瘦素诱导体外培养大鼠脂肪间充质干细胞凋亡

为观察瘦素诱导体外培养大鼠脂肪间充质干细胞凋亡的作用, 采用胶原酶消化法分离培养大鼠附睾脂肪垫间充质干细胞, 第3代细胞用于实验。细胞免疫荧光化学方法鉴定CD105、Vimentin表达阳性率约80%以上, 10-6 mol/L的瘦素作用细胞48 h、72 h后激光共聚焦显微镜观察分别可见早期及中晚期特征表现; 0 mol/L、10-8 mol/L、10-7 mol/L、10-6 mol/L瘦素分别作用于细胞48 h后, 应用AnnexinⅤ/PI双染色法流式细胞仪检测早期凋亡率分别为2.50%±0.72%、6.78%±1.99%、11.99%±1.58%、17.93%±4.82% (P<0.05); 随着瘦素浓度的增加和作用时间的延长, Caspase-3的活性逐渐增高, 至48 h时达到高峰。说明瘦素可以直接诱导脂肪间充质干细胞凋亡, 从数量上减少脂肪组织的含量。     

Electrospun poly-l-lactic acid/polyvinyl alcohol nanofibers improved insulin-producing cell differentiation potential of human adipose-derived mesenchymal stem cells

Combination of adipose-derived mesenchymal stem cells (ADSCs) and synthetic materials in terms of pancreatic tissue engineering can be considered as a treatment of diabetes. This study aimed to evaluate the differentiation of human ADSCs to pancreatic cells on poly-l -lactic acid/polyvinyl alcohol (PLLA/PVA) nanofibers as a three-dimensional (3D) scaffold. Mesenchymal stem cells (MSCs) were characterized for mesenchymal surface markers by flow cytometry. Then ADSCs were seeded on 3D scaffolds and treated with pancreatic differentiation medium. Immunostaining assay showed that ADSCs were very efficiently differentiated into a relatively homogeneous population of insulin-producing cells. Moreover, real-time RT-PCR results revealed that pancreas-specific markers were highly expressed in 3D scaffolds compared with their expression in tissue culture plates and this difference in expression level was significant. In addition, insulin and C-peptide secreted in response to varying concentrations of glucose in the 3D scaffold group was significantly higher than that in 2D culture. The results of the present study confirmed that PLLA/PVA scaffold seeded with ADSCs could be a suitable option in pancreatic tissue engineering.     

HMGA2 expression in white adipose tissue linking cellular senescence with diabetes

There is a clear link between overweight, gain of white adipose tissue, and diabetes type 2 (T2D). The molecular mechanism of the gain of adipose tissue is linked with the expression of high mobility group protein AT-hook 2 (HMGA2), and recent studies revealed an association with a SNP near HMGA2. In this study, we investigated the gene expression of HMGA2, p14^Arf, CDKN1A, and BAX in human abdominal subcutaneous white adipose tissue from 157 patients. We found a significant higher HMGA2 expression in obese individuals than in non-obese patients. Furthermore, the HMGA2 expression in white adipose tissue in patient with type 2 diabetes was significantly higher than in nondiabetic patients. There is an association between the DNA-binding nonhistone protein HMGA2 and the risk of developing T2D that remains mechanistically unexplained so far. Likewise, p14^Arf, an inducer of cellular senescence, has been associated with the occurrence of T2D. The data of the present study provide evidence that both proteins act within the same network to drive proliferation of adipose tissue stem and precursor cells, senescence, and increased risk of T2D, respectively.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-013-0354-6) contains supplementary material, which is available to authorized users.     

Differentiation of human adipose derived stem cells into Leydig‐like cells with molecular compounds

Leydig cells (LCs) are the primary source of testosterone in the testis, and testosterone deficiency caused by LC functional degeneration can lead to male reproductive dysfunction. LC replacement transplantation is a very promising approach for this disease therapy. Here, we report that human adipose derived stem cells (ADSCs) can be differentiated into Leydig‐like cells using a novel differentiation method based on molecular compounds. The isolated human ADSCs expressed positive CD29, CD44, CD59 and CD105, negative CD34, CD45 and HLA‐DR using flow cytometry, and had the capacity of adipogenic and osteogenic differentiation. ADSCs derived Leydig‐like cells (ADSC‐LCs) acquired testosterone synthesis capabilities, and positively expressed LC lineage‐specific markers LHCGR, STAR, SCARB1, SF‐1, CYP11A1, CYP17A1, HSD3B1 and HSD17B3 as well as negatively expressed ADSC specific markers CD29, CD44, CD59 and CD105. When ADSC‐LCs labelled with lipophilic red dye (PKH26) were injected into rat testes which were selectively eliminated endogenous LCs using ethylene dimethanesulfonate (EDS, 75 mg/kg), the transplanted ADSC‐LCs could survive and function in the interstitium of testes, and accelerate the recovery of blood testosterone levels and testis weights. These results demonstrated that ADSCs could be differentiated into Leydig‐like cells by few defined molecular compounds, which might lay the foundation for further clinical application of ADSC‐LC transplantation therapy.     

Construction and characterization of osteogenic and vascular endothelial cell sheets from rat adipose-derived mesenchymal stem cells

In this study, adipose-derived mesenchymal stem cells (ADSCs) were isolated from adipose tissues of rats. Flow cytometry identification showed that ADSCs of passage 3 highly expressed CD29 and CD44, but hardly expressed CD31 and CD45. Adipogenic, osteogenic, and chondrogenic differentiation were confirmed by the results of oil red O staining, alkaline phosphatase (ALP), and alcian blue staining, respectively. ADSCs at a density of 1 × 10⁶/cm² were cultured in the osteogenic medium and the osteogenic cell sheets could be obtained after 14 d. The cell sheets were positive with von kossa staining. The transmission electron microscopy (TEM) result showed that needle-like calcium salt crystals were deposited on the ECM. These results suggested that the osteogenic cell sheets may have potential osteogenesis ability. ADSCs at a density of 1 × 10⁶/cm² were cultured in the endothelial cell growth medium-2 and the endothelial cell sheets can be formed after 16 d of culture. The TEM image confirmed that the Weibel-Palade corpuscle was seen in the cells. The expression of CD31 was positive, suggesting that the endothelial cell sheets may have a strong ability to form blood vessels. In this study, two types of cell sheets with the potential abilities of osteogenesis and blood vessels formation were obtained by induced culture of ADSCs in vitro, which lays a foundation to build vascularized tissue engineered bone for the therapy of bone defects.     

Incorporation of DMSO and dextran-40 into a gelatin/alginate hydrogel for controlled assembled cell cryopreservation

A new cell cryopreservation strategy for cell-assembling constructs was proposed. With this strategy, different concentrations of dimethysulfoxide (DMSO) and dextran-40 were directly incorporated into the cell/gelatin/alginate systems, prototyped according to a predesigned structure, cryopreserved at −80 °C for 10 days and followed a thawing process at 17 °C. The rheological properties, bonding water contents and melting points of the gelatin/alginate hydrogel systems were changed with the addition of different amounts of DMSO. The microscopy analysis, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrasodium bromide (MTT) and hematoxylin and eosin (HE) staining indicated that the cell numbers were progressively in a selected DMSO concentration range. With DMSO 5% (v/v) alone, the metabolic rate in the construct attained (81.3 ± 5.7)%. A synergistic effect was achieved with the combination of the DMSO/gelatin/alginate and dextran-40/gelatin/alginate hydrogel systems. These results indicated that the inclusion of DMSO and dextran-40 in the hydrogel could effectively enhance the cell preservation effects. This cryopreservation strategy holds the ability to be widely used in organ manufacturing techniques.     

Knockdown of miR-423-5p simultaneously upgrades the eNOS and VEGFa pathways in ADSCs and improves erectile function in diabetic rats

This study aimed to explore the possibility of miR-423-5p modified adipose-derived stem cell (ADSCs) therapy on streptozotocin (STZ)-induced diabetes mellitus erectile dysfunction (DMED) rats. MiR-423-5p was knocked down in ADSCs. ADSCs, NC-miR-ADSCs and miR-ADSCs were co-cultured with human umbilical vein endothelial cells (HUVECs). Normal and high glucose media were supplemented. The supernatant and HUVECs were collected for assessment of eNOS and VEGFa expression, cell proliferation, and apoptosis. HUVECs co-cultured with ADSCs or miR-ADSCs exhibited higher eNOS and VEGFa protein expression levels compared to DM groups. MiR-ADSCs enhanced HUVEC proliferation compared to the ADSCs and NC-miR-ADSCs. Lower apoptotic rates were observed when HUVECs were co-cultured with miR-ADSCs, compared to ADSCs and NC-miR-ADSCs. Fifteen male Sprague-Dawley (SD) rats aged 12 weeks were induced to develop diabetes mellitus by intraperitoneal injection with STZ, and five healthy SD rats were used as normal controls. Eight weeks after developing diabetes, the rats received ADSCs and miR-ADSCs via injection into the corpora cavernosa, whereas normal controls and DM controls were injected with saline. Erectile function and histological assessment of penile tissues were performed 8 weeks after injection. The ICP/MAP indicated that erectile function was impaired in the DM rats compared with the normal group. Injection of ADSCs and miR-ADSCs improved erectile function significantly and was associated with the overexpression of eNOS and VEGFa. MiR-423-5p knockdown in ADSCs ameliorated high glucose-mediated damage to HUVECs and improved erectile function in DM rats by inducing eNOS and VEGFa overexpression, indicating that miR-423-5p may be a potential target in the treatment of DMED.