Hair follicle stem cells can be driven into a urothelial‐like phenotype: An experimental study

The aim of this study was to show that conditioned medium might induce transdifferentiation of hair follicle stem cells into urothelial‐like cells. Several conditioned media and culture conditions (skeletal muscle cell conditioned medium, smooth muscle cell conditioned medium, fibroblast conditioned medium, transforming growth factor‐conditioned medium, urothelial cell conditioned medium, and co‐culture of hair follicle stem cells and urothelial cells) were used. The hair follicle stem cells phenotype from rat whisker hair follicles was checked by using flow cytometry and immunofluorescence. Cytokeratins 7, 8, 15 and 18 were used as markers. Urothelial cell conditioned medium increased the expression of urothelial markers (cytokeratin 7, cytokeratin 8, cytokeratin 18), whereas it decreased a hair follicle stem cells marker (cytokeratin 15) after 2 weeks of culture. This process depended on the time of cultivation. This medium was able to sustain the epithelial phenotype of the culture. Other media including a co‐culture system failed to induce similar changes. Smooth muscle conditioned medium resulted in a loss of cells in culture. Hair follicle stem cells are capable of differentiating into urothelial‐like cells in vitro when exposed to a bladder‐specific microenvironment.     

Sox‐9 facilitates differentiation of adipose tissue‐derived stem cells into a chondrocyte‐like phenotype in vitro

The purpose of this study is to test whether ectopic expression of Sox‐9 can induce adipose tissue‐derived stem cells (ASCs) to function as real nucleus pulposus (NP) cells in vitro. Adenoviral vectors expressing Sox‐9 were reported to infect the chondroblastic and human disc cells, which resulted in increased Sox‐9 and type II collagen production. ASCs were isolated from rat inguinal adipose pad, characterized, and transduced in vitro with a retroviral vector encoding the Sox‐9 gene. Sox‐9‐engineered ASCs (ASCs/Sox‐9) were induced for the chondrocyte‐like cell differentiation by 3D cultured in alginate beads and TGF‐β3 for 2 weeks. Expression of exogenous Sox‐9 protein was detected. Type II collagen and Aggrecan gene expressions of induced ASCs/Sox‐9 were measured using real‐time PCR; proteoglycans expressions were measured by checking the glycosaminoglycan content and type II collagen production by enzyme‐linked immunosorbent assay. Isolated ASCs were CD 29⁺/CD44⁺/C‐Kit^?/Lin^?/CD34^?/CD45^?. ASCs/Sox‐9 expressed marked increase in exogenous Sox‐9 protein. After induction, type II collagen gene expression was sevenfold higher in mRNA levels, with an approximately twofold increase in protein levels of ASCs/Sox‐9 compared to ASCs. Type II collagen and proteoglycan productions were significantly increased in the ASCs/Sox‐9 compared to the ASCs. In addition, co‐culture of induced ASCs/Sox‐9 with matured NP cells resulted in enhanced increase in proteoglycan and type II collagen production. Constitutive retroviral expression of Sox‐9 could efficiently induce ASCs differentiation into chondrocyte‐like cells. This novel approach may provide a practicable system for a simple and rapid differentiation of ASCs into chondrocyte‐like cells which may be potentially used as a stem cell‐based therapeutic tool for the treatment of degenerative disc diseases. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1291–1297, 2011     

Olfactory stem cells can be induced to express chondrogenic phenotype in a rat intervertebral disc injury model

BackgroundIn humans, lower back pain is one of the most common causes of morbidity. Many studies implicate degeneration of intervertebral discs as the cause. In the normal intervertebral disc, the nucleus pulposus exerts a hydrostatic pressure against the constraining annulus fibrosus, which allows the disc to maintain flexibility between adjacent vertebrae, while absorbing necessary compressive forces. The nucleus pulposus performs this role because of its hydrophilic gel-like structure. The extracellular matrix of the nucleus pulposus is up to 80% hydrated, as a result of large amounts of the aggregating proteoglycan, chondroitin sulfate proteoglycan (CSPG). This proteoglycan is enmeshed in a randomly orientated network of fine collagen Type II (CT2) fibers.Study design and purposeA useful adult tissue-derived stem cell is that from the olfactory mucosa, the organ of smell. These cells, accessible in humans from nasal biopsies, are multipotent and are able to make many cell types from all germ layers. They are easily grown in vitro and can be expanded to large numbers and stored frozen. These qualities indicate the potential for autologous transplantation for disc repair. In this article, using a rat model, we explore the hypothesis that olfactory stem cells can differentiate into a nucleus pulposus chondrocyte phenotype in vitro, as well as in vivo after transplantation into the injured intervertebral disc.Patient sampleFemale rats (14 weeks) were anesthetized with xylazine/ketamine. The abdominal wall was shaved and injected with local anesthetic (lidocaine) before incision. The ventral part of the lumbar spine, including two intervertebral discs, was exposed. Disc degeneration was then induced in the two exposed discs by needle aspiration of the nucleus pulposus. The prominent spina iliaca posterior superior was used as an anatomical landmark for identification of the first disc. Two weeks later, one injured intervertebral disc was exposed in a second, similar, surgery and 20,000 olfactory neurosphere-derived cells were transplanted with a 25-G needle.Outcome measuresIn vitro induction of nucleus pulposus chondrocyte phenotype is measured by the percentage of cells expressing CT2 and CSPG. In vivo, a successful outcome is evidence of engraftment of donor-derived cells and their expression of CT2 and CSPG.MethodsIn this article, we tested two hypotheses: the first that progenitor cells within olfactory neurospheres could be induced to express markers distinctive of the nucleus pulposus when placed in vitro in a coculture experiment. The second hypothesis tested the same induction in genetically labeled transplanted cells within damaged vertebral discs in vivo. The two markers measured are those held by current literature to engender the necessary cushioning characteristics of nucleus pulposus, CT2 and CSPG.ResultsOur experiments demonstrated virtually 100% induction of these two markers in vitro. Also, this induction was achieved in donor-derived cells after delivery to the nucleus pulposus region of animals whose discs had previously been lesioned 2 weeks before transplant.ConclusionsThese results provide a rationale for moving toward more extensive larger animal studies for assessment of regeneration before human trials where relief of symptoms can be more easily assessed.     

体外人骨髓间充质干细胞向汗腺细胞表型转化的实验研究

目的:研究骨髓间充质干细胞(MSCs)向汗腺细胞分化的可行性.方法:体外分别分离培养、扩增并鉴定MSCs和汗腺细胞,将汗腺细胞置于47℃环境中1 h建立汗腺细胞体外休克模型,继续孵育3～5 d,收集上清液作为条件培养基对MSCs分化诱导,应用免疫组织化学和流式细胞仪法检测对比共培养10 d后MSCs细胞表型的改变.结果...     

骨髓间充质干细胞分化为血管内皮细胞的实验研究

目的：探讨骨髓间充质干细胞（MSCs)分化为肉芽组织中血管内皮细胞的可能性。及其参与创面修复的可能机制。方法：抽取小型香猪的骨髓，经密度梯度离心分离、纯化MSCs，体外培养扩增后，应用溴脱氧尿嘧啶（BrdU)标记技术标记细胞。于已抽取骨髓的小型香猪背部制作全层皮肤缺损创面，即刻以纤维蛋白胶为载体，将已标记的MSC回植到供体动物创面上。术后2、4、6、8、12周切取创面组织，行BrdU和因子Ⅷ（FⅧ）免疫组织化学染色，进行对比观察。结果：BrdU阳性的MSCs多聚集在创面肉芽组织中的小血管周围，且有个别血管内皮细胞也呈现BrdU 阳性。部分BrdU阳性的MSCs胞浆中亦有FⅧ表达。结论：创面愈合过程中，MSCs与肉芽组织中小血管的形成密切相关。在创面微环境下，MSCs可分化为血管内皮细胞，并参与创面修复。     

腺病毒介导的心肌营养素-1基因转染对神经干细胞作用的实验研究

目的:观察腺病毒介导的心肌营养素-1(Adv-CT-1)基因转染对大鼠神经干细胞(NSCs)的作用。方法:分离、培养胚胎大鼠NSCs,以Adv-CT-1基因转染NSCs,应用细胞集落与细胞突起计数、流式细胞仪凋亡检测等技术,检测NSCs的生长与凋亡情况。结果:Adv-CT-1基因转染培养大鼠NSCs可增加NSCs集落数量和诱导分化细胞突起的数量(P<0.05)。在H2O2诱导的NSCs凋亡模型中,转染Adv-CT-1基因的NSCs凋亡细胞数量较对照组减少(P<0.05),存活细胞数增加(P<0.01)。结论:CT-1基因转染可促进NSCs分裂增殖以及细胞突起的生长,减少超氧化损伤诱导的NSCs凋亡发生,促进损伤NSCs的存活。     

增强型绿色荧光蛋白(EGFP)转染对人骨髓基质细胞体外成骨诱导的影响

目的 研究增强型绿色荧光蛋白(EGFP)转染人骨髓基质细胞(MSC)，对其体外诱导后表达成骨表型的影响，探讨EGFP作为骨组织工程种子细胞示踪剂的可行性。方法 构建携带增强型绿色荧光蛋白(EGFP)重组逆转录病毒载体，并转染骨髓基质细胞，G418进行筛选。将转染后稳定表达绿色荧光蛋白的骨髓基质细胞(MSC-EGFP)进行成骨诱导扩增，以未转染的MSC为对照组，分别检测成骨诱导后碱性磷酸酶(AKP)活性，骨钙蛋白(OCN)含量和成骨细胞转录因子(Cbfal)的表达。结果 构建携带增强型绿色荧光蛋白(EGFP)重组逆转录病毒载体，转染后获得稳定表达绿色荧光蛋白的MSC-GFP，其体外经成骨诱导后，同样能表达成骨特征性表型，AKP，OCN，Cbfal表达和未转染组无明显差别。结论 MSC转染EGFP后，不影响其体外成骨诱导后成骨表型的表达，EGFP可作为骨组织工程种子细胞(MSC)良好的示踪剂。     

人脐带间充质干细胞在大鼠睾丸间质内向Leydig细胞分化的实验研究

目的:研究人脐带间充质干细胞(HUMSCs)在大鼠睾丸间质内向Leydig细胞分化的可行性。方法:贴壁法获得HUMSCs,分别通过流式细胞表面抗原染色与三系分化验证其纯度与多向分化能力,用CM-Dil标记HUMSCs后将其移植入大鼠睾丸间质内,并在移植后4周与8周对大鼠睾丸进行免疫荧光染色观察HUMSCs的存活与分化情况,移植后8周获得大鼠睾丸细胞悬液,通过流式细胞染色检测HUMSCs表达Leydig细胞标志物3β-HSD判断细胞分化的效率,通过流式细胞分选获得悬液内CM-Dil标记的在睾丸内分化后的HUMSCs,并对其进行培养,培养3 d后收集培养液,检测其睾酮水平。结果:Leydig细胞标志物CYP11a1在HUMSCs移植8周后有表达,而在移植4周后未见表达。3β-HSD流式染色显示其分化效率约为14.5%,流式细胞分选后细胞可存活,且其培养液内能检测出睾酮。结论:HUMSCs在大鼠睾丸间质内可向Leydig细胞分化。     

Autologous nucleus pulposus primes T cells to develop into interleukin‐4‐producing effector cells: An experimental study on the autoimmune properties of nucleus pulposus

An autoimmune response to herniated nucleus pulposus has been proposed to constitute a pathophysiologic mechanism for inducing sciatica based on the fact that nucleus pulposus under normal conditions is excluded from the development of immunological tolerance. The manifestation of an autoimmune response comprises different steps starting with antigen capture, continuing with activation of T helper (T_H) cells and ending with production of autoantibodies. Activated T_H cells differentiate into either T_H1 cells, predominately producing proinflammatory cytokines such as interferon γ (IFNγ) or a T_H2 subset mainly producing anti‐inflammatory cytokines such as interleukin‐4 (IL‐4). The aim of the present study was to examine if exposure of autologous nucleus pulposus (NP) to the immune system for 3 weeks is potent enough to prime T_H cells to differentiate into T_H2 cells. The study was performed in a pig model allowing the exposure of NP to the immune system. To assess the polarization of T_H cells the intracellular production of IFNγ and IL‐4 was measured in T cells by using flow cytometry. The revealed predominant production of IL‐4 together with low production of IFNγ in T cells after NP exposure to the immune system indicates that nucleus pulposus may prime T_H cells to develop into IL‐4‐producing T_H2 cells after being exposed to the immune system, for example, in association with disc herniation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:97–103, 2009     

定向诱导骨髓基质干细胞向神经元样细胞分化的实验研究

目的建立一套系统的体外分离、培养及诱导人骨髓基质干细胞（BMSCs）分化为神经元样细胞的方法。方法采用人淋巴细胞分离液从骨髓中分离出BMSCs，体外培养、扩增后，加以复合神经诱导剂进行诱导，诱导期间观察细胞形态的变化，并通过免疫细胞化学鉴定诱导细胞表面标志物的表达情况。结果人BMSCs在体外分离、培养、扩增后，经复合神经诱导剂诱导48h后，部分细胞出现胞体收缩和突起伸出，呈现神经元细胞样改变。免疫细胞化学染色显示巢蛋白或鼠抗人神经丝单克隆抗体阳性，兔抗人胶质纤维酸性蛋白阴性。结论本实验成功培养并诱导人BMSCs分化为神经元样细胞，并建立了一套系统的实验方法。     

Insight into curcumin nanomicelle‐induced derangements in male reproduction potential: An experimental study

This study was conducted to investigate the possible effects of nanomicelle curcumin (NMC) on spermatogenesis, sperm parameters and in vitro fertilisation potential. For this purpose, 24 mature male Wistar rats were divided into control and test groups. The animals in test groups received 7.5, 15 and 30 mg kg b.w^?1 of NMC (NO = 6 rats in each group). Following 48 days, the DNA integrity of testicular tissues, tubular differentiation (TDI) and spermiogenesis (SPI) indices, sperm parameters and DNA integrity were analysed. Finally, the in vitro fertilisation potential was investigated via evaluating pre‐implantation embryo generation. The NMC diminished the TDI and SPI ratios. The animals in NMC‐received groups exhibited a remarkable (p < .05) reduction in percentage of alive and motile spermatozoa. Moreover, the NMC enhanced the percentage of spermatozoa with decondensed chromatin and elevated the sperm DNA damage ratio. The testicles of NMC‐received groups exhibited severe DNA fragmentation. The percentages of zygote, 2‐cell, blastocysts and hatched embryos generation were decreased in NMC‐received groups compared to control animals. In conclusion, the NMC adversely affects the spermatogenesis and spermiogenesis processes, which in turn results in reducing the sperm quality. Ultimately, decreased sperm quality results in lower pre‐implantation embryo development.     

Differentiation of mesenchymal stem cells to germ‐like cells under induction of Sertoli cell‐conditioned medium and retinoic acid

The aim of this research was to find a way to differentiate germ cells from umbilical cord Wharton's jelly mesenchymal stem cells (MSCs) to support in vitro spermatogenesis. A small piece of Wharton's jelly was cultured in high‐glucose Dulbecco's modified Eagle's medium in present of 10% foetal calf serum. After the fourth passage, the cells were isolated and cultured in Sertoli cell‐conditioned medium under induction of two different doses of retinoic acid (10^?5, 10^?6 m ). The differentiation of MSC to germ‐like cells was evaluated by expression of Oct4, Nanog, Plzf, Stra8 and Prm1 genes during different days of culture through qPCR. The results showed that there were downregulation of Oct4 and Nanog and upregulation of pre‐meiotic germ cell marker (stra8) and haploid cell marker (Prm1) when MSCs are differentiated over time. The expression of Bax gene (an apoptotic marker) was significantly observed in high dosage of retinoic acid (RA). As a result, RA has positive effects on proliferation and differentiation of MSCs, but its effects are related to dosage. The success of this method can introduce umbilical cord MSC as a source of germ cells for treatment of infertility in future.