Stem cell-based therapy for erectile dysfunction

Objective  To review the effect of stem cells in erectile dysfunction as well as their application to the therapy of erectile dysfunction.

Data sources  The data used in the present article were mainly from PubMed with relevant English articles published from 1974 to 2011. The search terms were “stem cells” and “erectile dysfunction”.

Study selection  Articles regarding the role of stem cells in erectile dysfunction and their application to the therapy of erectile dysfunction were selected.

Results  Stem cells hold great promise for regenerative medicine because of their ability to self-renew and to differentiate into various cell types. Meanwhile, in preclinical experiments, therapeutic gene-modified stem cells have been approved to offer a novel strategy for cell therapy and gene therapy of erectile dysfunction.

Conclusion  The transplantation of stem cells has the potential to provide cell types capable of restoring normal function after injury or degradation inerectile dysfunction. However, a series of problems, such as the safety of stem cells transplantation, their application in cell therapy and gene therapy of erectile dysfunction need further investigation.

     

A surface-modified biodegradable urethral scaffold seeded with urethral epithelial cells

Background  Efficient cell adhesion and proliferation is a central issue in cell-based tissue engineering, which offers great promise for repair of urethral defects or strictures. This study evaluated the adhesion and growth of rabbit uroepithelium on a surface-modified three-dimensional poly-L-lactic acid (PLLA) scaffold.

Methods  Urethral mucosa were harvested from male New Zealand rabbits and the urothelium were dissociated and then cultured. Immunocytochemistry on cultured uroepithelium for pancytokeratin and uroplakin II and TE-7 confirmed pure populations. After in vitro proliferation, cells were seeded onto a surface-modified urethral scaffold with non-knitted filaments. The morphology and viability of the cells were examined by immunohistochemical and fluorescence staining. Inverted and scanning microscopes were used to document cell growth and adhesion.

Results  Three to five days after primary culture, the uroepithelial cells gradually became confluent, assuming a cobblestone pattern. The filaments of the urethral scaffold had excellent biocompatibility and allowed growth of the uroepithelium, without affecting viability. The uroepithelial cells adhered to and grew well on the scaffold. After 3–7 days, the cells grew vigorously and meshes of the scaffold were full of uroepitheliums.

Conclusions  The surface-modified urethral scaffold with non-knitted filaments allows the growth of uroepithelium and can serve as a carrier for the tissue engineering of urethra.

     

Three-dimensional cultures of human endometrial cells on Matrigel mimic in vivo morphology

Background  The regulation of endometrial physiology and morphogenesis by the paracrine effectors has been well established using in vivo studies. A more complete understanding of the endometrial function has been delayed due, in part, to a lack of appropriate culture models. In this study, we aimed to simulate the in vivo three-dimensional (3-D) growth pattern of endometrial cells using a 3-D in vitro culture system.

Methods  Isolated endometrial epithelial cells, stromal cells and RL95-2 cells were seeded into culture chambers coated with the extracellular matrix Matrigel and observed using light microscopy. Fluorescence staining and immunohistochemistry were used to assess the morphology.

Results  Depending on the culture conditions, epithelial cells and RL95-2 cells formed multicellular structures on Matrigel; stromal cells remained individually distinguishable or grew together to form 3-D lattice-like structures.

Conclusions  Matrigel provided a good microenvironment for culturing endometrial cells. The cells cultured in the Matrigel-coated chambers closely resembled those seen in vivo.

     

Derivation and characterization of Chinese human embryonic stem cell line with high potential to differentiate into pancreatic and hepatic cells

Background  Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background, which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use, especially in China due to its large population. This study contributes to isolating new Chinese human embryonic stem cell lines with clarified directly differentiation ability.

Methods  Donated embryos that exceeded clinical use in our in vitro fertilization-embryo transfer (IVF-ET) center were collected to establish human embryonic stem cells lines with informed consent. The classic growth factors of basic fibroblast growth factor (bFGF) and recombinant human leukaemia inhibitory factor (hLIF) for culturing embryonic stem cells were used to capture the stem cells from the plated embryos. Mechanical and enzymetic methods were used to propogate the newly established human embryonic stem cells line. The new cell line was checked for pluripotent characteristics with detecting the expression of stemness genes and observing spontaneous differentiation both in vitro and in vivo. Finally similar step-wise protocols from definitive endoderm to target specific cells were used to check the cell line’s ability to directly differentiate into pancreatic and hepatic cells.

Results  We generated a new Chinese human embryonic stem cells line, CH1. This cell line showed the same characteristics as other reported Chinese human embryonic stem cells lines: normal morphology, karyotype and pluripotency in vitro and in vivo. The CH1 cells could be directly differentiated towards pancreatic and hepatic cells with equal efficiency compared to the H1 cell line.

Conclusions  This newly established Chinese cell line, CH1, which is pluripotent and has high potential to differentiate into pancreatic and hepatic cells, will provide a useful tool for embryo development research, along with clinical treatments for diabetes and some hepatic diseases.

     

Preconditioning of stem cells for the treatment of myocardial infarction

Objective  Poor stem cell survival is one of the obstacles for cell regeneration therapy post myocardial infarction (MI) and responsible for unsatisfactory therapeutic effectiveness. Various approaches to improve the status of these cells and increase cell survival have become research foci. The following article is a mini-review on the utilization of cell preconditioning for stem cell survival.

Date sources  The data used in this review were mainly from the articles in Medline and PubMed published from 1990 to 2010. The search terms included “preconditioning, stem cell and myocardial infarction”.

Study selection  Original articles and critical reviews selected were relevant to the review’s theme.

Results  The harsh ischemic and inflammatory microenvironment in the infarcted myocardium offers a significant challenge to the transplanted donor stem cells. Survival of stem cells following transplantation is affected by many factors, such as limited blood supply, nutritional deficiency, hypoxia, oxidative stress, and inflammation. Preconditioning methods have potent cytoprotective effects, which enables cells to maintain a "standby state" through programmed initiation of cell survival pathways.

Conclusions  The findings suggest that cell preconditioning can be used as an effective anti-apoptotic strategy and enable cells to withstand and survive the harsh environment after transplantation.

     

Enhanced invasion in vitro and the distribution patterns in vivo of CD133+ glioma stem cells

Background  Recent studies have suggested that cancer stem cells cause tumor recurrence based on their resistance to radiotherapy and chemotherapy. Although the highly invasive nature of glioblastoma cells is also implicated in the failure of current therapies, it is not clear whether cancer stem cells are involved in invasiveness. This study aimed to assess invasive ability of glioma stem cells (GSCs) derived from C6 glioma cell line and the distribution patterns of GSCs in Sprague-Dawley (SD) rat brain tumor. 

Methods  Serum-free medium culture and magnetic isolation were used to gain purely CD133⁺ GSCs. The invasive ability of CD133⁺ and CD133^– C6 cells were determined using matrigel invasion assay. Immunohistochemical staining for stem cell markers and luxol fast blue staining for white matter tracts were performed to show the distribution patterns of GSCs in brain tumor of rats and the relationship among GSCs, vessels, and white matter tracts. The results of matrigel invasion assay were estimated using the Student’s t test and the analysis of Western blotting was performed using the one-way analysis of variance (ANOVA) test.

Results  CD133⁺ GSCs (number: 85.3±4.0) were significantly more invasive in vitro than matched CD133^– cells (number: 25.9±3.1) (t=14.5, P <0.005). GSCs invaded into the brain diffusely and located in perivascular niche of tumor-brain interface or resided within perivascular niche next to white fiber tracts. The polarity of glioma cells containing GSCs was parallel to the white matter tracts.

Conclusions  Our data suggest that CD133⁺ GSCs exhibit more aggressive invasion in vitro and GSCs in vivo probably disseminate along the long axis of blood vessels and transit through the white matter tracts. The therapies targeting GSCs invasion combined with traditional glioblastoma multiforme therapeutic paradigms might be a new approach for avoiding malignant glioma recurrence.

     

Co-culture with microglia promotes neural stem cells differentiation into astrocytes

Background  Neural stem cells (NSCs) are a self-renewing and multipotent population of the central nervous system (CNS), which are active during development and maintain homeostasis and tissue integrity throughout life. Microglias are an immune cell population resident in the CNS, which have crucial physiological functions in the developing and adult CNS. This study aimed to investigate that whether microglia co-cultured with NSCs could promote astrogliogenesis from NSCs.

Methods  Microglia and NSCs were co-cultured in 24-well insert plates. NSCs were plated in the bottom of the well and microglia in the insert. Fluorescent staining, Western blotting and RT-PCR were used to determine the effect of microglia on NSCs differentiation. 

Results  Co-culture of microglia and NSCs promoted astrogliogenesis from NSCs. Several key genes, such as Notch 1, Notch 2, Notch 3, Hes 5, and NRSF were downregulated, while the critical genes Id1 and Id2 were upregulated. BMP2 and FGF2 were upregulated.

Conclusion  Microglias act as a regulator of NSCs astrogliogenesis.

     

In vivo transplantation of bone marrow mesenchymal stem cells accelerates repair of injured gastric mucosa in rats

Background  Adult stem cells provide a promising alternative for the treatment of injured tissues. We aimed to investigate the effect of in vivo transplantation of bone marrow mesenchymal stem cells (BMMSCs) on injured gastric mucosa in rats.

Methods  The gastric ulcer in rats was induced by indomethacin. BMMSCs from male rats, labeled with the fluorescent cell linker 5,6-carboxyfluorescein diacetate succinimidyl ester (CFDA SE), were transplanted into the female rats via tail vein injection. The healing process of gastric ulcers was monitored by HE staining. The protein levels of vascular endothelial growth factor (VEGF) and the epidermal growth factor receptor (EGFR) in the injured gastric mucosa were determined by immunohistochemistry. 

Results  At 48 and 72 hours after BMMSCs transplantation, the CFDA SE labeled cells were found scattered in the injured gastric mucosa, but not in the gastric mucosa of control rats. At 72 hours after BMMSCs transplantation, the mean ulcer index was 12.67±2.16 in the BMMSCs transplanted group and 17.33±1.97 in vehicle-treated controls (P <0.01). Both VEGF and EGFR protein expression levels were significantly higher in the gastric section from the rats that received BMMSCs transplantation as compared to rats without BMMSCs transplantation.

Conclusion  Autologous BMMSCs transplantation can accelerate gastric ulcer healing in injured gastric mucosa in a rodent model.

     

Chemoresistance of CD133+ cancer stem cells in laryngeal carcinoma

Background  Mounting evidence suggests that tumors are histologically heterogeneous and are maintained by a small population of tumor cells termed cancer stem cells. CD133 has been identified as a candidate marker of cancer stem cells in laryngeal carcinoma. This study aimed to analyze the chemoresistance of CD133⁺ cancer stem cells.

Methods  The response of Hep-2 cells to different chemotherapeutic agents was investigated and the expression of CD133 was studied. Fluorescence-activated cell sorting analysis was used to identify CD133, and the CD133⁺ subset of cells was separated and analyzed in colony formation assays, cell invasion assays, chemotherapy resistance studies, and analyzed for the expression of the drug resistance gene ABCG2.

Results  About 1%–2% of Hep-2 cells were CD133⁺ cells, and the CD133⁺ proportion was enriched by chemotherapy. CD133⁺ cancer stem cells exhibited higher potential for clonogenicity and invasion, and were more resistant to chemotherapy. This resistance was correlated with higher expression of ABCG2.

Conclusions  This study suggested that CD133⁺ cancer stem cells are more resistant to chemotherapy. The expression of ABCG2 could be partially responsible for this. Targeting this small population of CD133⁺ cancer stem cells could be a strategy to develop more effective treatments for laryngeal carcinoma.

     

胶质瘤干细胞分离鉴定及其化疗敏感性研究

目的:探讨胶质瘤干细胞的分离、培养、鉴定及其与胶质瘤细胞化疗敏感性方面的差异.方法:将3株人胶质瘤细胞SHG-44、U251、U87MG分别接种于无血清培养基,从中分离出各自胶质瘤干细胞;扫描电镜观察胶质瘤干细胞的形态;RT-PCR法检测胶质瘤干细胞CD133、GFAP的表达;CCK8法检测化疗药物顺铂及长春新碱分别作用后胶质瘤细胞及其干细胞活性的变化.结果:从3株胶质瘤细胞中分离出的胶质瘤干细胞,可在体外增殖及传代,表达特异性标志物CD133,不表达GFAP.CCK8法检测出胶质瘤干细胞较胶质瘤细胞更为耐药(均P＜0.05).结论:胶质瘤干细胞较胶质瘤细胞化疗敏感性降低.     

CD133~+人脐血造血祖细胞的干性维持培养及鉴定

目的从人脐带血中分选出CD133+造血祖细胞,并进行长时间干性维持培养。方法通过免疫磁珠法分选出人脐带血中的CD133+造血祖细胞,经流式细胞仪检测免疫磁珠分选后的CD133+造血祖细胞。采用五种方法扩增培养该细胞,8周后,再通过细胞形态学、流式细胞术、免疫细胞化学和免疫荧光对细胞进行干性鉴定,探索最佳干性维持培养方法。结果通过免疫磁珠法可以从人脐带血中分选出80%以上的CD133+造血祖细胞。采用优化的无血清培养基培养8周之后,CD133+造血祖细胞可得到有效扩增。而其他的培养基会使CD133+造血祖细胞由半悬浮细胞分化为梭形贴壁细胞,并且细胞状态欠佳。结论利用免疫磁珠法分选出的CD133+造血祖细胞,采用优化的无血清培养基能够有效扩增该细胞,并可长期有效的维持其干性。     

替莫唑胺联合二甲双胍对胶质瘤干细胞的清除作用及其机制

目的:探讨替莫唑胺(TMZ)联合二甲双胍(MET)对体外胶质瘤干细胞(GSCs)的清除作用,探讨其作用机制。方法:神经干细胞培养基培养人胶质瘤U87细胞,免疫荧光法鉴定GSCs。收集GSCs,以对照液、不同浓度TMZ、MET和TMZ+MET作用细胞,分为对照组、TMZ组、MET组和TMZ+MET组。显微镜下计数各组二次神经球的数量;CCK-8法检测各组GSCs增殖抑制率;流式细胞术和Annexin-PI双染流式细胞术检测GSCs各细胞周期百分比和凋亡率。结果:与对照组比较,TMZ+MET组二次神经球数量以浓度依赖的方式减少;与TMZ和MET组比较,TMZ+EMT组二次神经球数量明显减少(P<0.01)。与对照组比较,TMZ和MET组GSCs增殖抑制率升高;TMZ+MET组GSCs增殖抑制率高于TMZ或MET组,联合指数(CI)小于1。流式细胞术,与TMZ和MET组比较,TMZ+MET组G₂/M期GSCs百分比明显升高(P<0.05);TMZ+MET组GSCs凋亡率明显高于TMZ和MET组(P<0.05)。结论:TMZ联合MET在体外能抑制GSCs的连续自我更新能力并清除GSCs,其机制可能与阻滞GSCs细胞周期进展和诱导细胞凋亡有关联。     

干扰素-α/β体外增敏替莫唑胺对MGMT阳性胶质瘤干细胞作用

【目的】探讨干扰素是否能增加替莫唑胺(TMZ)对O6甲基鸟嘌呤DNA甲基转移酶(MGMT)阳性胶质瘤干细胞的抗肿瘤作用及其可能机制。【方法】采用"悬浮克隆球形成法"对常规培养条件下MGMT阴性表达的胶质瘤细胞株U251、SKMG-4进行诱导,获得MGMT阳性的胶质瘤干细胞U251G、SKMG-4G;应用CCK-8法检测干扰素-α和干扰素-β联合替莫唑胺对MGMT阳性胶质瘤干细胞的杀伤效应;分别应用逆转录PCR(RT-PCR)、Western-blot检测干扰素-α/β作用后,MGMT阳性胶质瘤干细胞MGMT、NF-κB表达的变化。【结果】应用悬浮克隆球形成法,成功将U251、SKMG-4诱导为具有干细胞特征的胶质瘤干细胞U251G、SKMG-4G,Western-blot检测显示胶质瘤干细胞中MGMT蛋白表达明显增高。对MGMT阳性胶质瘤干细胞生长抑制实验显示,干扰素-α/β作用后提高了替莫唑胺的化疗敏感性,杀伤效应显著增强;RT-PCR、Western-blot检测结果表明,干扰素-α/β作用后,MGMT阳性胶质瘤干细胞NF-κB、MGMT在mRNA及蛋白水平表达均明显降低。【结论】对于MGMT阳性的胶质瘤干细胞,干扰素-α/β能够显著增加替莫唑胺的抗肿瘤效应,其机制可能是干扰素-α/β干预后,下调NF-KB的表达,从而降低了MGMT的转录表达,逆转替莫唑胺的化疗耐药。     

Study on the migration of nerve stem cell in vitro Induced by glioma cells

Objective： Previous studies have shown that glioma cells induced the migration of nerve stem cells （NSCs） in vivo. This study was exploring whether this situation could happen in vitro. Methods： Supematant from 05 glioma cell lines or astrocytes cultured in serum-free medium growing in logarithmic phase were separately placed in lower chambers and NSCs were placed in the upper chambers of Trans-well culture system. After 36 h co-incubation, these NSCs spheres occurred on the middle membrane were counted. Results： Results demonstrated that the supematant from 05 glioma cell culture, not from the astrocytes, enhanced the migration of NSCs （ P 〈0.01）. Conclusion： Some components in 05 glioma cell culture can attract the migration of NSCs.     

胶质瘤干细胞的培养和分化研究

目的探讨人类胶质瘤干细胞的体外培养、传代和分化。方法采用机械方法从胶质瘤组织中分离细胞，应用N2培养基进行培养，碱性成纤维细胞生长因子和表皮生长因子刺激细胞扩增；应用免疫组织化学染色对培养的细胞及其分化的细胞进行鉴定。结果从胶质瘤组织当中成功培养出人类的胶质瘤干细胞，培养条件下呈悬浮状态生长，形成神经球，绝大多数的细胞表达波形蛋白和巢蛋白两种神经干细胞的标志物；这种细胞可分化为神经元和星型胶质细胞。结论体外的培养条件下，可从胶质瘤组织中培养出干细胞，能够分化为神经元和星型胶质细胞，为胶质瘤的深入研究莫定基础。     

HMGA1在由胶质母细胞瘤细胞株U251分得的胶质瘤干细胞中的表达

目的分析人类胶质母细胞瘤(glioblastoma multiforme,GBM)细胞株U251和由此分离的胶质瘤干细胞中HMGA1的差异性表达。方法用MACS柱从U251中分离出表达表面标志物CD133的胶质瘤干细胞(glioblas-toma stem cells,GSCs),并用免疫荧光技术和流式细胞分析法对其进行分析。用实时反转录酶-聚合酶链反应技术(real-time PCR,RT-PCR)和蛋白质印迹技术分别探测到两类细胞的HMGA1基因在转录和翻译水平上表达的不同。结果从U251中分离出GSCs,U251中的GSCs约为0.32%。在GSCs中,HMGA1在转录和翻译水平上分别为U251蛋白的(6.13±0.25)倍和(2.75±0.99)倍。结论相比于U251,HMGA1在GSCs是过度表达的。HMGA1的过度表达可能与体内肿瘤干细胞的恶性扩增、侵袭和分化密切相关。HMGA1基因可望成为胶质母细胞瘤的一种生物标记物和治疗的靶向目标。     

BC200通过介导脑胶质瘤细胞干性的维持抑制替莫唑胺的促凋亡作用

目的：探讨血清长链非编码RNA BC200在脑胶质瘤干细胞（GSCs）中的作用及相关作用机制。方法：神经干细胞培养液培养脑胶质瘤U87 MG细胞,并提取成球生长的GSCs;采用免疫荧光法检测GSCs干细胞标志性分子CD133和Nestin;小分子RNA（siRNA）转染沉默GSCs中BC200表达;替莫唑胺（TMZ）200 μmol·L-1处理GSCs 24 h,光镜下观察TMZ对GSCs悬浮细胞球形态的影响;采用Western blot方法检测CD133、Bax、Bcl-2和cleaved caspase-3的表达变化。结果：分离提取的GSCs球状生长,细胞球表面CD133和Nestin呈阳性表达。TMZ处理GSCs 24 h,GSCs成球能力略微下降,未见细胞球状状态明显离散,Bax、cleaved cas-pase-3、Bcl-2未有明显变化（均P＞0.05）。沉默BC200后GSCs CD133表达显著下降（P＜0.05）。BC200沉默后,TMZ处理GSCs发现其成球能力明显减弱、细胞分散;cleaved caspase-3和Bax分别上调（45.36%±4.25%）和（63.23%±5.12%）,Bcl-2下调（31.22%±3.80%）,均P＜0.01。结论：BC200能够维持脑胶质瘤U87 MG细胞的干性,该效应可促进细胞对TMZ诱导凋亡的抵抗。     

CD133免疫磁珠分选脑肿瘤干细胞及其生物学特性

目的:从人脑肿瘤组织中分离、培养、纯化和鉴定脑肿瘤干细胞(BTSCs),探讨CD133免疫磁珠分选方法获取BTSCs的可行性及BTSCs的生物学特性.方法:留取人脑胶质母细胞瘤新鲜标本,分离获得脑肿瘤细胞.应用CD133免疫磁珠分选方法纯化BTSCs;流式细胞仪检测分选阳性率;神经球计数法分析CD133+/-亚群细胞神...