Isolation of Mesenchymal Stem Cells from Cryopreserved Human Umbilical Cord Blood

Umbilical cord blood (UCB) is well known to be a rich source of hematopoietic stem cells with practical and ethical advantages. Because mesenchymal stem cells (MSCs) from bone marrow have been regarded as good materials for cell/gene therapy as well as for tissue engineering because of their multidifferentiation potential, a number of trials have been undertaken to isolate MSCs from UCB. However, the results have been controversial, and little has become known about the effect of cryopreservation on the isolation of these stem cells. In this study, we examined the ability of cryopreserved UCB-derived cells to produce MSCs. Various culture conditions, including the seeding concentrations of cells and the media used, were investigated. We were able to obtain adherent cell populations after 3 to 5 weeks in our culture conditions from UCB-derived mononuclear cell fractions that had undergone cryopreservation for 0.1 to 5 years. These cells exhibited a fibroblast-like morphology and typical mesenchymal-like immunophenotypes. The results indicate that cryopreserved human UCB fractions can be used as an alternative source of MSCs for experimental and clinical applications as well as for tissue engineering.     

脐血间充质干细胞及其在脑梗死治疗中的作用

脐血间充质于细胞能分化为神经细胞、星形胶质细胞和少突胶质细胞,免疫原性低,在较宽的时间窗内通过静脉注射也能向脑梗死区迁移,减轻神经功能缺损。脐血间充质干细胞具有来源充足、无创和不涉及伦理学问题等优势。文章对脐血间充质细胞的特点、生物学特性及其在脑梗死治疗中的研究做了综述。     

脑瘫患儿应用脐血间质干细胞的临床安全性研究

目的评价人脐血间质干细胞临床应用于脑瘫患儿的安全性。方法脐血间质干细胞鞘内和静脉注射治疗44例脑瘫患儿,对治疗前、治疗后脑瘫患儿的血常规、肝肾功能、电解质、血清酶学、血清免疫蛋白及补体、T细胞亚群进行对比研究。结果患儿的血常规、肝肾功能、电解质、细胞与体液免疫各项指标治疗前后的检测结果无统计学意义(P>0.05)。结论脐血源神经干细胞的临床应用是安全可行的。     

诱导脐血间充质干细胞分化为神经细胞的研究进展

脐血间充质干细胞是从脐带血中分离和培养的一种多潜能成体干细胞,具有向神经细胞分化的潜能。近年来,脐血间充质干细胞治疗神经退行性疾病、神经损伤、卒中等神经系统疾病成为医学研究的热点之一,并取得了一定的进展。     

人脐带间充质干细胞向心肌样细胞定向分化及其临床移植研究进展

人脐带间充质干细胞是一种非常有潜力的干细胞资源,在一定条件下可分化为骨、软骨、脂肪、神经、肝、心肌等多种组织细胞。因与其他来源干细胞相比,其资源丰富,不存在伦理道德问题,免疫原性弱,并已应用临床移植研究。现就人脐带间充质干细胞向心肌细胞的定向分化及其临床移植应用研究做简要综述。     

骨髓间充质干细胞诱导为心肌样细胞的诱导剂概况

干细胞移植治疗疾病一直是研究的热点,通过诱导剂将骨髓间充质干细胞诱导为心肌样干细胞治疗心肌梗死更是心血管领域研究的热门话题。用于诱导骨髓间充质干细胞的诱导剂品种很多,现对目前常用的诱导剂做一个总体的评述。     

差速贴壁法体外培养人脐血内皮祖细胞的实验研究

目的建立一种经济可靠、稳定、高纯度内皮祖细胞的培养方法。方法采用 Ficoll 密度梯度离心法从人脐血中分离单个核细胞,于包被人纤连蛋白的培养皿中贴壁培养,收集48 h后的悬浮细胞重新贴壁培养至第7天,利用免疫组化、免疫荧光及流式细胞术对培养的细胞进行鉴定。结果培养的细胞呈短梭形、多角型、胞体小;可见到大量典型的内皮祖细胞克隆;vWF 和 flk-1免疫组化细胞阳性率>95%,免疫荧光 Dil-ac-LDL 和 FITC-UEA-1双染阳性的细胞阳性率>98%,流式分析 CD_(133)~+细胞的百分率为(7.0±1.8)%。结论差速贴壁法是一种经济可靠、稳定、高纯度内皮祖细胞的培养方法。     

脐血干细胞移植治疗血管性痴呆的可能性

血管性痴呆足常见的痴呆类型之一,其发生与脑血管病密切相关.脐血中含有丰富的可向神经细胞方向分化的干细胞.干细胞移植治疗缺血性脑血管病和神经系统变性疾病的研究已取得了一些成果,脐血干细胞也有可能用于血管性痴呆的治疗.     

Apoptosis,DAP-Kinase1 Expression and the Influences of Cytokine Milieu and Mesenchymal Stromal Cells on Ex Vivo Expansion of Umbilical Cord Blood-Derived Hematopoietic Stem Cells

Expansion of umbilical cord blood-derived CD34⁺ cells can potentially provide them in numbers sufficient for clinical applications in adult humans. In this study apoptosis rate of expanded cells, mRNA expression and promoter methylation status of DAPK1 were evaluated during cord blood hematopoietic stem cell (CB-HSC) ex vivo expansion using cytokines and a co-culture system with mesenchymal stromal cells (MSCs). Ex vivo cultures of CB-HSCs were performed in three culture conditions for 14 days: cytokines with MSCs feeder layer, cytokines without MSCs feeder layer and co-culture with MSCs feeder layer without cytokine. Total number of cells, CD34⁺ cells and colony forming unit assay were performed during expansion. Flow cytometric analysis by propidium iodide was performed to detection of apoptosis rate in expanded cells. Methylation status of the DAPK1 gene promoter was analyzed using methylation specific PCR, and DAPK1 mRNA expression was evaluated by real time-PCR. Maximum CB-CD34⁺ cells expansion was observed in day 10 of expansion. The highest apoptosis rate was observed in cytokine culture without feeder layer that showed significant difference with co-culture condition. The data showed that ex vivo expansion of CD34⁺ cells in all three culture conditions after 10 days resulted in, significant increased expression of DAPK1, also a significant difference between co-culture without cytokine and two other cytokine culture was observed (p < 0.01). DAPK1gene promoter of expanded CD34⁺ cells at days 5, 10 and 14 of culture remained in unmethylated form similar to fresh CD34⁺ cells. Expression of DAPK1 in hematopoietic cells was increased during 10 days expansion of CD34⁺ cells. Also no methylation of DAPK1 promoter was observed; otherwise it would be capable of initiating some leukemic cell progression or disruption in hematopoietic regeneration.     

人脐带血间充质干细胞治疗脑梗死的临床疗效研究

目的观察人脐带血间充质干细胞治疗脑梗死的临床疗效。方法选取我院2010年9月—2013年2月收治的脑梗死患者100例,将其随机分为治疗组和对照组,各50例。对照组给予常规药物治疗,同时予以康复训练;治疗组在对照组治疗基础上给予人脐带血间充质干细胞治疗。按照美国国立卫生院神经功能缺损评分(NIHSS)和简式Fugl-Meyer运功功能(FMA)评分评价治疗前,治疗后1、2、3个月时两组患者的神经功能缺损情况。结果治疗前、治疗后1个月、治疗后2个月,两组患者NHISS评分和FMA评分比较,差异均无统计学意义(P0.05);治疗后3个月,治疗组NHISS评分低于对照组,FMA评分高于对照组(P0.05)。治疗组有1例患者出现低热反应,余未发现其他不良反应。结论人脐带血间充质干细胞治疗脑梗死疗效明显,可以明显改善患者的临床症状,且不良反应少。     

Quality of Umbilical Cord Blood CD34<Superscript>+</Superscript> Cells in a Double-Compartment Freezing Bag Cryopreserved without a Rate-Controlled Programmed Freezer

The aim of this study was to evaluate how a simple method of cryopreservation influences the quality of CD34+ cells in umbilical cord blood (UCB). The cells were dispensed into a double-compartment freezing bag, cryopreserved at -85 degrees C without a rate-controlled programmed freezer, and stored in the liquid phase of nitrogen. The viability of the CD34+ cells before freezing and after thawing was assessed by flow cytometry with 7-aminoactinomycin D and by colony-forming assays. Twenty UCB units cryopreserved for a median of 92 days were analyzed. Mean CD34+ cell viabilities before freezing were 99.8% +/- 0.4% and after thawing were 99.5% +/- 0.8% in large chambers, 99.6% +/- 0.5% in small chambers, and 99.4% +/- 0.6% in sample tubes. The mean values from colony-forming assays of the viable CD34+ cells before freezing were 30.7 +/- 6.8 (colony-forming units-granulocyte-macrophage [CFU-GM] per 100 viable CD34+ cells) and 68.5 +/- 14.8 (total CFUs per 100 viable CD34+ cells). The CFU-GM and total CFU values after thawing were, respectively, 32.7 +/- 9.0 and 66.0 +/- 13.4 in large chambers, 32.4 +/- 8.1 and 64.5 +/- 16.1 in small chambers, and 30.9 +/- 5.4 and 64.7 +/- 12.4 in sample tubes. The results of the colony-forming assays before freezing and after thawing were not significantly different. Our findings overall indicated that our simple method for the cryopreservation of UCB cells without a rate-controlled programmed freezer does not impair the clonogenic capacity of UCB progenitor cells. This cryopreservation method could provide cellular products adequate for hematopoietic stem cell transplantation.     

In Vitro Study on Mesenchymal Stem Cells： Anti-apoptotic Effects on Cardiac Myocytes

Objectives To investigate the anti-apoptotic effects of mesenchymal stem cells （MSCs） on hypoxic injured cardiac myocytes in vitro. Methods MSCs were isolated from bone marrow of Sprague-Dawley （SD） rats, and cardiac myocytes from neonatal rats. The rat cardiac myocytes were co-cultured with MSCs or MSC-conditioned media in anoxia （95% N2 ±5% CO2） for 72 hours. Cell apoptosis was measured by Hoechst 33258 staining. The expression of Bcl-2 and Bax in cardiac myocytes was tested by Western Blot. Results The apoptotic rate was 51.6% ± 2.4% when cardiac myocytes were cultured in continuous hypoxia and was significantly decreased when cardiac myocytes were cocultured with MSCs or MSC-conditioned media （ 15.1% ± 5.4% and 24. 0% ± 4.2% respectively, P 〈 0. 001 ）. The decreased expression of Bax in the cardiac myocytes was greatly related to the decreasing of apoptosis, but there was no difference in Bcl-2 expression among these groups. Conclusions Co-cultured MSCs showed significant anti-apoptotic effects on cardiac myocytes in continuous hypoxia. The mechanism may be the interact of cell to cell and paracrine of cytokines which effected the expression of Bax in the cardiac myocytes.     

人脐血造血细胞体外扩增与表面标志的关系

目的：对脐血造血细胞体外扩增及移植的最佳时机选择进行探讨。方法：使用SCF，rIL－3，rIL－1β和rlL－6长期培养人脐血造血细胞，观察其体外扩增与表面标志的变化。结果：培养14天细胞增殖达高峰，28天下降，但仍为第7天的29．88倍。CD细胞第14天达高峰，28天后仍维持一定水平。HLA－DR+细胞培养14～21天达高峰，第35天仍较高。CD细胞培养第7天显著增多，14～21天达高峰，28天后明显下降。结论：此培养体系能够促进脐血造血细胞增殖、扩增，并维持其存活。14天造血细胞数量最多，状态最佳，适于移植。     

脐血间充质干细胞的研究进展

脐血干细胞作为间充质干细胞的一种,具有间充质干细胞的多向分化潜能、免疫调控及自我复制等特点,可以在体内或体外特定的诱导分化脂肪、骨、软骨、肌肉、肌腱、韧带、神经、肝、心肌、内皮等多种组织细胞,连续传代培养和冷冻保存后仍具有多向分化潜能且免疫原性低。     

叶酸诱导骨髓基质干细胞分化为神经细胞的浓度探索

目的 探讨叶酸对骨髓基质干细胞诱导分化为神经细胞的最佳浓度.方法 MSCs由正常成年人骨髓经密度梯度离心加贴壁离心获得,取第5代MSCs以1 mmol/L二巯基乙醇(BME)预诱导24 h后,用羟基茴香醚(BHA)、2%二甲基亚砜(DMSO)和3种不同浓度的叶酸诱导分化,采用免疫细胞化学法检测神经细胞特异性抗原标志神经元特异性烯醇化酶(NSE)、胶原纤维酸性蛋白GFAP的表达,MTT(四唑盐比色)法检测不同时间段对BMSCs增殖的影响.结果 不同剂量叶酸组分化为NSE阳性神经元样细胞的百分率都较对照组高(P<0.01) ,但以40 mg/L中等浓度的叶酸神经细胞最多,且神经元比例高于其他组.结论 叶酸诱导BMSCs向神经元分化以40 mg/L浓度为最佳.     

Gelatin-Based Hydrogels Promote Chondrogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells In Vitro

Due to the weak regeneration potential of cartilage, there is a high clinical incidence of articular joint disease, leading to a strong demand for cartilaginous tissue surrogates. The aim of this study was to evaluate a gelatin-based hydrogel for its suitability to support chondrogenic differentiation of human mesenchymal stem cells. Gelatin-based hydrogels are biodegradable, show high biocompatibility, and offer possibilities to introduce functional groups and/or ligands. In order to prove their chondrogenesis-supporting potential, a hydrogel film was developed and compared with standard cell culture polystyrene regarding the differentiation behavior of human mesenchymal stem cells. Cellular basis for this study were human adipose tissue-derived mesenchymal stem cells, which exhibit differentiation potential along the adipogenic, osteogenic and chondrogenic lineage. The results obtained show a promotive effect of gelatin-based hydrogels on chondrogenic differentiation of mesenchymal stem cells in vitro and therefore encourage subsequent in vivo studies.     

骨髓间充质干细胞向神经细胞分化的研究进展

骨髓间充质干细胞(BMSC)是一类具有多向分化潜能的细胞。近年来,许多学者已开始通过体内、体外实验研究BMSC是否具有定向分化为神经细胞的可能性。虽然对化学物质诱导BMSC的某些结果存在争议,但细胞移植和基因治疗的实验研究成果为未来神经系统疾病的治疗和康复展示了美好的前景。     

人骨髓及脐血间充质干细胞的分离培养及鉴定

间充质干细胞有多向分化潜能,以骨髓和脐血中含量最为丰富,但数量很少,须体外分离培养和扩增,但方法尚未统一,影响因素较多,尚缺乏鉴定的金标准.     

骨髓间质干细胞治疗脑梗死

脑梗死的发病率和致残率高,目前的治疗手段均未取得满意临床疗效。骨髓间质干细胞具有多向分化潜能,在体内和体外可转化为神经细胞,对脑梗死模型动物治疗后可取得神经功能改善。bcl-2具有抗凋亡作用,转染骨髓间质干细胞后,细胞内bcl-2高度表达可减少移植后干细胞凋亡。文章就骨髓间质干细胞治疗脑梗死的应用研究进展及前景进行了综述。     

大鼠骨髓间充质干细胞向心肌样细胞诱导分化的体外条件研究

目的研究体外条件下大鼠骨髓间充质干细胞(rat mesenchymal stem cells,rMSCs)向心肌样细胞分化的影响因素。方法采用免疫细胞化学方法检测rMSCs表面CD71、SH2、CD31、 CD34、CD45以及5-氮胞苷诱导后心肌细胞特异性蛋白α-sarcomeric actin、troponin T的表达;细胞计数法计算rMSCs倍增时间;观察不同传代数、不同时间及不同剂量5-氮胞苷、依地酸二钠(ethylenediami- netetraacetic acid tetrasodium salt,EDTA-2Na)及无血清培养液对rMSCs分化潜能的影响。结果rMSCs 表达CD71、SH2,而CD31、CD34和CD45呈阴性表达。第3代rMSCs经10μM 5-氮胞苷诱导后细胞呈现克隆、肌管等结,构,表达α-sarcomeric actin、troponin T等心肌细胞特异蛋白,细胞倍增时间为(100 ±8)h。第1、12代rMSCs诱导后细胞形态未发现明显改变、未出现上述结构,细胞倍增时间分别为 32±4 h、38±3 h。1、15、100μM 5-氮胞苷刺激12-72 h,2周后未发现克隆或肌管结构。5-氮胞苷刺激,以1 mM EDTA处理以及无血清培养液培养rMSCs形成的克隆、肌管结构不典型,α-sarcomeric ac- tin、troponin T表达较弱。结论rMSCs具有体外分化潜能,并且受细胞传代数、细胞倍增时间、诱导浓度和时间、细胞外钙离子浓度及培养条件等因素的影响。