Knockdown of long non-coding RNA MIR4697 host gene inhibits adipogenic differentiation in bone marrow mesenchymal stem cells / 北京大学学报(医学版)

OBJECTIVE@#To preliminarily investigate the role of long non-coding RNA (lncRNA) MIR4697 host gene (MIR4697HG) in regulating the adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).@*METHODS@#For adipogenic differentiation, BMSCs were induced in adipogenic media for 10 days. The mRNA expression levels of lncRNA MIR4697HG and adipogenic marker genes including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhanced binding protein α (CEBP/α) and adiponectin (ADIPQ) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) at different time points (0, 1, 2, 3, 5, 7, 10 days). The MIR4697HG stable knockdown-BMSC cell line was generated by infection of MIR4697HG shRNA-containing lentiviruses. To avoid off-target effect, two target sequences (shMIR4697HG-1, shMIR4697HG-2) were designed. And then cells were induced to differentiate in adipogenic medium. Oil red O staining, Western blot and qRT-PCR were used to detect the effect of MIR4697HG knockdown on adipogenic differentiation of BMSCs.@*RESULTS@#The mRNA expression level of MIR4697HG was significantly increased during adipogenic differentiation (P < 0.01), and adipogenic differentiation of BMSCs was evidenced by upregulated mRNA levels of specific adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ. Observed by fluorescence microscopy, more than 90% transfected target cells expressed green fluorescent protein successfully after shMIR4697HG-1 group, shMIR4697HG-2 group and shNC group transfection for 72 h. And the transfection efficiency of MIR4697HG examined by qRT-PCR was above 60%. Then the BMSCs were treated with adipogenic media for 7 days and showed that the mRNA expression levels of adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ were significantly decreased in the MIR4697HG knockdown group (P < 0.01), while the expression levels of PPARγ and CEBP/α proteins were decreased remarkably as well (P < 0.01). Consistently, MIR4697HG knockdown BMSCs formed less lipid droplets compared with the control BMSCs, which further demonstrated that MIR4697HG knockdown inhibited adipogenic differentiation of BMSCs.@*CONCLUSION@#lncRNA MIR4697HG played a crucial role in regulating the adipogenic differentiation of BMSCs, and MIR4697HG knockdown significantly inhibited the adipogenic differentiation of BMSCs. These data may suggest that lncRNA MIR4697HG could serve as a therapeutic potential target for the aberrant adipogenic differentiation-associated disorders including osteoporosis.

Mechanism of Bushen Huatan Prescription in Regulation of Osteogenesis and Adipogenic Differentiation and Improvement of Postmenopausal Bone Loss in Ovariectomized Rats with Osteoporosis / 中国实验方剂学杂志

ObjectiveTo explore the underlying mechanism of Bushen Huatan prescription in alleviating postmenopausal osteoporosis （PMOP） by maintaining the balance of osteogenesis and adipogenic differentiation in ovariectomized rats with osteoporosis. MethodSeventy-five 6-month-old non-pregnant female SD rats were randomly divided into sham-operation group， model group， atorvastatin group， liviol group， and Bushen Huatan prescription group. Bilateral ovaries were removed in the four groups except the sham-operation group， while only the same mass of adipose tissue around the ovaries was removed in the sham-operation group. On the 5th week after surgery， drugs were consecutively administrated for 8 weeks. Rats in the Bushen Huatan prescription group received 9.4 mg·kg-1 of the prescription， rats in the atorvastatin group received 0.92 mg·kg-1 of atorvastatin， rats in the Liviol group received 0.23 mg·kg-1 of liviol， and rats in the model group and the sham-operation group received saline once a day. Micro-computed tomography （Micro CT） was used to detect bone mineral density （BMD） of rat tibia in each group. Hematoxylin-eosin （HE） staining was used to detect the relative area of rat bone marrow adipose tissue （BMAT） in each group. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to detect the relative expression levels of Runt-related transcription factor 2 （Runx2）， peroxisome proliferator-activated receptor （PPARγ）， leptin （LPN）， and leptin receptor （OBR） in bone tissues. ResultAs compared with the sham operation group， the BMD of rats in the model group decreased （P<0.05）， while the relative area of BMAT increased （P<0.05）. In addition， the expression levels of LPN， OBR， and Runx2 decreased in the model group （P<0.05）， while the level of PPARγ increased （P<0.05）. As compared with the model group， the BMD of rats in the atorvastatin group， the Livial group， and the Bushen Huatan prescription group increased （P<0.05）， and the relative area of BMAT decreased （P<0.05）. The expression levels of LPN， OBR， and Runx2 in these groups increased （P<0.05）， while the expression level of PPARγ decreased （P<0.05）. ConclusionBushen Huatan prescription plays the anti-osteoporosis role in the rat model of PMOP through up-regulating LPN and OBR in bone tissues and maintaining the balance of osteogenesis and adipogenic differentiation， thereby reducing postmenopausal bone loss and playing a role in the prevention and treatment of PMOP.

Regulatory effect of uncarboxylated osteocalcin on osteogenic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells under high-glucose conditions / 中国组织工程研究

BACKGROUND: The method of promoting osteogenic differentiation of bone marrow mesenchymal stem cells under high-glucose conditions to inhibit adipogenic differentiation can provide prevention and treatment ideas for the treatment of bone metabolic diseases such as diabetic osteoporosis. OBJECTIVE: To explore the effects of uncarboxylated osteocalcin on adipogenic and osteogenic differentiation of mouse bone marrow mesenchymal stem cells under high-glucose conditions so as to reveal the action mechanism of uncarboxylated osteocalcin on the differentiation of bone marrow mesenchymal stem cells. METHODS: Mouse bone marrow mesenchymal stem cells were cultured by whole bone marrow culture and adherent purification. Cells were treated with uncarboxylated osteocalcin at different concentrations (0, 1, 3, 10, and 30 μg/L). Cell proliferation was detected by cell counting kit-8 to determine the best mass concentration. Passage 3 bone marrow mesenchymal stem cells were incubated with adipogenic (or osteogenic) differentiation medium, and assigned to four groups: control group, high glucose group, uncarboxylated osteocalci n group, and high glucose + uncarboxylated osteocalcin group. Corresponding groups received the addition of 25.5 mmol/L exogenous glucose and 3 μg/L uncarboxylated osteocalcin. Lipid droplets and calcium nodules were detected by oil red and alizarin red staining. Quantitati ve reverse transcription-polymerase chain reaction was used to detect the relative expression levels of adipogenic marker genes (Fabp4, PPARγ, Adipsin and FAS) and osteogenic differentiation marker genes (Runx2, Osx, alkaline phosphatase, and type I collagen). Kits were used to detect alkaline phosphatase activity and type I collagen levels. The relative expression levels of P-Erk and P-AMPKα were detected using signal pathway specific inhibitors (PD98059 and BML) and western blot assay. RESULTS AND CONCLUSION: (1) Uncarboxylated osteocalcin 3 μg/L promoted cell proliferation (P < 0.01). (2) Uncarboxylated osteocalcin promoted the formation of calcium nodules (P < 0.01) in bone marrow mesenchymal stem cells under high-glucose conditions but inhibited the formation of lipid droplets (P < 0.05), down-regulating the relative expression levels of adipogenic marker genes (PFabp4 < 0.01; PPPARγ < 0.05; PAdipsin < 0.01; PFAS < 0.01), but increasing the relative expression levels of osteogenic differentiation marker genes (PRunx2 < 0.05; POsx < 0.05; PALP < 0.01; PCOLI < 0.01). Uncarboxylated osteocalcin increased alkaline phosphatase activity (P < 0.01) and type I collagen level (P < 0.05). (3) Uncarboxylated osteocalcin up-regulated the expression levels of P-Erk (P < 0.01) and P-AMPKα (P < 0.01) under high-glucose conditions. (4) These results indicate that uncarboxylated osteocalcin promoted osteogenic differentiation of bone marrow mesenchymal stem cells under high-glucose conditions through Erk/AMPKα signaling pathway and inhibited adipogenic differentiation.

Effects of astragalus polysaccharide on radiation-induced adipogenic differentiation of bonemarrow mesenchymal stem cells / 西安交通大学学报(医学版)

Objective: To study the effects of astragalus polysaccharide (APS) on adipogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs) irradiated by X-ray. Methods: Human bone marrow mesenchymal stem cells irradiated by 2 Gy X-ray were intervened with 50 μg/mL of APS. The number and size of lipid droplets of stem cells were observed by oil red O staining and the expressions of CEBPα and PPAR-γ were detected by Western blot after induction with special medium. Results: The number of adipocytes differentiated from bone marrow mesenchymal stem cells decreased, and the area of lipid droplets in adipocytes decreased significantly after irradiation (P<0.05). The area of lipid droplets in the bone marrow mesenchymal stem cells treated with APS in advance increased compared with that in radiation alone (P<0.05). Similarly, the expressions of PPAR-γ and recombinant human CCAAT enhancer binding protein (CEBPα ) decreased after X-ray irradiation, while the expressions of PPAR-γ and CEBPα increased after the intervention of APS (P<0.05). Conclusion: X-ray can damage the directional adipogenic differentiation of human bone marrow mesenchymal stem cells, and APS has a protective effect on the adipogenic differentiation of human bone marrow mesenchymal stem cells after X-ray irradiation.

MicroRNA-138 Suppresses Adipogenic Differentiation in Human Adipose Tissue-Derived Mesenchymal Stem Cells by Targeting Lipoprotein Lipase

PURPOSE: Adipogenic differentiation of adipose tissue-derived mesenchymal stem cells (AMSCs) is critical to many disease-related disorders, such as obesity and diabetes. Studies have demonstrated that miRNA-138 (miR-138) is closely involved in adipogenesis. However, the mechanisms affected by miR-138 remain unclear. This work aimed to investigate interactions between miR-138 and lipoprotein lipase (LPL), a key lipogenic enzyme, in AMSCs. MATERIALS AND METHODS: Human AMSCs (hAMSCs) isolated from human abdomen tissue were subjected to adipogenic differentiation medium. Quantitative real-time polymerase chain reaction and Western blot assay were applied to measure the expressions of miR-138, LPL, and the two adipogenic transcription factors cytidine-cytidine-adenosine-adenosine-thymidine enhancer binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ). The relationship between miR-138 and LPL was predicted utilizing the miRTarBase database and validated by dual luciferase reporter assay. RESULTS: Showing increases in C/EBPα and PPARγ expression levels, hAMSCs were induced into adipogenic differentiation. During adipogenesis of hAMSCs, miR-138 expression was significantly downregulated. Overexpression of miR-138 by transfection inhibited hAMSCs adipogenic differentiation in vitro. Mechanically, LPL was a target of miR-138. LPL expression was upregulated during adipogenesis of hAMSCs, and this upregulation was reversed by miR-138 overexpression. Functionally, silencing of LPL by transfection exerted similar inhibition of the expressions of C/EBPα and PPARγ. Meanwhile, LPL ectopic expression was able to partly abolish the suppressive effect of miR-138 overexpression on adipogenic differentiation of hAMSCs. CONCLUSION: Upregulation of miR-138 inhibits adipogenic differentiation of hAMSCs by directly downregulating LPL.

Relationship between Wnt10b and bone morphogenetic protein-9 induced osteogenic differentiation of mesenchymal stem cells / 中国药理学通报

Aim To study the relationship between WntlOb and bone morphogenetic protein-9 (BMP9)-induced osteogenic differentiation of mesenchymal stem cells (MSCs), and the molecular mechanisms underlying this process. Methods PCR, Western blot and histochemical staining were used to detect the effect of BMP9 on WntlOb and the effect of WntlOb on BMP9-induced osteogenic differentiation in MSCs. Meanwhile, real-time PCR, Western blot, oil red O staining, and flow cytometry assay were used to analyze the potential mechanism of Wnt10b affecting the function of BMP9. Results Wnt10b could be detected in C3H10T1/2, C2C12, MEFs and MC3T3-E1 cells. BMP9 up-regulated the expression of WntlOb in C3H10T1/2 cells. WntlOb enhanced the capability of BMP9 to increase the level of OCN and mineralization in C3H10T1/2 cells, and silencing Wnt10b attenuated these effects of BMP9. Wnt10b exhibited no substantial effect on cell cycle affected by BMP9, but it enhanced the effect of BMP9 on inducing phosphorylation of Smadl/5/8. While silencing Wnt10b attenuated this effect of BMP9. In addition, Wnt10b inhibited BMP9-induced adipogenic differentiation in C3H10T1/2 cells, and silencing Wnt10b promoted this effect of BMP9. Conclusions Wnt10b can promote BMP9-induced osteogenic differentiation of MSCs, which may be mediated through enhancing BMP/Smad signaling and reducing adipogenic differentiation.

Research progress on the balance regulation of osteogenic differentiation and adipogenic differentiation in bone marrow mesenchymal stem cell / 解剖学报

Osteoporosis (OP), a systemic bone metabolism disease, mainly manifested in the decrease of bone mass, the increase of bone fragility and the microstructure degeneration of the bone. Along with the in-depth research of the pathogenesis of osteoporosis, the imbalance differentiation of bone marrow mesenchymal stem cell (BMSCs) (Osteogenic differentiation decrease and adipogenic differentiation increase) is the main reason that causes osteoporosis. In this paper, we summarize the signal pathways of osteogenic differentiation and adipogenic differentiation of BMSCs. Better understand these signal pathways is conducive to elucidate and treat osteoporosis.

Experimental research of adipogenic differentiative ability of adipose tissue-derived stromal cells in patients with type 2 diabetes mellitus / 重庆医学

Objective To investiagte the adipogenic differentiative ability of adipose tissue-derived stromal cells(ADSCs) between the patients with type 2 diabetes mellitus(T2DM) and healthy persons.Methods The adipose tissues were taken from the adipose tissue in T2DM patients and healthy persons for separating and culturing ADSCs.The cells of third generation were taken for inoculation.The difference in cellular phenotype and growth speed were compared between the two groups.Adding adipogenesis inducing fluid,the adipogenic differentiative situation was observed in the two groups.The oil red O was added on 14 d for conducting the cell staining and observation.The oil red O was extracted by isopropanol,and the cellular absorbances were compared between two groups.Meanwhile,the expression of PPAR-γ,C/EBP-α and C/EBP-β on 14 d of adipogenic differentiation were compared between two groups by using qPCR method.Results The cellular phenotype and growth speed of ADSCs had no statisticat difference between T2DM patients and healthy persons.On 14 d of adipogenic differentiation,the oil red O absorbance value of AD-SCs in T2DM patients was significantly higher than that in the healthy persons,and the expression of PPAR-γ,C/EBP-α and C/EBP-β were significantly higher than those in the healthy persons.Conclusion The adipogenic differentiative ability of ADSCs in T2DM patients is obviously higher than that in healthy persons,which may be one of causes easy to be obese in T2DM patients.

Molecular mechanism of quercitrin on osteogenic differentiation and adipogenic differentiation of rat bone marrow stromal stem cells (rBMSCs) / 中草药·英文版

Objective: The study was designed to investigate the molecular mechanism of quercitrin on osteogenic differentiation and adipogenic differentiation of rBMSCs. Methods: rBMSCs were harvested from SD rats, and determination of alkaline phosphatase (ALP) activity, quantification of mineralization by Alizarin Red S staining, and the mRNA expression of osteogenic differentiation markers (Runx2, BMP-2, and OSX) by RT-PCR after rBMSCs stimulated by osteogenic induction with (0.1–10) µg/mL of quercitrin, quantification of Lipid droplet by Oil Red O staining and the mRNA expression of adipogenic differentiation marker (PPARγ C/EBPα and aP2) by RT-PCR after rBMSCs stimulated by adipogenic induction with (0.1-10) µg/mL of quercitrin. Results: Quercitrin can up-regulate the mRNA expression of osteogenic differentiation markers (Runx2, BMP-2, and OSX) and increase ALP activity and mineralization after osteogenic induction, on the other hand quercitrin can suppress the mRNA expression of adipogenic differentiation markers (PPARγ C/EBPα and aP2) and decrease lipid droplet after adipogenic induction. Conclusion: This study suggested that quercitrin not only stimulated osteogenic differentiation but also inhibited adipogenic differentiation of rBMSCs, which was associated with the up-regulation of Runx2, BMP-2, and OSX mRNA expression and the down-regulation of PPARγ C/EBPα and aP2 mRNA expression.

Effect of sinapine on H2O2-induced adipogenic differentiation of bone marrow mesenchymal stem cells / 中国病理生理杂志

AIM:To investigate the effects of sinapine,an effective monomer of Chinese medicine,on hydro-gen peroxide(H2O2)-induced adipogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).METHODS:The undifferentiated rat BMSCs were identified and screened by flow cytometry.The adipogenic differentiation of BMSCs was induced by H2O2,and the toxicity of sinapine on BMSCs was tested by CCK-8 assay.After the modeling method and the concentration range of sinapine were determined,the lipid droplets in the cells were detected by Oil Red O semi-quanti-tative assay,and the optimal drug concentration was selected.Finally,Oil Red O assay was observed 24 h after drug inter-vention,and the expression of adipogenic differentiation-related proteins,adipocyte protein 2(aP2),peroxisome prolifera-tor-activated receptor γ(PPARγ)and glucose transporter 4(Glut4),at mRNA and protein levels in the BMSCs was deter-mined by qPCR and Western blot.RESULTS:Treatment with H2O2at 200 μmol/L for 1 h induced BMSCs to differentiate into adipocytes.Below the concentration of 40 μmol/L,sinapine had no toxicity to BMSCs.The best inhibitory concentra-tion of sinapine on adipogenic differentiation was at 15 μmol/L.The number of lipid droplets in sinapine(15 μmol/L) group was significantly lower than that in model group.In sinapine group,the expression of aP2,PPARγand Glut4 at mR-NA and protein levels was lower than that in model group(P<0.01).CONCLUSION: Sinapine inhibits H2O2-induced adipogenic differentiation of rat BMSCs.The mechanism may be related to the PPARγ/AMPK signaling pathway.

Effect of pinostrobin chalcone on adipogenic differentiation of mesenchymal stem cell C3H10T1/2 / 中国中药杂志

Chalcones is a flavonoid wildly presented in many herbs. It has the effect to inhibit cells adipogenic differentiation. In order to study the effect of pinostrobin chalcone extracted and isolated from leaves of hickoryes on the adipogenic differentiation of murine embryonic mesenchymal stem cell (C3H10T1/2), MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium] method was used to detect the cell proliferation; adipogenic differentiation was characterized by oil red O staining and isopropanol extraction; the triglyceride content was detected by GAP-PAP enzyme method; and the C3H10T1/2 cell differentiation into adipocytes was also examined by the mRNA and protein expression of PPARγ, C/EBPα and FABP4 by RT-PCR and Western blot respectively. Results indicated that pinostrobin chalcone almost had no effect on cell proliferation activity when the concentration was less than or equal to 50 μmol•L⁻¹; the oil red O staining, isopropanol extraction and GAP-PAP enzyme method showed that pinostrobin chalcone significantly decreased the C3H10T1/2 adipogenic differentiation and triglyceride content in the cytoplasm of adipocytes; the RT-PCR and Western blot analysis showed that pinostrobin chalcone can down-regulate the mRNA and protein levels of FABP4, PPARγ and C/EBPα in C3H10T1/2 cells(P<0.05 or P<0.01). The experiment results suggest that pinostrobin chalcone can inhibit C3H10T1/2 adipogenic differentiation.

Effects of Astragalus polysaccharides on adipogenic differentiation of bone marrow mesenchymal stem cells in low oxygen environment / 生物工程学报

To study the effect of Astragalus polysaccharide (APS) on adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) cultured in hypoxic environment. The optimal APS concentration, which could promote the proliferation of BMSCs, was screened by methyl thiazolyl tetrazolium method. The concentration was used to intervene in BMSCs-induced by adipogenic differentiation fluid growing in different oxygen concentrations (3%, 6%, 10% and 20%). The formation of lipid droplets in the BMSCs-intervened was observed by oil red O staining under the optical microscope. The mRNA and protein levels of the lipid relating genes peroxisome proliferator activated receptor gamma 2 (PPAR-γ₂) and lipoprotein lipase (LPL) were detected by Real-time PCR and Western blotting, respectively. The results showed that, comparing with the control group, 40 μg/mL APS could significantly promote the proliferation of BMSCs under low oxygen concentration. A large amount of lipid droplets existed in BMSCs growing in the adipogenic inducing fluid containing 40 μg/mL APS and the hypoxic environment, and the protein and mRNA levels of PPAR-γ₂ and LPL also raised. It was worth noting that the phenomenon was more significant in 10% oxygen concentration, and the difference was statistically significant (P<0.05). 40 μg/mL APS had effect on promoting the proliferation and adipogenic differentiation of BMSCs cultured in hypoxic environment, and the effect was related to the concentration of oxygen of BMSCs-cultured.

The comparison on pluripotent differentiation between human gingival fibroblasts and human periodontal ligament cells in vitro / 天津医药

Objective To investigate the the multi-directional differentiation potential between pluripotent of human gingival fibroblasts (HGFs) and human periodontal ligament cells (HPDLCs). Methods HPDLCs and HGFs were obtained from the primary culture. HPDLCs and HGFs at 3rd-4th passage were cultured in osteogenic, adipogenic or chondrogenic me-dium. Cells without differentiation were taken as control. Alizarin red, Alcian blue and oil red O staining were performed to detect osteogenic differentiation, chondrogenic and adipogenic differentiation in vitro, respectively. Reverse transcription polymerase chain reaction (RT-PCR) was applied to examine the expression of osteocalcin (OCN), runt-related transcription factor 2 (RUNX2) and collagen 1 (Col 1), peroxisome proliferator-activated receptor gamma 2 (PPARγ2) and collagen 10 (Col 10). Results HPDLCs and HGFs cultured in osteogenic medium showed massive calicium nodulus at day 28, but HP-DLCs formed more calicium nodulus than those of HGFs. The expressions of OCN, RUNX2 and Col 1 were significantly high-er in HPDLCs than those in HGFs (P<0.05). In chondrogenic medium both cells were found blue deposit at day 14, and the expression of Col 10 was significantly higher in HGFs than that of HPDLCs (P<0.01). Furthermore, in adipogenic medium HGFs showed more lipid-filled droplets stained with oil red O than HPDLCs at day 21. The expression of PPARγ2 was sig-nificantly higher in HGFs than that of HPDLCs (P<0.01). Conclusion HPDLCs has the better potency of osteogenic differ-etiation than HGFs, however, HGFs has the better potency of adipogenic and chondrogenic differentiation.

Isolation,cultivation and identification of dental pulp stem cells from canine / 实用口腔医学杂志

Objective:To culture canine dental pulp stem cells(cDPSCs)in vitro.Methods:Canine pulp cells were isolated and cultured by enzyme digestion and explanted tissue culture respectively.Cell morphology was observed under phase-contrast micro-scope.The clone forming unit(CFU)of the cells was examined by plate clone formation assay.Cell markers and protein-expression were examined by flow cytometry(FC)and immunofluorescence.Odontogenic and adipogenic potential were evaluated by alizarin red staining and oil red O staining.Results:Short spindle fibroblast-like and steadily growing cells were obtained by both methods.The clone assay showed that CFU was 1 5.1 7% ±2.79%.FC observasion showed that the CD90,STRO-1 and CD24 positive cells were 24.43% ±7.1 0%,20.67% ±1 .42% and 2.03% ±0.06% respectively,but CD34 was negative.Immunofluorescence analysis showed positive expression of Nestin,Vimentin,weak expression of ALP and negative expression of DSP of the cells.Differentiation ex-periment confirmed the odontogenic and adipogenic differentiation potential of the cells.Conclusion:cDPSCs can be cultured in vitro.

Parathyroid hormone on the adipogenic potential of rat bone marrow mesenchymal stem cells / 中华风湿病学杂志

Objective To observe the effect of different concentrations of parathyroid hormone 1-34 on the adipogenic potential of rat bone marrow mesenchymal stem cells (BMSCs).Methods ① Rat bone marrow mesenchymal cells were separated and expanded by adherent culture.The morphology of cells was observed and cell surface markers were examined by flow cytometry.② The multi-lineage differentiation capability of cells was examined by culturing cells under conditions favorable for adipogenic and osteogenic differentiation.③ Taken P3 of BMSCs for test,different concentrations of PTH1-34 (0,10-10,10-9,10-8 mol/L) were used to stimulate BMSCs respectively,14 days later,lipoprotein lipase (LPL) activity were measured by enzyme linked immuno-sorbent assay (ELISA),mRNA expression of alkaline phosphatase (ALP) and PPARγ-2 were measured by realiime polymerase chain reaction (PCR).④ Statistical analysis:data were presented as x±s.All statistical analysis was performed with windows Statistical Praduct and Serice Solutions (SPSS) 13.0.One-way analysis of variance (ANOVA) was applied to determine the difference between groups.Least signisicant difference (LSD) was used to determine the difference between the two randomized groups.Differences were considered significant at a value of P＜0.05.Results The cells expressed CD44,CD29 but without expression of CD45.By culturing in adipogenic medium for 3 weeks and in osteogenic medium for 4 weeks respectively,and then identified by oil red O and Alizarin red,the cells were successfully induced to adipocytes and osteogenesis.Expressions of LPL were 11.20±0.16,7.62±0.48,5.84±0.57,5.32±0.52,mRNA expressions of PPARγ-2 were 2.80±0.05,1.36±0.23,0.94-±0.11,0.78±0.04,ALP activity were 0.191 ±0.016,0.333±0.024,0.549±0.025,0.684±0.021 respectively.Compared with the control group,different concentrations of PTH1-34 groups could decrease mRNA expression of LPL and PPARγ-2.ALP activity were increased(P＜0.05).Conclusion PTH1-34 inhibits BMSCs of adipogenic differentiation and promotes osteogenic differentiation in a dose-dependent manner.

Improved adipogenic in vitro differentiation: comparison of different adipogenic cell culture media on human fat and bone stroma cells for fat tissue engineering / 대한해부학회지

To date there is no sufficient in vitro fat tissue engineering and a protocol has not been well established for this purpose. Therefore, we evaluated the in vitro influence of two different adipogenic growth media for their stimulation potential on different cell lineages to clearly define the most potent adipogenic growth media for future in vitro tissue engineering approaches. The samples for differentiation were composed of human adipogenic-derived stroma cells (hADSCs) and human bone marrow mesenchymal stroma cells (hMSCs). A normal adipogenic medium (NAM) and a specific adipogenic medium (SAM) were tested for their adipogenic stimulation potential. After 10 days and 21 days the relative gene expression was measured for the adipogenic marker genes PPARgamma2, C/EBPalpha, FABP4, LPL, and GLUT4 detected through real time reverse transcriptase polymease chain reaction (RT-PCR). Other study variables were the comparison between NAM and SAM and between the used cells hADSCs and hMSCs. Additionally an Oil-Red staining was performed after 21 days. Our results revealed that only SAM was significantly (P<0.05) superior in the differentiation process in contrast to NAM for 10 days and 21 days. As well was SAM superior to differentiate the used cell lineages. This was evaluated by the detected marker genes PPARgamma2, C/EBPalpha, FABP4, LPL, and GLUT4 through real time RT-PCR and by Oil-Red staining. In addition, the hMSCs proofed to be equal donor cells for adipogenic differentiation especially when stimulated by SAM. The results suggest that the SAM should be established as a new standard medium for a more promising in vitro adipogenic differentiation.

Aislamiento, caaacterización y potencial de difrenciación de células madre messenquimales caninas derivadas de tejido adiposo / Islation, characcterization and poential differentiation of caninne mesenchymal stem cells derived from adipose tissue

Se caracterizaron células obtenidas del tejido adiposo subcutáneo de caninos para demostrar su naturaleza de células madre mesenquimales (ad-MSCs). Las células se cultivaron y expandieron bajo condiciones in vitro. Se determinó la presencia de marcadores de superficie CD73, CD90 y CD105 por citometría de flujo y PCR en tiempo real, siendo idéntico el perfil de expresión obtenido al reportado en células madre mesenquimales humanas. Se demostró su potencial de diferenciación mediante análisis de expresión génica por PCR en tiempo real luego de inducirlas a procesos de diferenciación osteogénica y adipogénica. La capacidad de diferenciación osteogénica se evidenció a través de la expresión de genes de tejido óseo (colágeno tipo I y osteonectina) en una cinética de expresión concordante con los eventos moleculares y celulares del desarrollo óseo in vivo. El potencial adipogénico se evaluó por la formación de vacuolas lipídicas intracelulares y la expresión del gen de lipoprotein-lipasa, acorde con el proceso in vivo. Se concluye que las células mesenquimales obtenidas del tejido adiposo de caninos cumplen los criterios morfológicos, de expresión de marcadores de superficie específicos y de capacidad de diferenciación mesodérmica, confirmando su naturaleza de células madre mesenquimales. Los resultados del presente trabajo indicaron que las ad-MSCs de los caninos son un excelente modelo preclínico para el desarrollo de aplicaciones en medicina regenerativa, tanto en medicina veterinaria, como en medicina humana.

Cells obtained from the canine subcutaneous adipose tissue were characterized to demonstrate the nature of mesenchymal stem cells (adMSCs). The cells were cultured and expanded in vitro conditions. The presence of the surface markers CD73, CD90 and CD105 by flow cytometry and real time PCR was determined. The expression profile is identical to that reported in human mesenchymal stem cells. The differentiation potential was demonstrated by gene expression by real time PCR after induction osteogenic and adipogenic. Osteogenic differentiation was evidenced by the expression of genes of bone tissue (type I collagen and osteonectin) in expression kinetics consistent with the molecular and cellular events of bone development in vivo. In the adipogenic differentiation, were the formation of intracellular lipid vacuoles and expression of lipoprotein lipase gene (LPS), according to the in vivo process. We conclude that mesenchymal, cells derived from adipose tissue of dog, meet the morphological criteria, expression of specific surface markers and mesodermal differentiation capacity, confirming the nature of mesenchymal stem cells. The canine mesenchymal stem cells are an excellent preclinical model for developing applications in regenerative medicine that can be used both in veterinary and human medicine.

Mesengenic Differentiation: Comparison of Human and Rat Bone Marrow Mesenchymal Stem Cells

BACKGROUND AND OBJECTIVES: Cellular therapies using Mesenchymal Stem Cells (MSCs) represent a promising approach for the treatment of degenerative diseases, in particular for mesengenic tissue regeneration. However, before the approval of clinical trials in humans, in vitro studies must be performed aimed at investigating MSCs' biology and the mechanisms regulating their proliferation and differentiation abilities. Besides studies on human MSCs (hMSCs), MSCs derived from rodents have been the most used cellular type for in vitro studies. Nevertheless, the transfer of the results obtained using animal MSCs to hMSCs has been hindered by the limited knowledge regarding the similarities existing between cells of different origins. Aim of this paper is to highlight similarities and differences and to clarify the sometimes reported different results obtained using these cells. METHODS AND RESULTS: We compare the differentiation ability into mesengenic lineages of rat and human MSCs cultured in their standard conditions. Our results describe in which way the source from which MSCs are derived affects their differentiation potential, depending on the mesengenic lineage considered. For osteogenic and chondrogenic lineages, the main difference between human and rat MSCs is represented by differentiation time, while for adipogenesis hMSCs have a greater differentiation potential. CONCLUSIONS: These results on the one hand suggest to carefully evaluate the transfer of results obtained with animal MSCs, on the other hand they offer a clue to better apply MSCs into clinical practice.

Isolation, expansion and adipogenic differentiation of human keloid-derived precursor cells / 中华医学美学美容杂志

Objective To explore the isolation,amplification methods and adipogenic differentiation under specific culture medium of human keloid-derived precursor cell (KPC) in vitro,in order to study their possibility of being new seed cells of tissue engineering fat.Methods KPCs were isolated from human keloid tissue of 4 different patients in our hospital and were cultured in the modified L-DMEM culture medium.Their cloning efficiency and growth curve were tested.The subcultured cells were tested of the mesenchymal stem cell (MSC)-related gene expression by flow cytometry.In addition,they were cultured in H-DMEM medium (containing 1 μmol/L dexamethasone,0.5 mmol/L 3-isobutyl-1-methyl-xanthine 10 mg/L of bovine insulin,100 mmol/L indomethacin,and 10 ％ FBS)and were later observed in oil red O staining under phase contrast microscope to determine whether lipid droplets generation was formed,using skin-derived precursors (SKP) as control.Results More than 95 ％ KPC expressed many antigens of MSC,such as CD29,CD44,CD90 and CD105 while few of them expressed CD34,CD45(1.0 ％-2.5 ％).And the cells increased in size gradually after inducted the same time,changing from spindle into round or polygonal in shape.The lipid droplets were seen in 72 hours and expressed a positive rate of 78.6 ％ in Day 19 in oil red O staining while the same rate was 54.6 ％ in SKP.Conclusions Human keloid-derived precursor cells can express a variety of MSC-related surface markers without expressing hematopoietic stem cell (HSC) related markers.Furthermore,they can be differentiated into fat cells under certain conditions,which may make them as a new source of seed cells for tissue engineering fat.

Platelet-rich plasma effects on grainy fat transplantation / 中国组织工程研究

BACKGROUND:The higher long-term absorption rate greatly influence the widely application of fat transplantation. Platelet-rich plasma contains a high concentration of growth factors, which benefits to the tissue healing and regeneration. OBJECTIVE:To observe the effects of platelet-rich plasma on grainy fat transplantation and to investigate the mechanisms preliminarily. METHODS:Ten 6-week-old nude mice were prepared. The right or left dorsal subcutaneous tissues were randomly selected as the platelet-rich plasma group (0.5 mL fat granule+0.1 mL platelet-rich plasma), and the contralateral side was regarded as the control group (0.5 mL fat granule+0.1 mL phosphate-buffered saline). At 10 and 90 days after implantation, five nude mice were selected from each group, and then the mice were sacrificed to obtain the grafts in each group for general appearance observation, volume determination and histological detection. Furthermore, we isolated adipose-derived stem cells from human subcutaneous fat tissue during the in vitro experiment. Cel counting kit-8 and real-time PCR were used to evaluate the influence of platelet-rich plasma on adipose-derived stem cel proliferation and adipogenic differentiation in vitro, respectively. RESULTS AND CONCLUSION:Comparison of the grafts obtained at 10 and 90 days after implantation, the residual volume in the platelet-rich plasma group was significantly larger than that in the control group (P<0.05), Moreover, more normal adipocytes and capil ary formation were observed in the platelet-rich plasma group (P<0.05). For in vitro experiment, platelet-rich plasma could significantly improve adipose-derived stem cel proliferation, and the expressions of adipogenic-related genes were up-regulated in platelet-rich plasma-induced adipose-derived stem cells. Al results demonstrate that platelet-rich plasma can improve the survival of fat grafts,which might be closely related to that platelet-rich plasma can promote the proliferation and adipogenic differentiation of adipose-derived stem cells and the revascularization in grafted fat tissue.