Research progress in quality research and evaluation of human mesenchymal stem cells / 国际生物医学工程杂志

As adult stem cells, human mesenchymal stem cells (hMSCs) have the potential for self-replication, renewal, and multidirection differentiation. Their unique biological function determines their wide clinical indications. Researchers can define the quality attributes of hMSCs according to clinical expectations. The quality study of hMSCs should consider microbiological safety, biological safety, cell biological properties, and biological effectiveness. Quality evaluation is a common physical, chemical, and biological evaluation method for hMSCs. Traditional product safety evaluation strategies cannot fully adapt to current technology and product usage characteristics. Researchers have developed new, effective evaluation methods based on current technology. In terms of product efficacy evaluation strategies, an efficacy evaluation system has been gradually established and standardized according to the intended clinical use and based on quality studies, which can enable researchers to evaluate hMSCs products more comprehensively at different stages and processes. In this paper, the progress of quality research and evaluation of human mesenchymal stem cells was reviewed to provide a reference for the utilization of stem cells in the field of regenerative medicine.

Recovery of motor function in rats with complete spinal cord injury following implantation of collagen/silk fibroin scaffold combined with human umbilical cord-mesenchymal stem cells

SUMMARY OBJECTIVE: This study aimed to assess the effect of the collagen/silk fibroin scaffolds seeded with human umbilical cord-mesenchymal stem cells on functional recovery after acute complete spinal cord injury. METHODS: The fibroin and collagen were mixed (mass ratio, 3:7), and the composite scaffolds were produced. Forty rats were randomly divided into the Sham group (without spinal cord injury), spinal cord injury group (spinal cord transection without any implantation), collagen/silk fibroin scaffolds group (spinal cord transection with implantation of the collagen/silk fibroin scaffolds), and collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group (spinal cord transection with the implantation of the collagen/silk fibroin scaffolds co-cultured with human umbilical cord-mesenchymal stem cells). Motor evoked potential, Basso-Beattie-Bresnahan scale, modified Bielschowsky's silver staining, and immunofluorescence staining were performed. RESULTS: The BBB scores in the collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group were significantly higher than those in the spinal cord injury and collagen/silk fibroin scaffolds groups (p<0.05 or p<0.01). The amplitude and latency were markedly improved in the collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group compared with the spinal cord injury and collagen/silk fibroin scaffolds groups (p<0.05 or p<0.01). Meanwhile, compared to the spinal cord injury and collagen/silk fibroin scaffolds groups, more neurofilament positive nerve fiber ensheathed by myelin basic protein positive structure at the injury site were observed in the collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group (p<0.01, p<0.05). The results of Bielschowsky's silver staining indicated more nerve fibers was observed at the lesion site in the collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group compared with the spinal cord injury and collagen/silk fibroin scaffolds groups (p<0.01, p< 0.05). CONCLUSION: The results demonstrated that the transplantation of human umbilical cord-mesenchymal stem cells on a collagen/silk fibroin scaffolds could promote nerve regeneration, and recovery of neurological function after acute spinal cord injury.

DNA methylation of HDAC4 gene affects the trans-differentiation process of hMSCs into sweat gland like cells / 中国应用生理学杂志

OBJECTIVE@#To investigate the changesof DNA methylation in histone deacetylases 4 gene (HDAC4) and its effectduring the trans-differentiation process of human mesenchymal stem cells (hMSCs) into sweat gland like cells (SGLCs).@*METHODS@#Selected cell lines of human mesenchymal stem cells (hMSCs) were cultured and expended , the third generation ofhMSCs and heat-shocked sweat gland cells were picked up, and were co-culturedwith adding inducible factor in the transwell chamber. The sweat gland like cells (SGLCs)in experiment group and the hMSCs in control group were collected, the changes of DNA methylation degree of CpG dinucleotide sitesin histone deacetylases 4 gene (HDAC4) promotor were detected by methylation specific PCR (MSP)andMaldi-TOF Mass Array. And then, the hMSCs in experiment group were treated with 5-aza-CdR (5-aza-2-deoxycytidine, 10 μmol/L), while the hMSCsin control group were culturedwith PBS at the same time. ThemRNA expressions of HDAC4 gene and carcino-embryonic antigen (CEA)gene in the two groups were measured by RT-PCR.@*RESULTS@#The methylation of HDAC4gene in hMSCs was in high level before induction, the methylation degreeof CpG dinucleotide sites located in cg2463009 was 0.901, and the methylation degree of HDAC4gene in SGLCs was markedly decreased by 37% after induction, which was 0.531. The methylationlevel of CpG dinucleotide sites located in cg14823429was changed from 0.687to 0.386 after induction. The mRNA expression of HDAC4 gene was upregulated in test group after treated with 5-aza-CdR for 48 hours, the mRNA expression of CEA gene related with transdifferentiation was enhanced too at the same term, there was significantly statistic difference compared with control group (<0.05).@*CONCLUSIONS@#Methylation of HDAC4 gene participates in the regulation of the trans-differentiation of hMSCs into sweet gland like cells.

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.

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.

Long Term Exposure to Myrtucommulone-A Changes CD105 Expression and Differentiation Potential of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) represent a heterogeneous group of multipotent stem cells that could be found in various somatic tissues. MSCs are defined by molecular and functional features including spindle-shape morphology, adherence to plastic surfaces, expression of specific surface markers and differentiation potential to chondrocytes, adipocytes and osteocytes. The surface markers were proposed to affect the differentiation potential of MSCs by a limited number of studies. Endoglin (CD105) is defined to be a significant marker for osteogenic and chondrogenic differentiation ability of MSCs. Low CD105 expression is associated with increased osteogenic potential while high CD105 expression is correlated with strong chondrogenic potential. Myrtucommulone-A (MC-A) is an active compound with various biological effects on different cell types but its effect on MSC differentiation has not been described yet. In the present study we aimed at investigating the longterm effects of MC-A on hMSCs. MC-A-treatment reduced CD105 expression in distinct human mesenchymal stem cell (hMSC) lines and gave rise to CD105(low) population but did not change CD44, CD90 or CD73 expression. The decrease in CD105 expression reduced the chondrogenic potential of hMSCs subsequently while adipogenic or osteogenic differentiation was not affected dramatically. MC-A-treatment also suppressed the NF-κB p65 activation which might be responsible for the reduced chondrogenic potential. Our findings suggest thatMC-Acould be used to enrichCD105(low)hMSCs without the need for cell sorting or changing culture conditions which could be utilised in targeted differentiation studies.

Low-Dose Ionizing γ-Radiation Promotes Proliferation of Human Mesenchymal Stem Cells and Maintains Their Stem Cell Characteristics

Mesenchymal stem cells (MSCs), which are multipotent and have self-renewal ability, support the regeneration of damaged normal tissue. A number of external stimuli promote migration of MSCs into peripheral blood and support their participation inwound healing. In an attempt to harness the potential beneficial effects of such external stimuli, we exposed human MSCs (hMSCs) to one such stimulus-low-dose ionizing radiation (LDIR)-and examined their biological properties. To this end, we evaluated differences in proliferation, cell cycle, DNA damage, expression of surface markers (CD29, CD34, CD90, and CD105), and differentiation potential ofhMSCs before and after irradiation with γ-rays generated using a ¹³⁷ CSirradiator.At doses less than 50 mGy, LDIR had no significant effect on the viability or apoptosis of hMSCs. Interestingly, 10 mGyofLDIR increased hMSC viability by 8% (p<0.001) comparedwith non-irradiatedhMSCs.At doses less than 50 mGy, LDIR did not induceDNA damage, including DNA strand breaks, or cause cellular senescence or cell-cycle arrest. Surface marker expression and in vitro differentiation potential of hMSCs were maintained after two exposures to LDIR at 10 mGy per dose. In conclusion, a two-dose exposure to LDIR at 10 mGy per dose not only facilitates proliferation of hMSCs, it alsomaintains the stem cell characteristics of hMSCswithout affecting their viability.These results provide evidence for the potential ofLDIRas an external stimulus for in vitro expansion of hMSCs and application in tissue engineering and regenerative medicine.

The effect of biomechanical stimulation on osteoblast differentiation of human jaw periosteum-derived stem cells / 대한악안면성형재건외과학회지

BACKGROUND: This study was to investigate the effect of biomechanical stimulation on osteoblast differentiation of human periosteal-derived stem cell using the newly developed bioreactor. METHODS: Human periosteal-derived stem cells were harvested from the mandible during the extraction of an impacted third molar. Using the new bioreactor, 4% cyclic equibiaxial tension force (0.5 Hz) was applied for 2 and 8 h on the stem cells and cultured for 3, 7, and 14 days on the osteogenic medium. Biochemical changes of the osteoblasts after the biomechanical stimulation were investigated. No treatment group was referred to as control group. RESULTS: Alkaline phosphatase (ALP) activity and ALP messenger RNA (mRNA) expression level were higher in the strain group than those in the control group. The osteocalcin and osteonectin mRNA expressions were higher in the strain group compared to those in the control group on days 7 and 14. The vascular endothelial growth factor (VEGF) mRNA expression was higher in the strain group in comparison to that in the control group. Concentration of alizarin red S corresponding to calcium content was higher in the strain group than in the control group. CONCLUSIONS: The study suggests that cyclic tension force could influence the osteoblast differentiation of periosteal-derived stem cells under optimal stimulation condition and the force could be applicable for tissue engineering.

Comparative Evaluation for Potential Differentiation of Endothelial Progenitor Cells and Mesenchymal Stem Cells into Endothelial-Like Cells

Understanding the mechanisms of vascular remodeling could lead to more effective treatments for ischemic conditions. We aimed to compare between the abilities of both human Wharton jelly derived mesenchymal stem cells (hMSCs) and human cord blood endothelial progenitor cells (hEPCs) and CD34+ to induce angiogenesis in vitro. hMSCs, hEPCs, and CD34+ were isolated from human umbilical cord blood using microbead (MiniMacs). The cells characterization was assessed by flow cytometry following culture and real-time PCR for vascular endothelial growth factor receptor 2 (VEGFR2) and von Willebrand factor (vWF) to prove stem cells differentiation. The study revealed successful isolation of hEPCs, CD34+, and hMSCs. The hMSCs were identified by gaining CD29+ and CD44+ using FACS analysis. The hEPCs were identified by having CD133+, CD34+, and KDR. The potential ability of hEPCs and CD34+ to differentiate into endothelial-like cells was more than hMSCs. This finding was assessed morphologically in culture and by higher significant VEGFR2 and vWF genes expression (p<0.05) in differentiated hEPCs and CD34+ compared to differentiated hMSCs. hEPCs and CD34+ differentiation into endothelial-like cells were much better than that of hMSCs.

miR-708-5p accelerates migration of human mesenchymal stem cells by repressing TMEM88 expression / 中国病理生理杂志

AIM: To investigate the effect of microRNA-708-5p (miR-708-5p) on the migration of human mesenchymal stem cells (hMSCs).METHODS:The expression of miR-708-5p was determined by miRNA arrays and re-al-time PCR.By transfection of miR-708-5p mimic or inhibitor, the up-regulation or down-regulation of miR-708-5p ex-pression in hMSCs was evaluated .The cell scratch and Transwell tests were used to detect the migration capability of hM-SCs.The effects of transmembrane protein 88 (TMEM88), a miR-708-5p target gene, onβ-catenin expression and migra-tion of hMSCs were detected .RESULTS:The expression of miR-708-5p was down-regulated in the old hMSCs compared with the young hMSCs.Up-regulation of miR-708-5p resulted in increasing migration of hMSCs.Conversely, down-regula-tion of miR-708-5p resulted in decreasing cell migration .The expression of TMEM88 was up-regulated in the old hMSCs compared with the young hMSCs , while the expression of β-catenin was down-regulated.Directly repression of TMEM88 expression increased the β-catenin expression and migration of hMSCs .The regulation of miR-708-5p on hMSCs was atten-uated by inhibiting the expression of miR-708-5p and TMEM88 together.CONCLUSION:miR-708-5p increases β-cate-nin expression and Wnt/β-catenin activity by repressing TMEM 88, thus enhancing the migration of hMSCs .

miR-486-5p enhances senescence of human mesenchymal stem cells by de-creasing telomerase activity / 中国病理生理杂志

AIM:To investigate the effects of microRNA-486-5p (miR-486-5p) on the senescence of human mesenchymal stem cells ( hMSCs).METHODS: The expression of miR-486-5p was determined by miRNA arrays and real-time PCR.By transfection of miR-486-5p mimic or inhibitor, up-regulation or down-regulation of miR-486-5p expres-sion in hMSCs was established.The effect of miR-486-5p and silence information regulator 1 (SIRT1) on hMSC telomerase activity and senescence were detected byβ-galactosidase staining.RESULTS:The expression of miR-486-5p was up-regu-lated in the old hMSCs compared with the young hMSCs.Up-regulation of miR-486-5p resulted in increasing senescence of hMSCs.Conversely, down-regulation of miR-486-5p resulted in decreasing cell senescence.The expression of SIRT1 and telomerase reverse transcriptase ( TERT) was down-regulated in the old hMSCs compared with the young hMSCs.Directly repression of SIRT1 expression inhibited the hMSC TERT protein expression and telomerase activity, but increased cell se-nescence.The regulation of miR-486-5p on hMSC senescence was attenuated by inhibiting the expression of miR-486-5p and SIRT1 together.CONCLUSION:miR-486-5p enhances senescence of hMSCs by decreasing the expression of SIRT1 and telomerase activity.

Galectin-9 is Involved in Immunosuppression Mediated by Human Bone Marrow-derived Clonal Mesenchymal Stem Cells

Bone marrow-derived mesenchymal stem cells (MSCs) have immunomodulatory properties and can suppress exaggerated pro-inflammatory immune responses. Although the exact mechanisms remain unclear, a variety of soluble factors are known to contribute to MSC-mediated immunosuppression. However, functional redundancy in the immunosuppressive properties of MSCs indicates that other uncharacterized factors could be involved. Galectin-9, a member of the beta-galactoside binding galectin family, has emerged as an important regulator of innate and adaptive immunity. We examined whether galectin-9 contributes to MSC-mediated immunosuppression. Galectin-9 was strongly induced and secreted from human MSCs upon stimulation with pro-inflammatory cytokines. An in vitro immunosuppression assay using a knockdown approach revealed that galectin-9-deficient MSCs do not exert immunosuppressive activity. We also provided evidence that galectin-9 may contribute to MSC-mediated immunosuppression by binding to its receptor, TIM-3, expressed on activated lymphocytes, leading to apoptotic cell death of activated lymphocytes. Taken together, our findings demonstrate that galectin-9 is involved in MSC-mediated immunosuppression and represents a potential therapeutic factor for the treatment of inflammatory diseases.

A Cannabinoid Receptor Agonist N-Arachidonoyl Dopamine Inhibits Adipocyte Differentiation in Human Mesenchymal Stem Cells

Endocannabinoids can affect multiple cellular targets, such as cannabinoid (CB) receptors, transient receptor potential cation channel, subfamily V, member 1 (TRPV1) and peroxisome proliferator-activated receptor gamma (PPARgamma). The stimuli to induce adipocyte differentiation in hBM-MSCs increase the gene transcription of the CB1 receptor, TRPV1 and PPARgamma. In this study, the effects of three endocannabinoids, N-arachidonoyl ethanolamine (AEA), N-arachidonoyl dopamine (NADA) and 2-arachidonoyl glycerol (2-AG), on adipogenesis in hBM-MSCs were evaluated. The adipocyte differentiation was promoted by AEA whereas inhibited by NADA. No change was observed by the treatment of non-cytotoxic concentrations of 2-AG. The difference between AEA and NADA in the regulation of adipogenesis is associated with their effects on PPARgamma transactivation. AEA can directly activate PPARgamma. The effect of AEA on PPARgamma in hBM-MSCs may prevail over that on the CB1 receptor mediated signal transduction, giving rise to the AEA-induced promotion of adipogenesis. In contrast, NADA had no effect on the PPARgamma activity in the PPARgamma transactivation assay. The inhibitory effect of NADA on adipogenesis in hBM-MSCs was reversed not by capsazepine, a TRPV1 antagonist, but by rimonabant, a CB1 antagonist/inverse agonist. Rimonabant by itself promoted adipogenesis in hBM-MSCs, which may be interpreted as the result of the inverse agonism of the CB1 receptor. This result suggests that the constantly active CB1 receptor may contribute to suppress the adipocyte differentiation of hBM-MSCs. Therefore, the selective CB1 agonists that are unable to affect cellular PPARgamma activity inhibit adipogenesis in hBM-MSCs.

Caracterización fenotípica de células madre mesenquimales humanas de médula ósea y tejido adiposo. Resultados preliminares / Phenotypic characterization of mesenchymal human stem cell from bone marrow and adipose tissue. preliminary results

Introducción:las células madre mesenquimales (CMM) poseen características fenotípicas y funcionales que les confieren un amplio potencial terapéutico por su posible uso en la terapia celular regenerativa, en el rechazo del trasplante alogénico y en enfermedades inflamatorias crónicas. Objetivo: evaluar la expresión de moléculas de membrana que permiten identificar la expresión de patrones moleculares característicos de CMM humanas mantenidas en cultivo. Métodos: se estudió la expresión fenotípica de células mononucleares procedentes de médula ósea obtenidas mediante aspiración medular, separadas por gradiente de Ficoll y cultivadas ex vivo entre los pases o subcultivos 3 y 16; y adipocitos cultivados procedentes de la extracción enzimática de tejido adiposo de donantes sanos. Se realizó doble marcaje para las moléculas CD34/CD45, CD34/CD90, CD34/CD117, y CD34/CD44. Resultados: en los resultados preliminares obtenidos se observó que las células cultivadas procedentes de médula ósea, entre los pases 4 y 8 de cultivo expresaron 45,13 por ciento de células CD34-/CD45- (doblemente negativas), lo que correspondió con el 25,24 por ciento de células CD34-/CD90+ y el 96,90 por ciento de CD34-/CD117-. En las células procedentes de cultivo de adipocitos se observó el 52,3 por ciento de CD34-/CD45- (doblemente negativas),12,31 por ciento de CD34-/CD90+,43,31 por ciento de CD34-/CD117- y 64,68 por ciento de CD34-/CD44+.Estos resultados sugieren que ambos cultivos se diferenciaron a CMM. Las CMM procedentes de adipocitos mostraron el 64,68 por ciento de células con expresión de la molécula de adhesión CD44 a la que se atribuyen propiedades funcionalescomo el asentamiento tisular. Conclusiones: estos resultados preliminares permiten corroborar que ambos métodos experimentales de cultivo son efectivos para la obtención de CMM con fines terapéuticos

Introduction:mesenchymal stem cells (MSCs) have phenotypic and functional characteristics whichgives them a broad therapeutic potential for possible use in regenerative cell therapy, allogeneic transplant rejection and chronic inflammatory diseases. Objective: to evaluate the expression of moleculemembranes expression to identify molecular patterns characteristic of human MSCs maintained in culture. Methods:the phenotypic expression of mononuclear cells from bone marrow wereobtained by bone marrow aspiration, separated by Ficoll and cultured ex vivo between passages or subcultures 3 and 16 and adipocytes cultured obtained from enzyme extraction of adipose tissue of healthy donor. Double staining was performed for molecules CD34/CD45, CD34/CD90, CD34/CD117 and CD34/CD44. Results:preliminary results showed that cultured mononuclear cells from bone marrow between passage 4 and 8 of culture expressed 45,13 percent CD34-/CD45- cells (double-negative), corresponding to 25,24 percent CD34-/CD90+ cells and 96,90 percent of CD34-/CD117-. Adipocytes from culture cells showed 52,3 percent CD34-/CD45- (double-negative), 12,31 percent cells CD34-/CD90+, 43,31 percent CD34-/CD117- (double-negative). Our results suggest that both cultures were differentiated to MSCs. Adipocytes from MSCs showed 64,68 percent of cells with expression of CD44 adhesion molecule conferring functional homing properties Conclusions:these preliminary results corroborate that the experimental methods used in cultivation are effective for obtaining MSCs with therapeutic purposes

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.

MicroRNA-378*enhances apoptosis of human mesenchymal stem cells by repressing expression of CTGF / 中国病理生理杂志

[ ABSTRACT] AIM:To investigate the effects of microRNA-378*( miR-378*) on the survival and apoptosis of human mesenchymal stem cells ( hMSCs ) .METHODS: The expression of miR-378* was determined by microRNA arrays and quantitative real-time PCR ( qRT-PCR) .H2 O2 was used to induce hMSCs apoptosis.By transfection of miR-378*mimic or inhibitor, we up-regulated or down-regulated miR-378* expression in hMSCs.The effect of miR-378*and connective tissue growth factor ( CTGF) on hMSC survival and apoptosis were detected by MTT, LDH, caspase-3/7 and TUNEL assays.RESULTS:The expression of miR-378*was up-regulated in the old hMSCs compared with the young hMSCs.H2 O2 increased the expression of miR-378*, decreased the expression of CTGF.Up-regulation of miR-378*re-sulted in increasing apoptosis and decreasing survival of hMSCs.Conversely, down-regulation of miR-378*resulted in de-creasing cell apoptosis and increasing survival.The regulation of miR-378*on hMSC apoptosis and survival was attenuated by inhibiting the expression of miR-378* and CTGF together.Direct repression of CTGF expression inhibited the hMSC survival and increased apoptosis.CONCLUSION:miR-378*enhances apoptosis of hMSCs by repressing the expression of CTGF.

Survival of hypoxic human mesenchymal stem cells is enhanced by a positive feedback loop involving miR-210 and hypoxia-inducible factor 1

The use of mesenchymal stem cells (MSCs) has emerged as a potential new treatment for myocardial infarction. However, the poor viability of MSCs after transplantation critically limits the efficacy of this new strategy. The expression of microRNA-210 (miR-210) is induced by hypoxia and is important for cell survival under hypoxic conditions. Hypoxia increases the levels of hypoxia inducible factor-1 (HIF-1) protein and miR-210 in human MSCs (hMSCs). miR-210 positively regulates HIF-1alpha activity. Furthermore, miR-210 expression is also induced by hypoxia through the regulation of HIF-1alpha. To investigate the effect of miR-210 on hMSC survival under hypoxic conditions, survival rates along with signaling related to cell survival were evaluated in hMSCs over-expressing miR-210 or ones that lacked HIF-1alpha expression. Elevated miR-210 expression increased survival rates along with Akt and ERK activity in hMSCs with hypoxia. These data demonstrated that a positive feedback loop involving miR-210 and HIF-1alpha was important for MSC survival under hypoxic conditions.

Bone regeneration of mouse critical-sized calvarial defects with human mesenchymal stem cells in scaffold / 한국실험동물학회지

Combination of tissue engineering and cell therapy represents a promising approach for bone regeneration. Human mesenchymal stem cells (hMSCs) have properties that include low immunogenicity, high proliferation rate, and multi-differentiation potential; therefore, they are an attractive seeding source for tissue engineering therapy. Here we found that hMSCs with a scaffold did not affect cell viability and osteogenic differentiation. We also investigated regenerative effect of hMSCs with the scaffold in a calvarial bone defect model. Formation of new bone was evaluated by micro-CT, histology and expression of osteogenic markers. The results clearly showed interesting evidence indicating that hMSCs with scaffold increased the formation of new bone and expression of osteogenic markers, compared to the empty and scaffold only groups. Overall, our results suggest that hMSCs with scaffold are suitable for stimulation of intense bone regeneration in critical-sized bone defects.

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.

Preparation of a novel porous scaffold from poly(lacticco-glycolic acid)/hydroxyapatite.

Background: Scaffolds for bone tissue engineering must meet functional requirements, porosity, biocompatibility, and biodegradability. Different polymeric scaffolds have been designed to satisfy these properties. Composite materials could improve mechanical properties compared with polymers, and structural integrity and flexibility compared with brittle ceramics. Objective: Fabricate poly (lactic-co-glycolic acid) (PLGA) /hydroxyapatite (HA) porous scaffolds by freezeextraction method, and evaluate the possibility for optimizing their biocompatibility by changing their HA content. Methods: Porous PLGA/HA composites structure were prepared by freezing a polymer solution, and then the solvent was extracted by a non-solvent and subsequently air-dried. The scaffolds were coated with triblock copolymer and sterilized by ultraviolet light. Human mesenchymal stem cells were cultured on the prepared scaffolds and were studied after three days by 4, 6-diamidino-2-phenylindole (DAPI) fluorescence microscopy. Results: Microstructural studies with SEM showed the formation of about 50 micrometer size porous structure and interconnected porosity so that cells were adhered well into the structure of the coated samples. DAPI fluorescence microscopy showed more cell adhesion to the coated scaffolds and cell diffusion into the pores are visible. Direct assay of cell proliferation performed with MTT test showed cell growing on the scaffold similar to or more than on control samples. Conclusion: The triblock-coated PLGA/HA porous scaffolds may provide cell adhesion and proliferation, demonstrating their potential application in bone engineering.