in both cases at levels similar to that induced by DEX alone. Expression knock-down using small interfering RNA against RGS2 resulted in decreased differentiation efficiency during both adipogenesis and osteogenesis. On the other hand, expression knock-down of RGS4 also resulted in decreased adipogenic differentiation but increased osteogenic differentiation. CONCLUSIONS: RGS2 and RGS4 are differentially regulated during adipogenic and osteogenic differentiation of hMSCs. In addition, both RGS2 and RGS4 play positive roles during adipogenesis but opposing roles during osteogenesis, with RGS2 as a positive regulator and RGS4 as a negative regulator. These results imply that members of RGS proteins may play multifaceted roles during human adipogenesis and osteogenesis to balance or counterbalance each other's function during those processes.

Extracorporeal shockwaves induce osteogenic differentiation of human mesenchymal stem cells by ATP release / 医用生物力学

Objective To investigate whether extracorporeal shockwave could induce differentiation of human mesenchymal stem cells (hMSCs) into osteoprogenitor cells by ATP release and the activation of P2X7 receptors. Methods Cultured bone marrow-derived hMSCs were subjected to shockwave treatment and ATP release was assessed. Osteogenic differentiation and mineralization of hMSCs were evaluated by examining alkaline phosphatase (ALP) activity, osteocalcin (OC) production, and calcium nodule formation. The mRNA expression of P2X7 receptors was determined with real-time RT-PCR. P2X7-siRNA, apyrase, and P2 receptor antagonists were used to evaluate the roles of ATP release and P2X7 receptors in shockwave-induced osteogenic hMSCs differentiation. Results Shockwave treatment released significant amounts of ATP from hMSCs. Shockwaves and exogenous ATP induced hMSC differentiation. Removal of ATP with apyrase, targeting of P2X7 receptors with P2X7-siRNA or selective antagonists prevented osteogenic differentiation of hMSCs. Conclusions Shockwaves can contribute to osteogenic differentiation of hMSCs by realeasing cellular ATP that activate signaling. These research findings provide the theoretical basis for shockwave therapy in treating fracture healing and bone nonunion.

Effect of perfusion flow rate on proliferation and osteoblastic differentiation of human mesenchymal stem cells / 医用生物力学

Objective To investigate the effect of different perfusion flow rates on proliferation and osteoblastic differentiation of human mesenchymal stem cells (hMSCs) in large scale β-TCP (tricalcium phosphate) scaffold at perfusion bioreactor. Methods hMSCs isolated from iliac bone marrow aspiration were loaded into large scale β-TCP scaffold and cultured in perfusion bioreactor at the perfusion flow rate of 3, 6 or 9 mL/min for 15 days. The culture media were collected for D-glucose consumption assay every 3 days. After perfusion culture for 15 days, the cell-scaffold composites were harvested for assessment of cell viability by MTT colorimetric method, SEM observation and osteogenic gene expression by real-time PCR. Results The proliferation of hMSCs assayed by daily glucose consumption showed that at early stage of culture, cells proliferated faster at flow rate of 9 mL/min than at 3 or 6 mL/min (P<0.001); while at late stage of culture, cells proliferated faster at flow rate of 6 mL/min (P<0.05). The cell viability indicated that the cell-scaffold composites at flow rate of 6 mL/min exhibited the most viable cells (P<0.001). SEM indicated that all the macropores of the scaffold at different flow rates were filled with cellular layers. All cellular layers at flow rate of 3 mL/min were incompact, but that at 9 mL/min were compact; at flow rate of 6 mL/min, the cellular layers were either compact or incompact. Real-time PCR revealed that after perfusion culture for 15 days, the mRNA expression of osteobalstic genes including ALP and OP, were enhanced significantly at flow rate of 6 and 9 mL/min as compared to that at 3 mL/min (P<0.01); however, the 9 mL/min group presented the higher OC expression than 3 and 6 mL/min group (P<0.001). Conclusions At early stage of perfusion culture, the proliferation of hMSCs was promoted at flow rate of 9 mL/min, while at late stage, there was more viable cells in scaffolds at flow rate of 6 mL/min. The osteoblastic differentiation of hMSCs was facilitated with the increase of perfusion flow rate, which was attributed to the increased flow shear stress.

Action Potential of Cardiomyocyte-like Cells Differentiated from Human Marrow Mesenchymal Stem Cells in Vitro / 中国康复理论与实践

@#Objective To detect whether the cardiomyocyte-like cells differentiated from human marrow mesenchymal stem cells (hMSCs) could produce action potential (AP). Methods Isolated and cultured hMSCs were induced into cardiomyocyte-like cells with 5-Azacytine in vitro. They were measured for their AP by patch clamp technique, and compared with those of hMSCs of the same generation and beating cardiomyocytes (CMs) derived from 2 day-old SD rats. Results 6/30 cardiomyocyte-like cells produced AP. The CMs produced significant AP, hMSCs appeared no AP, and cardiomyocyte-like cells appeared weak AP. Conclusion The hMSCs manifested the potential to differentiate into CMs in the electrophysiology characteristics following 5-Azacytine induction.

Inducing Human Bone Marrow Mesenchymal Stem Cells to Cardiomyo-Like Cells with Supernatant of Normal or Injured Rat Cardiomyocytes in Vitro / 中国康复理论与实践

@# Objective To observe whether human bone marrow mesenchymal stem cells (hMSCs) could differentiate into cardiomyo-like cells by culturing with supernatant of normal or injured rat cardiomyocytes (CMs) in vitro. Methods hMSCs were cultured with supernatant from normal or injured rat CMs for 27~30 d. The morphologies of induced hMSCs were observed with inverted microscope and the special cardio-markers cTnI and Desmin were identified with immunocytochemisry. Results A few cells cultured with supernatant from normal CMs enlarged and expressed cTnI, but little Desmin. While more cells cultured with supernatant from injured CMs enlarged and expressed cTnI, and parts of them expressed Desmin. The incidence of cTnI or Desmin positive cells were significantly different between these two groups (P<0.01). Conclusion Supernatant from both normal and injured CMs can induce hMSCs into cardio-like cells in vitro, and that from injured CMs is more effectively.

Effects of the Human-Mesenchymal Stem Cells Transplantation on Spinal Cord Injury of the Rats / 대한해부학회지

Two sources of adult stem cells that have aroused great interest are human bone marrow-derived mesenchymal stem cells (hMSCs) and human umbilical cord blood cells. hMSCs have been reported to maintain their ability to differentiate into neuronal lineage cells in the central nervous system. Therefore, transplantation of hMSCs represents an attractive new form of cellular therapy for clinical application in spinal cord injury (SCI). The aim of this study was to investigate how transplanted hMSCs from the venous circulation moved into a target zone of compression injury in the spinal cord of rats, and if they ameliorated the behavioral impairments associated with SCI. SCI in rats was induced by compressing the spinal cord for 30 s with an aneurysm clip. hMSCs labeled with cholera toxin subunit B conjugated to fluorescein isothiocyanate (CTX B-FITC) were injected intravenously through the tail vein or directly on the SCI site using a 27-g needle. Suspensions of hMSCs collected from adult humans were delivered at concentrations (1x10(6)cells/200 microliter) in 1 or 5 d after experimental SCI. After transplantation of hMSCs, the SCI regions displayed some endogenous background fluorescence, but CTX B-FITC-labeled hMSCs were clearly identifiable. They were observed in injured but not in intact areas; they were usually round or slightly elongated with a prominent nucleus. Only a few hMSCs were found in the spinal cord in each case but there were more cells in the rats injected at day one than at day five. This study confirmed that these were indeed transplanted hMSCs using antisera recognizing human-specific nuclei or human-specific mitochondria. Double immunofluorescence analysis showed the production of some neuronal and glial cell markers in the SCI lesions. Behavioral test scores of SCI rats treated with hMSCs at day one were significantly better than those for rats treated at day five and for the untreated SCI group. Thus, hMSCs appear to be beneficial in reversing the behavioral effects of SCI in this rat model, even when infused one day after injury. They might be a viable source of stem cells for the treatment of human neurological disorders.

Extracorporeal Shock Wave Promotes Postnatal Human Bone Marrow Stromal Cells Osteogenesis In vivo / 生物化学与生物物理进展

It is known that extracorporeal shock wave (SW) may promote healing of fracture. A previous study reported that SW promoted human bone marrow stromal cells (hMSCs) towards osteoblasts in vitro. To study the osteogenesis ability of hMSCs treated by shock wave in porous hydroxyapatite (HA) in vivo, primary hMSCs of SW group and control group were cultured in the porous HA for 2 weeks and then implanted into subcutaneous sites of nude mouse. These implants were harvested and prepared for the biochemical analysis of alkaline phosphatase activity by AKP kit, histological analysis of decalcified and undecalcified sections and morphology by scan electric microscope (SEM), as well as osteocalcin mRNA expression by RT- PCR 4 weeks and 8 weeks after implantation. It showed that cells of SW and control group almost covered the rough surface of HA before implantation and the extracelluar matrix of SW group was abundant by SEM photomicrograph . The histological analysis and SEM photomicrograph showed active bone formation 4 weeks and 8 weeks after implantation, as well as tetracycline labeling under fluoroscopy analysis in SW group. Alkaline phosphatase in supernatants of the implants detected 4 weeks and 8 weeks after implantation in SW group was higher than in control group (P

Expression and signififcance of IGF-Ⅰ TGF-?1 in human mesenchymal stem cells after exposured to extracoporeal shock wave / 中国矫形外科杂志

[Objective]To demonstrate mechanism of ESW in curing osteogetic disorders,we studied expression of some osteogenetic factors in human mesenchymal stem cells(hMSCs)when exposed to ESW.[Method]After success in marrow aspiration,isolation and obtainment optimal experimental energy,a dose of 5kV and 100 times of ESW was applied to hMSCs of passage 1.The expression of IGF-Ⅰ and TGF-?1 were examined by immunocytochemical staining.[Result]The cytochemical staining results showed that expression of IGF-Ⅰ and TGF-?1 appeared at different passage of hMSCs after ESW intervention.Appearance of IGF-Ⅰ was earlier than TGF-?1 which didnt express until passage 7.At the same interval,the expression of IGF-Ⅰ and TGF-?1 in control group difference is lower than ESW group,respectively(P

Culture,proliferation and differentiation of human marrow mesenchymal stem cells to cardiomyocytes in vitro / 中国免疫学杂志

95% purity were treated for 24 h with 1,5,10 and 20 ?mol/L 5-azacytidine to induce cellular differentiation.Expression of the cardiac-specific marker-troponin Ⅰ and desmin,identified by immunohistochemistry,was used to identify cardiac muscle differentiation.Results:hMSCs expressed a high level of CD44(hMSCs 93.26%?2.48% vs 3.42%?1.09% in a control group,P