Advanced Properties of Urine Derived Stem Cells Compared to Adipose Tissue Derived Stem Cells in Terms of Cell Proliferation,Immune Modulation and Multi Differentiation

Adipose tissue stem cells (ADSCs) would be an attractive autologous cell source. However, ADSCs require invasive procedures, and has potential complications. Recently, urine stem cells (USCs) have been proposed as an alternative stem cell source. In this study, we compared USCs and ADSCs collected from the same patients on stem cell characteristics and capacity to differentiate into various cell lineages to provide a useful guideline for selecting the appropriate type of cell source for use in clinical application. The urine samples were collected via urethral catheterization, and adipose tissue was obtained from subcutaneous fat tissue during elective laparoscopic kidney surgery from the same patient (n = 10). Both cells were plated for primary culture. Cell proliferation, colony formation, cell surface markers, immune modulation, chromosome stability and multi-lineage differentiation were analyzed for each USCs and ADSCs at cell passage 3, 5, and 7. USCs showed high cell proliferation rate, enhanced colony forming ability, strong positive for stem cell markers expression, high efficiency for inhibition of immune cell activation compared to ADSCs at cell passage 3, 5, and 7. In chromosome stability analysis, both cells showed normal karyotype through all passages. In analysis of multi-lineage capability, USCs showed higher myogenic, neurogenic, and endogenic differentiation rate, and lower osteogenic, adipogenic, and chondrogenic differentiation rate compared to ADSCs. Therefore, we expect that USC can be an alternative autologous stem cell source for muscle, neuron and endothelial tissue reconstruction instead of ADSCs.     

Mesenchymal stromal cells form vascular tubes when placed in fibrin sealant and accelerate wound healing in vivo

Non-healing, chronic wounds are a growing public health problem and may stem from insufficient angiogenesis in affected sites. Here, we have developed a fibrin formulation that allows adipose-derived mesenchymal stromal cells (ADSCs) to form tubular structures in vitro. The tubular structures express markers of endothelium, including CD31 and VE-Cadherin, as well as the pericyte marker NG2. The ability for the MSCs to form tubular structures within the fibrin gels was directly dependent on the stoichiometric ratios of thrombin and fibrinogen and the resulting gel concentration, as well as on the presence of bFGF. Fibrin gel formulations that varied in stiffness were tested. ADSCs that are embedded in a stiff fibrin formulation express VE-cadherin and CD31 as shown by PCR, FACS and immunostaining. Confocal imaging analysis demonstrated that tubular structures formed, containing visible lumens, in the stiff fibrin gels in vitro. There was also a difference in the amounts of bFGF secreted by ADSCs grown in the stiffer gels as compared to softer gels. Additionally, hAT-MSCs gave rise to perfusable vessels that were VE-cadherin positive after subcutaneous injection into mice, whereas the softer fibrin formulation containing ADSCs did not. The application of ADSCs delivered in the stiff fibrin gels allowed for the wounds to heal more quickly, as assessed by wound size, amount of granulation tissue and collagen content. Interestingly, following 5 days of healing, the ADSCs remained within the fibrin gel and did not integrate into the granulation tissue of healing wounds in vivo. These data show that ADSCs are able to form tubular structures within fibrin gels, and may also contribute to faster wound healing, as compared with no treatment or to wounds treated with fibrin gels devoid of ADSCs.     

绿色荧光蛋白标记的犬脂肪干细胞体外复合珊瑚材料的生长特性

目的研究犬脂肪干细胞(AdiposeDividedStromalCells,ADSCs)作为种子细复合珊瑚材料体外培养的生长特性。方法分离犬ADSCs,成骨诱导并转染绿色荧光蛋白(GFP)后,复合珊瑚材料培养。检测细胞总数,激光共聚焦观察细胞形态。结果ADSCs-珊瑚复合物体外增殖良好。激光共聚焦显示培养2周后细胞呈老化形态。结论GFP转染不影响细胞传代、增殖。细胞-材料复合物体外培养7天后回植较为适宜。     

兔脂肪干细胞增强脂肪移植效果的实验研究

目的:建立兔耳脂肪移植模型,观察兔脂肪干细胞（adipose-derived stem cells,ADSCs）复合脂肪颗粒(adipose granule,AG）和富血小板纤维蛋白(platelet-rich fibrin,PRF)移植后的形态学变化,为临床脂肪干细胞移植提供实验依据。方法:以健康新西兰家兔为实验动物,共取24只,随机分成4组(n=6):A组 移植物为AG;B组 AG+PRF;C组 AG+ADSCs;D组 AG+PRF+ADSCs。在术后1、3、6个月,用B超、游标卡尺测量移植脂肪后的体积变化。透射光活体观察各时间点新生血管的形成及HE染色分析其形态学改变。结果:术后1、3、6个月在脂肪存活率、免疫组化、透光观察等方面D组与A、B、C三组比较均有显著性差异（P＜0.05）。结论:ADSCs复合PRF、AG能够显著提高移植脂肪组织的成活率,可为临床脂肪干细胞移植提供实验依据。     

rhBMP-2和rhVEGF转染ADSCs后体外表达及诱导骨缺损成骨修复的实验研究

目的观察重组人骨形成蛋白2（rhBMP-2）和血管内皮生长因子（rhVEGF）转染脂肪源性干细胞（ADSCs）后的体外表达情况,并以含该转染体系的组织工程骨进行大动物负重骨缺损的修复研究。方法将外源性基因rhBMP-2和rhVEGF通过脂质体Lipofectamine^TM 2000介导的方式转染第四代ADSCs,以G418筛选出阳性克隆并扩大培养后,用RT-PCR和SABC免疫组化法在转录和蛋白质水平观察其转染后第2天和第4周表达情况。建立小香猪自体前肢尺骨中段1.5cm骨缺损模型,分3组进行对比试验：（A组）转染了生物活性因子的ADSCs与脱细胞骨基质材料（ACBM）复合而成新型组织工程骨植入修复组;（B组）未转染生物活性因子的ADSCs复合ACBM植入修复组;（C组）未处理组。以x线和组织切片评价其修复效果。结果转基因ADSCs在转染后瞬时和4周时都可表达rhBMP-2和rhVEGF。大动物骨缺损修复实验中,前2周内3组动物均未出现明显的急性炎症反应;连续x线观察和骨切片显示：A组在术后第3个月已完全修复,且修复效果和进程明显优于B组,C组缺损则主要由纤维结缔充填。结论rhBMP2及rh-VEGF转染ADSCs后可获得4周内稳定的表达。携带该缓释体系的新型组织工程骨是一种具有显著成骨能力的优良骨缺损修复材料。     

富血小板纤维蛋白对人脂肪干细胞增殖和成脂分化的影响

目的 探讨自体富血小板纤维蛋白(platelet-rich fibrin,PRF)对体外培养人脂肪来源干细胞(adipose-derived stem cells,ADSCs)增殖及成脂分化的影响.方法 将自愿捐献由脂肪抽吸术获取的脂肪组织进行分离培养ADSCs并鉴定.将第3代ADSCs分为空白对照组和1个PRF膜片组(1PRFM组)和2个PRF膜片组(2PRFM组).倒置显微镜观察细胞生长情况,培养后1、2、3、4、5、6、7d采用四甲基偶氮噻唑蓝比色法(MTT)法检测细胞增殖活性.在第3、5、7、9、11和14天时采用油红O染色法检测细胞成脂分化情况.结果 随着PRFM剂量的增加,细胞增殖数量和成脂率增加,3组差异具有显著统计学意义.结论 PRF能明显促进ADSCs增殖和成脂分化,可以作为自体材料应用于脂肪组织工程的研究.     

人脂肪间充质干细胞静脉移植修复脊髓损伤：相关因子及行为学评价

背景：研究证实脂肪间充质干细胞在体外经丹参等诱导剂诱导后可分化为神经元样细胞,因此有可能成为治疗脊髓损伤新的种子细胞。 目的：探讨脂肪间充质干细胞尾静脉移植后,对急性闭合性脊髓损伤大鼠行为学及损伤脊髓组织中各因子表达的影响。 方法：无菌条件下体外分离培养人脂肪间充质干细胞,传至第4代,将细胞收集并制成浓度为1×109 L-1细胞悬液。盐水对照组、细胞移植组大鼠建立脊髓损伤模型,造模成功后1周,细胞移植组经尾静脉注射1 mL干细胞悬液,盐水对照组同法注射等体积的生理盐水,模型对照组不做任何处理。 结果与结论：与模型对照组和盐水对照组比较,细胞移植组大鼠后肢运动功能明显恢复,BBB评分明显升高(P < 0.05);胶质纤维酸性蛋白阳性表达明显减少(P < 0.05),神经元特异性烯醇化酶、巢蛋白的阳性表达均明显升高(P < 0.05)。移植后3 d及1周,在损伤区及临近的脊髓节段可见经荧光染料标记的脂肪间充质干细胞,主要聚集在受损伤脊髓节段1 cm范围内,呈不均匀分布。提示急性闭合性脊髓损伤大鼠经尾静脉移植人脂肪间充质干细胞后,其行为学得到改善,受损脊髓节段局部神经元细胞分化明显增多,修复速度加快。     

Exosomes from ovarian cancer cells induce adipose tissue-derived mesenchymal stem cells to acquire the physical and functional characteristics of tumor-supporting myofibroblasts

Objective

Most tumor tissue is composed of parenchymal tumor cells and tumor stroma. Mesenchymal stem cells (MSCs) can function as precursors for tumor stromal cells, including myofibroblasts, which provide a favorable environment for tumor progression. A close relationship between tumor cells and MSCs in a tumor microenvironment has been described. Exosomes are small membrane vesicles that are enriched with a discrete set of cellular proteins, and are therefore expected to exert diverse biological functions according to cell origin.

Methods

In the current study, we determined the biological effect of exosomes from two ovarian cancer cell lines (SK-OV-3 and OVCAR-3) on adipose tissue-derived MSCs (ADSCs).

Results

Exosome treatment induced ADSCs to exhibit the typical characteristics of tumor-associated myofibroblasts, with increased expression of α-SMA, and also increased expression of tumor-promoting factors (SDF-1 and TGF-β). This phenomenon was correlated with an increased expression of TGF-β receptors I and II. Analysis of TGF-β receptor-mediated downstream signaling pathways revealed that each exosome activated different signaling pathways, showing that exosomes from SK-OV-3 cells increased the phosphorylated form of SMAD2, which is essential in the SMAD-dependent pathway, whereas exosomes from OVCAR-3 cells increased the phosphorylated form of AKT, a representative SMAD-independent pathway. Taken together, exosomes from ovarian cancer cells induced the myofibroblastic phenotype and functionality in ADSCs by activating an intracellular signaling pathway, although the activated pathway could differ from exosome-to-exosome.

Conclusion

The current study suggested that ovarian cancer-derived exosomes contribute to the generation of tumor-associated myofibroblasts from MSCs in tumor stroma.     

CGF与脂肪干细胞联合应用修复大鼠全层皮肤缺损的研究

目的:探讨以明胶海绵为支架材料,复合浓缩生长因子(concentrated growth factor,CGF)诱导的大鼠脂肪干细胞(adipose-derived stem cells,ADSCs)得到的组织工程皮肤替代物,对大鼠全层皮肤缺损愈合的作用。方法:取健康雄性大鼠脂肪组织分离、培养并鉴定脂肪干细胞,观察CGF对其增殖、分化的影响。在健康雌性大鼠背部制造一直径3 cm全层皮肤缺损,其上覆盖复合脂肪干细胞的明胶海绵(A组)、无细胞的明胶海绵(B组)或不作处理单纯包扎(C组)。术后观察各组缺损的愈合情况,并于7、14、21 d及完全愈合后,取背部全层皮肤作组织学观察。结果:CGF能促进体外培养的ADSCs增殖;术后伤口渗出物、红肿等炎症反应A组ADSCs在体外的增殖;复合CGF与ADSCs的组织工程皮肤替代物利于大鼠皮肤创伤愈合;移植的ADSCs能够存活,并分化为上皮细胞。