Effect of Human Bone Marrow Mesenchymal Stem Cells with Ectopic High OCT4 Expression on T Lymphocyte Function / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of human bone marrow mesenchymal stem cells (MSCs) with ectopic high OCT4 expression on T-cell proliferation, activation and secretion in vitro.@*METHODS@#Peripheral blood mononuclear cells were isolated from healthy children. Anti-CD3 and anti-CD28 monoclonal antibodies were used to activate T lymphocytes, which were stimulated by interleukin (IL)-2 for one week in vitro. Then MSCs with ectopic high OCT4 expression (MSC-OCT4) were co-cultured with activated T lymphocytes. After one week of co-culture, the supernatant was collected and the levels of Th1/Th2 cytokines [IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α and interferon (IFN)-γ] were determined by flow cytometry. The lymphocytes after one week of co-culture were collected and counted by Countstar software. After the proportions of activated/inactivated T cell subsets were determined by flow cytometry, the absolute lymphocyte counts were calculated and expressed as mean ± standard deviation.@*RESULTS@#Compared with control T cell alone culture group, the proliferation of CD3+ T cells, CD3+CD4+ T cells, and CD3+CD8+ T cells were significantly inhibited in MSC group and MSC-OCT4 group. Compared with MSC, MSC-OCT4 could inhibit CD3+CD8+ T cell proliferation better (P =0.049), and mainly inhibited early T cell activation. Compared with control T cell alone culture group, the levels of IL-2 and INF-γ were significantly down-regulated both in MSC group and MSC-OCT4 group.After co-culture with T cells for one week, the level of IL-6 significantly increased in MSC group and MSC-OCT4 group compared with that before co-culture. Compared with control MSC group, MSC-OCT4 group had higher viable cell numbers after 1 week of co-culture (P =0.019), and could resist the inhibition of proliferation by higher concentration of mitomycin C.@*CONCLUSION@#Both MSC and MSC-OCT4 can inhibit the proliferation and activation of IL-2-stimulated T cells in vitro. After overexpression of OCT4, MSC has better proliferation ability in vitro and can inhibit the proliferation of CD3+CD8+ T cells more effectively, which may have a better and more lasting immunosuppressive ability to regulate the balance of Th1/Th2.

Effect of Wnt/β-catenin signaling pathway in neural differentiation of human bone marrow mesenchymal stem cells / 中国修复重建外科杂志

OBJECTIVE@#To explore the effect of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and the combination of bFGF and EGF in the neural differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and the role of Wnt/β-catenin signaling pathway in this process.@*METHODS@#The identified 4th-generation hBMSCs were divided into five groups according to different induction conditions, namely control group (group A), EGF induction group (group B), bFGF induction group (group C), EGF and bFGF combined induction group (group D), and EGF, bFGF, and Dickkopf-related protein 1 (DKK-1) combined induction group (group E). After 7 days of continuous induction, the cell morphology was observed by inverted fluorescence phase contrast microscopy, levels of genes that were related to neural cells [Nestin, neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP-2), and glial fibrillary acidic protein (GFAP)] and key components of the Wnt/β-catenin signaling pathway (β-catenin and Cyclin D1) were detected by RT-PCR, and the levels of proteins that were related to neural cells (Nestin and GFAP) as well as genes that were involved in Wnt/β-catenin signaling pathway [β-catenin, phosphorylation β-catenin (P-β-catenin), Cytoplasmic β-catenin, and Nuclear β-catenin] were explored by cellular immunofluorescence staining and Western blot.@*RESULTS@#When compared to groups A and B, the typical neuro-like cell changes were observed in groups C-E, and most obviously in group D. RT-PCR showed that the relative expressions of Nestin, NSE, and MAP-2 genes in groups C-E, the relative expressions of GFAP gene in groups D and E, the relative expression of NSE gene in group B, the relative expressions of β-catenin gene in groups C and D, and the relative expressions of Cyclin D1 gene in groups B-D significantly increased when compared with group A ( P<0.05). Compared with group E, the relative expressions of Nestin, NSE, MAP-2, GFAP, β-catenin, and CyclinD1 genes significantly increased in group D ( P<0.05); compared with group C, the relative expression of Nestin gene in group D significantly decreased ( P<0.05), while NSE, MAP-2, and GFAP genes significantly increased ( P<0.05). The cellular immunofluorescence staining showed that the ratio of NSE- and GFAP-positive cells significantly increased in groups C-E than in group A, in group D than in groups C and E ( P<0.05). Western blot assay showed that the relative expression of NSE protein was significantly higher in groups C and D than in group A and in group D than in groups C and E ( P<0.05). In addition, the relative expression of GFAP protein was significantly higher in groups C-E than in group A and in group D than in group E ( P<0.05). Besides, the relative expressions of β-catenin, Cytoplasmic β-catenin, Nuclear β-catenin, and the ratio of Nuclear β-catenin to Cytoplasmic β-catenin were significantly higher in groups C and D than in group A and in group D than in group E ( P<0.05), whereas the relative expression of P-β-catenin protein was significantly lower in groups C and D than in group A and in group D than in group E ( P<0.05).@*CONCLUSION@#Different from EGF, bFGF can induce neural differentiation of hBMSCs. In addition, EGF can enhance the hBMSCs neural differentiation of bFGF, while the Wnt/β-catenin signaling pathway may play a positive regulatory role in these processes.

Exosome-derived circ_0009362 mediates osteogenic differentiation of human bone marrow mesenchymal stem cells by regulating miR-29b-3p / 中华内分泌外科杂志

Objective:To investigate the effect and potential mechanism of serum exosome-derived cirC_0009362 on the osteogenic differentiation of human bone marrow mesenchymalstem cells (hBMSCs) .Methods:Serum samples from patients with osteoporosis (OP) were collected and exosomes were isolated. The expression level of circ_0009362 in exosomes was detected by qRT-PCR. hBMSCs osteogenesis was induced and the expression of circ_0009362 and miR-29b-3p was detected. The interaction between circ_0009362 and miR-29b-3p was detected by dual luciferase reporter assay. Alkaline phosphatase (ALP) kit was used to detect ALP activity and alizarin red (ARS) staining was used to detect calcium deposition.Results:Compared with healthy control group, the expression of circ_0009362 in serum exosomes of OP patients was increased, and the expression of circ_0009362 was decreased after inducing hBMSCs osteogenesis (all P<0.05) . The ALP activity and the percentage of calcium deposition in hBMSCs were decreased by exosomes, and this effect was achieved by secreting circ_0009362. The effect of exosomes was partially offset by circ_0009362 expression in knockdown exosomes (all P<0.05) . The expression of miR-29b-3p was increased after inducing hBMSCs osteogenesis ( P<0.05) . Circ_0009362 had a targeting relationship with miR-29b-3p, and exosomes inhibited the expression of miR-29b-3p by secreting circ_0009362. The ALP activity and the percentage of calcium deposition in hBMSCs were promoted by overexpression of miR-29b-3p, which was partially offset by exosomes (all P<0.05) . Conclusion:Serum exosomes of OP patients inhibit the osteogenic differentiation of hBMSCs by secreting circ_0009362 to down-regulate the expression of miR-29b-3p.

Biocompatibility and effect on bone formation of a native acellular porcine pericardium: Results of in vitro and in vivo / 北京大学学报(医学版)

OBJECTIVE@#To examine the morphology and biocompatibility of a native acellular porcine pericardium (APP) in vitro and to evaluate its barrier function and effects on osteogenesis when used in guided bone regeneration (GBR) in vivo.@*METHODS@#First, the morphology of APP (BonanGenⓇ) was detected using a scanning electron microscope (SEM). Next, for biocompatibility test, proliferation of human bone marrow mesenchymal stem cells (hBMSCs) were determined using cell counting kit-8 (CCK-8) after being seeded 1, 3 and 7 days. Meanwhile, the cells stained with phalloidine and 4, 6-diamidino-2-phenylindole (DAPI) were observed using a confocal laser scanning microscopy (CLSM) to view the morphology of cell adhesion and pattern of cell proliferation on day 5. A 3-Beagle dog model with 18 teeth extraction sockets was used for the further research in vivo. These sites were randomly treated by 3 patterns below: filled with Bio-OssⓇand coverd by APP membrane (APP group), filled with Bio-OssⓇand covered by Bio-GideⓇmembrane (BG group) and natural healing (blank group). Micro-CT and hematoxylin-eosin (HE) were performed after 4 and 12 weeks.@*RESULTS@#A bilayer and three-dimensional porous ultrastructure was identified for APP through SEM. In vitro, APP facilitated proliferation and adhesion of hBMSCs, especially after 7 days (P < 0.05). In vivo, for the analysis of the whole socket healing, no distinct difference of new bone ratio was found between all the three groups after 4 weeks (P>0.05), however significantly more new bone regeneration was detected in APP group and BG group in comparison to blank group after 12 weeks (P < 0.05). The radio of bone formation below the membrane was significantly higher in APP group and BG group than blank group after 4 and 12 weeks (P < 0.05), however, the difference between APP group and BG group was merely significant in 12 weeks (P < 0.05). Besides, less resorption of buccal crest after 4 weeks and 12 weeks was observed in APP group of a significant difference compared in blank group (P < 0.05). The resorption in BG group was slightly lower than blank group (P>0.05).@*CONCLUSION@#APP showed considerable biocompatibility and three-dimentional structure. Performing well as a barrier membrane in the dog alveolar ridge preservation model, APP significantly promoted bone regeneration below it and reduced buccal crest resorption. On the basis of this study, APP is a potential osteoconductive and osteoinductive biomaterial.

A novel hydroxyapatite fiber scaffold for three-dimensional cell culture / 中国组织工程研究

BACKGROUND: The dense microstructure of biological scaffolds and the limitation of cell growth microenvironment are the two major difficulties in the application of biological scaffolds in bone tissue repair. OBJECTIVE: To prepare fluffy hydroxyapatite/polylactic acid composite fiber scaffold, so that cells can easily enter into the scaffold and to realize three-dimensional culture of bone marrow mesenchymal stem cells. METHODS: Fluffy hydroxyapatite/polylactic acid composite scaffold was prepared by using modified electrospinning technology combined with biomineralization. The physical and chemical properties of the fiber scaffold were measured and observed. Human bone marrow mesenchymal stem cells were inoculated on the fluffy hydroxyapatite/polylactic acid composite scaffold and traditional hydroxyapatite/polylactic acid composite scaffold. Cell proliferation, adherence and morphological changes were detected. RESULTS AND CONCLUSION: (1) The thickness of hydroxyapatite coating in the fluffy hydroxyapatite/polylactic acid composite scaffold was about 8.3 µm, most of hydroxyapatite fibers were in discrete state with a diameter of 8-14 µm. The fibers were connected by pores, and the pore diameter was (65±35) µm. The surface area, porosity and water absorption of the scaffold were significantly higher than those of the traditional scaffold (P < 0.01). (2) After 12 hours of culture, the adherence of bone marrow mesenchymal stem cells on the two scaffolds was similar, 83% and 81% cells adhered on the traditional and fluffy scaffolds, respectively. (3) After 7 days of culture, the number of proliferated cells in the fluffy hydroxyapatite/polylactic acid composite scaffold was significantly more than that in the traditional hydroxyapatite/polylactic acid composite scaffold (P < 0.01). (4) After 7 days of culture, FDA staining and scanning electric microscopy showed that cell-cell independent shape appeared in the traditional scaffold. A large number of cells appeared in the fluffy scaffold and grew into cell clusters with high cell activity, which formed a cell-fiber construction. These results indicate that this new type hydroxyapatite/polylactic acid composite scaffold is beneficial for cell entry and proliferation.

In vitro hematopoietic support of human umbilical cord blood cd34+ cells by human skeletal muscle-derived pericytes/perivascular cells / 中国组织工程研究

BACKGROUND: Perivascular cells have been shown to be the precursor cells of mesenchymal stem cells, which regulate the behavior of hematopoietic stem cells and support hematopoiesis through cell-to-cell contact or paracrine effects. Hematopoietic support of human skeletal muscle-derived pericytes/perivascular cells (hMD-PCs) remains to be studied. OBJECTIVE: To identify the biological characteristics of hMD-PCs isolated from human skeletal muscle and to study their supporting effect on umbilical cord blood CD34+ cells in vitro. METHODS: (1) hMD-PCs with phenotype CD146+ CD56-CD34-CD144-CD45- were sorted from human skeletal muscle by enzymatic digestion and multiparameter fluorescence-activated cell sorting, and their biological characteristics were identified. (2) The in vitro culture system of umbilical cord blood CD34+ cells co-cultured with human CD146+ hMD-PCs (experimental group) or with human bone marrow mesenchymal stem cells (positive control group) was established. After 1, 2 and 4 weeks of co-culture, the number of cells, the colony formation ability and immunophenotype were measured and statistically analyzed. RESULTS AND CONCLUSION: (1) CD146+ hMD-PCs were sorted by multiparameter fluorescence-activated cell sorting and the purity was (91.5±1.85)% (n=5). CD146+ hMD-PCs expressed mesenchymal surface markers CD73, CD90, CD105, CD44, and did not express hematopoietic cell and endothelial cell markers CD45, CD34, and CD31. After induced culture, CD146+ hMD-PCs could differentiate into osteoblasts, chondrogenesis, adipocytes and myoblasts. (2) There were no significant differences in the cell number, colony f ormation ability or immunophenotype (CD45+, CD34+ CD33-, CD14+, CD10+/CD19+) between experimental and positive control groups (P > 0.05, n=6). The number of cells in the blank control group without feeder was significantly decreased at 1 week of culture, and there was almost no cell survival at 2 weeks of culture. (3) In summary, CD146+ hMD-PCs, like human bone marrow mesenchymal stem cells, have hematopoietic support capacity in vitro.

Effect of micro RNA-335-5p regulating bone morphogenetic protein 2 on osteogenic differentiation of human bone marrow mesenchymal stem cells / 中国修复重建外科杂志

Objective: To investigate the effect of micro RNA (miR)-335-5p regulating bone morphogenetic protein 2 (BMP-2) on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Methods: hBMSCs were cultured in vitro and randomly divided into control group (group A), miR-335-5p mimics group (group B), miR-335-5p mimics negative control group (group C), miR-335-5p inhibitor group (group D), and miR-335-5p inhibitor negative control group (group E). After grouping treatment and induction of osteogenic differentiation, the osteogenic differentiation of cells in each group was detected by alkaline phosphatase (ALP) and alizarin red staining; the expressions of miR-335-5p and BMP-2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) mRNAs were detected by real-time fluorescence quantitative PCR analysis; the expressions of Runx2, OPN, OCN, and BMP-2 proteins were detected by Western blot. Results: Compared with group A, the relative proportion of ALP positive cells and the relative content of mineralized nodules, the relative expressions of BMP-2, miR-335-5p, OPN, OCN, Runx2 mRNAs, the relative expressions of Runx2, OPN, OCN, and BMP-2 proteins in group B were significantly increased ( P0.05). Conclusion: miR-335-5p can up-regulate BMP-2 expression and promote osteogenic differentiation of hBMSCs.

A novel hydroxyapatite fiber scaffold for three-dimensional cell culture / 中国组织工程研究

BACKGROUND: The dense microstructure of biological scaffolds and the limitation of cell growth microenvironment are the two major difficulties in the application of biological scaffolds in bone tissue repair. OBJECTIVE: To prepare fluffy hydroxyapatite/polylactic acid composite fiber scaffold, so that cells can easily enter into the scaffold and to realize three-dimensional culture of bone marrow mesenchymal stem cells. METHODS: Fluffy hydroxyapatite/polylactic acid composite scaffold was prepared by using modified electrospinning technology combined with biomineralization. The physical and chemical properties of the fiber scaffold were measured and observed. Human bone marrow mesenchymal stem cells were inoculated on the fluffy hydroxyapatite/polylactic acid composite scaffold and traditional hydroxyapatite/polylactic acid composite scaffold. Cell proliferation, adherence and morphological changes were detected. RESULTS AND CONCLUSION: (1) The thickness of hydroxyapatite coating in the fluffy hydroxyapatite/polylactic acid composite scaffold was about 8.3μ m, most of hydroxyapatite fibers were in discrete state with a diameter of 8-14μm. The fibers were connected by pores, and the pore diameter was (65±35) μm. The surface area, porosity and water absorption of the scaffold were significantly higher than those of the traditional scaffold (P<0.01).(2) After 12 hours of culture, the adherence of bone marrow mesenchymal stem cells on the two scaffolds was similar, 83% and 81% cells adhered on the traditional and fluffy scaffolds, respectively. (3) After 7 days of culture, the number of proliferated cells in the fluffy hydroxyapatite/polylactic acid composite scaffold was significantly more than that in the traditional hydroxyapatite/polylactic acid composite scaffold (P<0.01).(4) After 7 days of culture, FDA staining and scanning electric microscopy showed that cell-cell independent shape appeared in the traditional scaffold. A large number of cells appeared in the fluffy scaffold and grew into cell clusters with high cell activity, which formed a cell-fiber construction. These results indicate that this new type hydroxyapatite/polylactic acid composite scaffold is beneficial for cell entry and proliferation.

The role of mir-124-3p in inhibiting osteogenic differentiation of bone marrow mesenchymal stem cell and reducing bone quality in senile osteoporosis / 中华内分泌代谢杂志

Objective To investigate different expression levels between young and old bone marrow mesenchymal stem cells in microRNAs (miRNAs) that are significantly conserved between humans and mice.Additional studies have been conducted to discover changes in miRNA expression in old mice relative to that in young adults and discussed the roles of miRNAs in primary osteoporosis.Methods MiRNAs that are highly conserved between human and mice,and are expressed at significantly different levels in the bone marrow mesenchymal stem cells of young and old people were identified by searching the Gene Expression Omnibus (GEO) database.Human bone mesenchymal stem cells (hBMSCs) were transfected with miRNA mimics,and their relative alkaline phosphatase (ALP) activity levels were then determined.Micro-CT scanning was employed to quantitatively characterize cortical and cancellous bones of young and old mice,and to confirm that these mice accurately modeled natural aging osteoporosis.Simultaneously,we investigated differences in expression levels of miRNAs that influence ALP activity in hBMSCs in the two groups of mice.Correlations between miRNA expression levels,and parameters of bone mass and bone strength were studied.Results 28 miRNAs were found to be more than 2 fold up-regulated (down-regulated) with statistical significance (P＜0.05) in the GEO database.We also found that ALP activity was lower in hBMSCs transfected with 4 miRNAs (mir-124-3p,mir-126-3p,mir-128-3p,mir-424-5p,P＜0.05 or P＜ 0.01).The micro-CT scans indicated that the mice are accurately modeled natural aging osteoporosis.Expression of mir-124-3p increased significantly in older mice.This upregulation correlated positively with trabecular separation,and negatively with trabecular pattern factor in trabecular bone.However,in cortical bone,its expression correlated positively with trabecular separation,and negatively with bone volume fraction,trabecular number,and bone mineral density (P＜ 0.05).Conclusion Hsa-mir-124-3p,which is expressed differently in young and old bone marrow stromal cells,inhibited the osteogenic differentiation of hBMSCs.Upregulation of this miRNA in the bone tissue of aged mice may be related to the development of osteoporosis.

Biological characteristics of human bone marrow mesenchymal stem cells in the microenvironment of rabbit bladder acellular matrix graft / 西安交通大学学报(医学版)

Objective: To explore the effects of the microenvironment of rabbit bladder acellular matrix graft (BAMG) on proliferation, cell surface markers, and molecular protein level of human bone marrow mesenchymal stem cells (hBMSCs). Methods: We prepared BAMG immersion fluid medium and detected its effect on the proliferation of hBMSCs by MTT method. The expressions of CD44, CD45, CD73 and PDGFRβ were detected by flow cytometry. The expressions of PPAR, OCN and α-SMA were detected by RT-PCR, and the expression of OCT was detected by Western blot. Results: hBMSCs had good compatibility with BAMG. The MTT method showed that BAMG and BAMG immersion medium did not affect the proliferation capacity of hBMSCs. The surface of hBMSCs cells cultured with immersion fluid still expressed CD44, CD73 and PDGFRβ, but not CD45. RT-PCR showed that OCN, PPAR, and α-SMA were all expressed. Western blot test also showed the positive expression of OCT-4. Conclusion: hBMSCs can still keep their original biological characteristics in the microenvironment of rabbit BAMG. It can be the seed cells and combined substrate materials for urinary system tissue engineering.

Inhibitory Effect of Lnc-1708 on Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells in vitro / 中山大学学报(医学科学版)

@#【Abstract】 【Objective】To investigate the differential expression of long non-coding RNA-1708(lncRNA-1708)in osteogenic differentiation of human bone marrow mesenchymal stem cells(hBMSC)and its effect on osteogenic differentiation of hBMSC.【Methods】Purchased 3 groups of hBMSC from different sources and cultured in vitro. qRT-PCR was used to detect the expression of lncRNA-1708 after osteogenic differentiation of three groups of hBMSC,and the relationship between lncRNA-1708 and hBMSC osteogenic differentiation was analyzed. LncRNA-1708 overexpressing lentivirus and lncRNA-1708 interferenced plasmid were transfected respectively,to obtain stable hBMSC cell line. After 14-day osteogenic differentiation on transfected hBMSC,RT-PCR was used to detect the expressions of runt-related transcription factor 2(RUNX2)and alkaline phosphatase(ALP)mRNA,and alkaline phosphatase staining was made.【Results】The expression of lnc-1708 decreased after osteogenic differentiation of hBMSC for 7 d(P < 0. 001).Two cell lines,which respectively express high lncRNA-1708 and low lncRNA-1708,were constructed successfully. In lnc-1708-overexpressed BMSC,the mRNA levels of osteogenic markers RUNX2 and ALP were both significantly down-regulated(0.46±0.03 vs.1.00±0.02,0.15±0.07 vs. 1.02±0.28,P < 0.01). On the contrary,in the lnc-1708-silencing BMSC,the expressions of RUNX2 and ALP mRNA level were significantly up-regulated(1.62±0.18 vs. 1.00±0.04,1.58±0.11 vs. 1.01±0.18,P < 0.01).【Conclusion】LncRNA-1708 may have an inhibitory effect on osteogenic differentiation of hBMSC.

CCL3 promotes hBMSC proliferation and inhibits exosome secretion / 中国病理生理杂志

AIM:To explore the regulatory effect of chemokine CCL 3 on exosome secretion from human bone marrow mesenchymal stem cells(hBMSCs).METHODS: hBMSCs were stimulated with chemokine CCL 3 at different concentrations in vitro.The proliferation of hBMSCs was measured by CCK-8 assay and viable cell counting.Exosome se-cretion from hBMSCs was qualitatively analyzed by transmission electron microscope(TEM)and flow cytometry, and the quantitative analysis was carried out by flow cytometry and nanoparticle tracking analysis(NTA).RESULTS:Compared with control group,the viability of the hBMSCs detected by CCK-8 assay was increased when hBMSCs were treated with CCL3(P<0.05).The results of viable cell counting demonstrated that the number of hBMSCs was raised in CCL 3 group in a dose-dependent manner(P<0.05).The results of flow cytometry showed that hBMSCs expressed 3 CCL3-related spe-cific receptors,CCR1,CCR5 and CCR9.Compared with control group,the fluorescence intensity of CCR9 in CCL3 group was obviously enhanced.However,no significant difference of fluorescence intensity for CCR 5 and CCR1 was observed be-tween the 2 groups.The results of NTA demonstrated that the secretion capacity of CCL 3-induced hBMSCs was far less than that in control group(P<0.05).However, the microvesicles larger than 100 nm in CCL3 groups were increased(P<0.05).The above results indicated that the higher concentration of CCL 3 induced the lower secretion of exosomes.In addi-tion,the results of flow cytometry demonstrated that CCL 3-induced hBMSCs showed lower quantity of CD 9 +exosomes than those in control group(P<0.01).CONCLUSION:CCL3 promotes the proliferation of hBMSCs but depresses the secre-tion of exosomes in a dose-dependent manner.CCL3 affects the size distribution of exosomes and increases the number of nonfunctional microvesicles of larger than 100 nm in size.CCL3 induces the expression of CCR9 in hBMSCs.

Small peptide KR-12-a5 promotes the osteogenic differentiation of human bone marrow mesenchymalstem cells / 上海交通大学学报（医学版）

Objective: To investigate the effect of KR-12 analog, KR-12-a5, on osteogenic differentiation of human bone marrow mesenchymal stem cells (HBMSCs). Methods: Osteogenic differentiation-related experiments were performed using HBMSCs. KR-12-a5 was used as additional stimulation under osteogenesis inducing environment. By alkaline phosphatase (ALP) staining assay, ALP quantitative assay, alizarin red staining and elution quantitative analysis, and real-time quantitative polymerase chain reaction (RT-qPCR), the effect of KR-12-a5 on osteogenic differentiation of HBMSCs was verified. Results: As the concentration of KR-12-a5 increased, the staining intensity of ALP and alizarin red also increased. The strongest staining effect was exhibited at the highest concentration (50 μg/mL) of KR-12-a5. Quantitative tests showed similar results. After 3 days of stimulation with KR-12-a5, the mRNA level of RUNX2 increased in a dose-dependent manner. On the 7th day, the expression levels of ALP, COL1A1, BSP and BMP2 in the KR-12-a5-treated group showed significant changes. On the 14th day, OSX, OCN and OPN levels increased significantly. Conclusion: KR-12-a5 promoted the osteogenic differentiation of HBMSCs in a dose-dependent manner.

Angiogenic ability of 3 different tissues-derived mesenchymal stem cells on endothelial progenitor cells / 中南大学学报(医学版)

Objective:To compare the ability between bone marrow-derived mesenchymal stem cells (MSCs) (BM-MSCs) and adipose-derived MSCs (AD-MSCs) or umbilical cord-derived MSCs (UC-MSCs) on promotion of vessels formation and vessds stabilization relevant to the functions of EPCs.Methods:In vitro,co-culture blood vessel test was performed to compare the angiogenic ability between BM-MSCs,AD-MSCs or UC-MSCs.In vivo,angiogenic assay dependent on basement membrane matrix Matrigel and immunohistochemistry were performed to compare the ability of vessels formation functions between BM-MSCs and AD-MSCs or UC-MSCs.Results:The lengths and dots of vascular structures formed by EPCs on AD-MSCs layer are greater than those by EPCs on BM-MSCs layer and UC-MSCs layer in angiogenic assay in vitro.The stability of the capillary-like structures formed by EPCs with AD-MSCs on Matrigel was more stable than that by the BM-MSCs,UC-MSCs or EPCs.AD-MSCs and EPCs could form abundant functional vessels with blood perfusion in Matrigelin vivo;UC-MSCs and EPCs could form a few functional vessels with blood perfusion in Matrigelin vivo;BM-MSCs and EPCs could form broken vessels with hemocytes leakage in Matrigel in vivo.Conclusion:AD-MSCs have the stronger ability to promote the angiogenesis and stabilize the vessels compared with BM-MSCs or UC-MSCs ex vivo and in vivo.

Hypoxia promotes proliferation of bone marrow mesenchymal stem cells and maintains their potential of differentiation / 中国比较医学杂志

Objective To observe the promoting effect of hypoxia on proliferation of human bone marrow mesenchymal stem cells (BMMSCs) and maintaining their potential of multi-directional differentiation in vitro.Methods BMMSCs were isolated from bone marrow blood samples of patients with bone fracture, and cultured under hypoxic (group A) or normoxic (group B) conditions.The morphology, proliferation and osteogenic and adipogenic potential of the BMMSCs were observed.Results BMMSCs in the group A showed a long spindle shape and a fish shoal-like distribution, and were well-grown, while the morphology of cells in the group B appeared polygonal or flat.The quantity and growth rate of BMMSCs in the group A were increased compared with the group B (P< 0.05), with an osteogenic and adipogenic potential.Conclusions Hypoxia can promote the proliferation of BMMSCs in vitro and maintain their multi-directional differentiation potential.

α2-macroglobulin alleviates X-ray induced obstacle on osteogenic differen-tiation of human bone marrow mesenchymal stem cells / 中国病理生理杂志

AIM:To evaluate the effect of α2-macroglobulin(α2M) against X-ray induced obstacle on osteo-genic differentiation of human bone marrow mesenchymal stem cells(hBMMSCs). METHODS:hBMMSCs were cultured in vitro. The 4th generation of hBMMSCs was irradiated with 8 Gy X-ray,then induced osteogenic differentiation and trea-ted with different concentrations of α2M(0.5 and 1.0 g/L). The alkaline phosphatase(ALP) activity and the mRNA ex-pression of runt-related transcription factor-2 (RUNX2) were detected on day 7 after osteogenic induction. The protein ex-pression of osteoglycin (OGN) was evaluated by Western blot on day 14 after osteogenic induction. The formation of calci-um nodules was detected by alizarin red staining on day 21 after osteogenic induction. The activity of superoxide dismutase (SOD) and the protein expression of MnSOD of irradiated hBMMSCs with 8 Gy X-ray were determined at 24 h after α2M treatment. RESULTS:Compared with 8 Gy X-ray group,the activity of ALP,the mRNA expression of RUNX2,the pro-tein expression of OGN and MnSOD,as well as SOD activity were higher than those in the hBMMSCs treated with α2M at 0.5 and 1.0 g/L after 8 Gy X-ray irradiation,and the calcium nodules were also increased. CONCLUSION:α2M signifi-cantly improves the osteogenic differentiation ability,the SOD activity and MnSOD protein expression of hBMMSCs after ra-diation injury.

Cellular Response of Human Bone Marrow Derived Mesenchymal Stem Cells to Titanium Surfaces Implanted with Calcium and Magnesium Ions

Surface characteristics and cellular response to titanium surfaces that had been implanted with calcium and magnesium ions using plasma immersion ion implantation and deposition (PIIID) were evaluated. Three different titanium surfaces were analyzed: a resorbable blast media (RBM) surface (blasted with hydroxyapatite grit), a calcium ionimplanted surface, and a magnesium ion-implanted surface. The surface characteristics were investigated by scanning electron microscopy (SEM), surface roughness testing, X-ray diffraction (XRD), and Auger electron spectroscopy (AES). Human bone marrow derived mesenchymal stem cells were cultured on the 3 different surfaces. Initial cell attachment was evaluated by SEM, and cell proliferation was determined using MTT assay. Real-time polymerase chain reaction (PCR) was used to quantify osteoblastic gene expression (i.e., genes encoding RUNX2, type I collagen, alkaline phosphatase, and osteocalcin). Surface analysis did not reveal any changes in surface topography after ion implantation. AES revealed that magnesium ions were present in deeper layers than calcium ions. The calcium ion- and magnesium ion-implanted surfaces showed greater initial cell attachment. Investigation of cell proliferation revealed no significant difference among the groups. After 6 days of cultivation, the expression of RUNX2 was higher in the magnesium ion-implanted surface and the expression of osteocalcin was lower in the calcium ion-implanted surface. In conclusion, ion implantation using the PIIID technique changed the surface chemistry without changing the topography. Calcium ion- and magnesium ion-implanted surfaces showed greater initial cellular attachment.

Human bone marrow mesenchymal stem cells into vascular endothelial cells before and after differentiation induced differential expression of eNOS/activity and its metabolites / 西安交通大学学报(医学版)

Objective To investigate the differences in eNOS gene expression,activity and its metabolites before and after human bone marrow mesenchymal stem cells (hBMSCs) are induced into vascular endothelial cells.Methods hBMSCs were induced into vascular endothelial cells.The morphological changes of the cells were observed under inverted microscope.Transwell assay was used to detect the cells' migration ability.The protein expression of eNOS was detected by immunofluorescence and Western blot.The activity of eNOS was detected by ELISA and the content of NO in cell culture supernatant was determined by nitrate reduction method.Results Compared with those in the undifferentiated group,the morphological changes of the differentiated cells were obvious.Cell migration ability increased by 238.10％ (73.000±7.002 vs.21.000±4.359,P＜0.05).The expression of eNOS protein increased by 114.72％ (0.423±0.011 vs.0.197±0.079,P＜0.05).The activity of eNOS was enhanced by 157.49％ (4.967±0.073 vs.1.929±±0.103,P＜0.05).The synthesis and release of NO increased by 155.67％ (184.909±1.853 vs.72.323±0.426,P＜0.05).Conclusion After hBMSCs are induced into endothelial cells,the expression of eNOS gene increases,their activities increase,synthesis and release of the metabolite NO increase.It may provide a basis for prevention and treatment of cardiovascular diseases with stem cells.

Effect of dasatinib on biological properties of human bone marrow mesenchymal stem cells / 中国病理生理杂志

AIM:To explore the effect of dasatinib on the viability, migration, cell cycle and apoptosis of human bone marrow mesenchymal stem cells (hBMSCs), as well as the underlying signal pathway to evaluate the influence of dasatinib on hematopoietic microenvironment clinically.METHODS:The cell viability was measured by CCK-8 assay.The migration ability was detected by wound healing assay.The cell cycle and apoptosis were analyzed by flow cytometry.Acridine orange/ethidium bromide staining was also used to detected apoptosis.The secretion of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) were measured by ELISA.The protein levels of cleaved caspase-3, protein kinase B (Akt) and phosphorylated Akt were determined by Western blot.RESULTS:Compared with control group, dasatinib at 1~10 nmol/L suppressed the viability and migration ability of hBMSCs, and dasatinib at concentration of 7 nmol/L was adopted in the following assays.Dasatinib promoted apoptosis, and blocked the cell cycle in G1 phase.In addition, the secretion of TGF-β1 and TNF-α was increased markedly.The protein levels of cleaved caspase-3 was increased, but the protein levels of Akt and phosphorylated Akt were decreased.CONCLUSION:Dasatinib inhibits the viability and migration ability of hBMSCs in a dose-dependent manner, promotes the secretion TGF-β1 and TNF-α, and induces cell cycle arrest and apoptosis.Dasatinib might regulate the biological behaviors of hBMSCs observed above by modulating the expression and phosphorylation of Akt.

Effects of interleukin-34 expressed by human bone marrow derived mesenchymal stem cells on THP-1 cells / 生物工程学报

To construct recombinant eukaryotic expression plasmid vector of human IL-34 gene, and to study the effects of IL-34 expressed by human bone marrow-derived mesenchymal stem cells (hBM-MSCs) on THP-1 cells. Full-length IL-34 encoding sequence was amplified by PCR. And this fragment was cloned into the plasmid pIRES2-EGFP. Western blotting and ELISA were used to analyze the expression of IL-34 in hBM-MSCs. THP-1 cells were cultured with hBM-MSCs medium containing IL-34 protein. Real-time PCR detected the effects of IL-34 on the expression of IL-10 and TNFα in THP-1 cells. Restrictive enzyme analysis and sequencing demonstrated that IL-34 eukaryotic expression vector was successfully constructed. IL-34 protein expressed by hBM-MSCs could promote IL-10 and TNFα expression in THP-1 cells. Those results show that IL-34 expressed by hBM-MSCs has regulating effect on THP-1 cells.