以人皮肤成纤维细胞作为饲养层细胞培养人胚胎干细胞

目的：比较人皮肤成纤维细胞（humandermalfibroblasts,HDFs）与小鼠胚胎成纤维细胞（Mouseembryonicfibroblasts,MEFs）的增殖能力及研究人皮肤成纤维细胞作为饲养层支持人胚胎干细胞（humanembryonicstemcells,hESCs）未分化生长的能力。方法：利用组织贴壁法从人皮肤中分离出HDFs,通过细胞形态的观察和生长曲线的绘制比较HDFs与MEFs的体外增殖能力。将HDFs作为饲养层细胞与hESCs共培养,传代12代后,检测hESCs碱性磷酸酶（AKP）、表面特异性标志及胚胎干细胞特异性转录因子。结果：HDFs可连续传代培养15代以上,10代以下的HDFs增殖迅速,而MEFs自第4代起,增殖能力就明显下降;hESCs在HDFs饲养层上可传代培养12代以上,克隆边界清晰,细胞排列紧密,碱性磷酸酶、表面标志物检测均呈阳性,表达了hESCs特异性转录因子。结论：HDFs比MEFs具有更强的增殖能力;HDFs可作为培养hEscs的饲养层细胞。     

人孤雌胚胎干细胞在人包皮成纤维细胞饲养层上的生长状态

人孤雌胚胎干细胞(human parthenogenetic embryonic stem cells,hPESCs)体外培养常需饲养层的支持以保持干细胞特性.通过原代培养获得人包皮成纤维细胞(human foreskin fibroblasts,hFFs)并将其制备成饲养层,使hPESCs在hFFs上进行体外培养及传代.倒置显微镜下观察hPESCs的生长状态,采用碱性磷酸酶(alkalinephosphatase,AKP)检测、核型分析和体内分化实验研究hPESCs的生物学特性及分化潜能,以探索hFFs能否长期支持hPESCs的生长并维持其未分化状态.经原代培养成功获得了hFFs,通过形态学观察和免疫细胞化学染色鉴定符合成纤维细胞的生物学特性;在hFFs上生长的hPESCs克隆形态规则,不易分化;已成功在体外培养20余代,hPESCs仍能够保持基本生物学特性和正常核型,在裸鼠体内可形成含有3个胚层组织成分的畸胎瘤.作为人源性饲养层,hFFs可长期支持hPESCs的生长并维持其未分化状态.     

人脐带间充质干细胞研究进展及应用前景

人脐带问充质干细胞（hUCMSC）是来源于发育早期中胚层和外胚层、存在于脐带沃顿胶和血管周围组织中的一类具有自我更新、增殖和多向分化潜能的干细胞。当前主要通过分离、扩增传代培养,然后超低温保存的方法提取保存hUCMSC。与其他来源的干细胞相比,hUCMSC具有来源广泛、可塑性强、对供者无不利影响、无伦理争论限制等优势,并且具有很强的向多组织分化的潜能,因此hUCMSC成为在组织工程、造血干细胞移植及基因治疗等研究领域具有巨大潜力的种子细胞,在临床应用方面有十分广阔的前景。     

人滑膜间充质干细胞与人脐带间充质干细胞的生物学性状比较

该文旨在比较人滑膜间充质干细胞(human synovial mesenchymal stem cells,hSMSCs)与人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)的生物学性状.流式细胞仪鉴定hSMSCs和hUC-MSCs.比较两种间...     

人脐带间充质干细胞体内移植的安全性研究

目的:探讨人脐带间充质干细胞(hUCMSC)的成瘤性及其对荷瘤鼠肿瘤生长的影响。方法:分离培养hUCMSC,取第6代细胞裸鼠皮下移植,观察其成瘤性;对荷瘤鼠尾静脉注射移植hUCMSC,观察其对肿瘤生长的影响;体外共培养hUCMSC和MCF-7肿瘤细胞,观察hUCMSC对MCF-7细胞克隆形成率的影响。结果:hUCMSC裸鼠皮下移植30 d,未观察到有肿瘤形成;尾静脉注射移植hUCMSC对荷瘤鼠肿瘤的生长无明显影响;体外共培养结果表明,hUCMSC对MCF-7肿瘤细胞的克隆形成无明显影响。结论:hUCMSC体内移植无成瘤性;静脉移植后对肿瘤生长无显著影响。     

生长因子诱导人脐带/胎盘间充质干细胞定向分化

近年来,间充质干细胞(mesenchymal stem cell,MSC)已成为干细胞领域的研究热点,其不仅支持造血系统,还可在特定的培养条件下向多种组织细胞分化。人脐带和胎盘来源的MSC取材容易,较骨髓间充质干细胞有更广泛的应用前景。本文就含有特定生长因子的培养基诱导人脐带MSC和人胎盘MSC定向分化的研究进展作一简要的综述。     

人脐带间充质干细胞分离培养方法的研究

目的探讨人脐带间充质干细胞(MSCs)体外分离培养的最佳方法。方法无菌条件下采集早产儿(不足37周)和足月儿的脐带,分离MSCs,比较胎龄、脐带新鲜程度、分离方法和不同培养基对脐带MSCs原代培养过程的影响,通过免疫荧光法检测脐带MSCs表面标记物的表达情况,观察脐带MSCs的生物学特性。结果足月分娩,新鲜脐带,采用组织块平铺法和MesencultTM培养基,脐带MSCs原代培养成功率较高。相同条件下,早产儿脐带MSCs原代培养成功率低于足月分娩脐带。人脐带MSCs高表达CD44、CD90和CD29。结论筛选出一种人脐带MSCs体外分离培养的最佳方法。     

人脐带间充质干细胞抑制卵巢癌的初步研究

目的:从足月剖腹产分娩新生儿脐带中分离出人脐带间充质干细胞(UC-MSCs),探讨其在体外促进SKOV3卵巢癌细胞凋亡,抑制其增殖的作用。方法:新鲜人脐带洗净后剥离动静脉及脐带外膜,得到脐带Wharton's胶。采用组织块贴壁法分离、纯化得到UC-MSCs细胞,光镜下观察UC-MSCs细胞的形态及贴壁生长情况。收集UC-MSCs细胞培养上清,加入SKOV3细胞共培养后,观察不同作用时间(12 h,24 h,36 h,48 h,60 h,72 h)其体外促进SKOV3卵巢癌细胞凋亡,抑制其增殖的作用。结果:光镜下UC-MSCs细胞成长梭状,单核,并成放射或漩涡状排列。PI染色提示,随着UC-MSCs细胞培养上清对SKOV3卵巢癌细胞作用时间的增加,其发生凋亡的细胞数量增多,且具有统计学意义(P0.05)。MTT实验提示SKOV3细胞增殖活力随UC-MSCs细胞培养上清作用时间的增加而显著下降(P0.05),共培养24 h,48 h,72 h的抑制率分别为17.08%,35.36%,46.83%。结论:UC-MSCs在体外具有明显促进SKOV3卵巢癌细胞凋亡,抑制其增殖的作用。     

人脐带间充质干细胞抗肿瘤机制的研究进展

人脐带间充质干细胞(HUMSCs)是一种具有高度自我更新能力和多向分化潜能的干细胞群,具有分泌特定细胞因子、诱导肿瘤细胞凋亡、适用于基因编辑、安全性好及肿瘤趋向性等特性。有较多的研究者研究HUMSCs对肿瘤的作用,试图将HUMSCs作为肿瘤治疗的新方法。就HUMSCs抗肿瘤作用的研究进展作一综述。     

人脐带间充质干细胞重复静脉注射动物毒性试验

摘要 目的：评价多次尾静脉注射脐带间充质干细胞（hUC-MSCs）对小鼠的体内毒性作用。方法：48只健康ICR小鼠,按性别和体重随机分为4组（即对照组、低剂量组、中剂量组和高剂量组）。小鼠通过微静脉注射不同剂量hUC-MSCs悬浮液,间隔3天给药1次,共给药4次。记录小鼠摄食量、体重、体温,给药结束后恢复两周后牺牲动物作大体解剖,检查各个器官器质性病变;利用流式细胞仪分别检测CD3、CD4、CD8阳性细胞亚群数量;ELISA试剂盒检测血清IgM、IgG、C3、C4指标;对肺脏、脾脏、肾脏行组织病理学检查。结果：实验组与对照组相比较,注射不同剂量干细胞后一般观察、体重、体温、摄食量、IgM以及C3在给药期和恢复期均未发生显著变化。在恢复期,注射中、高剂量hUC-MSCs组血清IgG和C4水平略有降低,但未达到显著水平P<0.05;CD4阳性T细胞集群数量以及CD4/CD8系数在hUC-MSCs中、高剂量组显著上升（P<0.05）。大体剖检,除脾脏相比溶媒对照组略显增大外其它各器官均未发现肉眼可见明显异常;称重后发现hUC-MSCs高剂量组脾重量与溶媒对照组相比显著升高（P<0.05）。脾脏、肺脏、肾脏病理学检测未见明显异常。结论：健康ICR小鼠尾静脉注射临床剂量hUC-MSCs（1×10⁶ cells/kg）可能调动动物免疫反应,此外,未观察到hUC-MSCs对小鼠有明显毒副作用。     

Proliferative feeder cells support prolonged expansion of human embryonic stem cells

Long-term proliferation of human embryonic stem (hES) cells is currently achieved by co-culturing with mitotically inactive primary mouse embryonic fibroblasts (mEFs), which serve as feeder cells. This study explores the possibility that proliferative mEFs can be used as feeder cells to maintain the prolonged expansion of hES cells. All undifferentiated hES cell clumps were re-plated on six different densities of proliferative mEFs. hES colonies cultured on 1 x 10(5) - 5 x 10(5) proliferative mEFs amplified over 130 days of continuous culture and remained undifferentiated, as did those cultured on mitotically inactive mEFs. This suggests that certain densities of proliferative mEFs can maintain the propagation of hES cells, which may be helpful for identifying the cytokines and adhesion molecules that are required for their self-renewal.     

人脐带间充质干细胞移植治疗脊髓损伤的研究进展

脊髓损伤（SCI）由于复杂病理生理和神经修复再生困难,至今仍旧是难以攻克的医学难题,而干细胞因其神经再生和神经保护特性被认为是治疗SCI最有希望的方法。其中人脐带间充质干细胞（HUC-MSCs）近年培养分化方法不断改进、神经修复机制初步阐明,联合移植等综合治疗方案也不断实践,使HUC-MSCs移植治疗效果提高。另外关于HUC-MSCs治疗SCI的临床试验逐渐开展,术后患者神经功能恢复改善且无严重并发症出现,表明干细胞移植应用于人体是安全有效的。本文就HUC-MSCs治疗SCI的研究状况及进展进行综述。     

Me2SO- and serum-free cryopreservation of human umbilical cord mesenchymal stem cells using electroporation-assisted delivery of sugars

Cryopreservation is the universal technology used to enable long-term storage and continuous availability of cell stocks and tissues for regenerative medicine demands. The main components of standard freezing media are dimethyl sulfoxide (hereinafter Me₂SO) and fetal bovine serum (FBS). However, for manufacturing of cells and tissue-engineered products in accordance with the principles of Good Manufacturing Practice (GMP), current considerations in regenerative medicine suggest development of Me₂SO- and serum-free biopreservation strategies due to safety concerns over Me₂SO-induced side effects and immunogenicity of animal serum.In this work, the effect of electroporation-assisted pre-freeze delivery of sucrose, trehalose and raffinose into human umbilical cord mesenchymal stem cells (hUCMSCs) on their post-thaw survival was investigated. The optimal strength of electric field at 8 pulses with 100 μs duration and 1 Hz pulse repetition frequency was determined to be 1.5 kV/cm from permeabilization (propidium iodide uptake) vs. cell recovery data (resazurin reduction assay).Using sugars as sole cryoprotectants with electroporation, concentration-dependent increase in cell survival was observed. Irrespective of sugar type, the highest cell survival (up to 80%) was achieved at 400 mM extracellular concentration and electroporation. Cell freezing without electroporation yielded significantly lower survival rates. In the optimal scenario, cells were able to attach 24 h after thawing demonstrating characteristic shape and sugar-loaded vacuoles. Application of 10% Me₂SO/90% FBS as a positive control provided cell survival exceeding 90%. Next, high glass transition temperatures determined for optimal concentrations of sugars by differential scanning calorimetry (DSC) suggest the possibility to store samples at −80 °C. In summary, using electroporation to incorporate cryoprotective sugars into cells is an effective strategy towards Me₂SO- and serum-free cryopreservation and may pave the way for further progress in establishing clinically safe biopreservation strategies for efficient long-term biobanking of cells.     

Human umbilical cord mesenchymal stem cell transplantation restores damaged ovaries

Ovarian injury because of chemotherapy can decrease the levels of sexual hormones and potentia generandi of patients, thereby greatly reducing quality of life. The goal of this study was to investigate which transplantation method for human umbilical cord mesenchymal stem cells (HUMSCs) can recover ovarian function that has been damaged by chemotherapy. A rat model of ovarian injury was established using an intraperitoneal injection of cyclophosphamide. Membrane‐labelled HUMSCs were subsequently injected directly into ovary tissue or tail vein. The distribution of fluorescently labelled HUMSCs, estrous cycle, sexual hormone levels, and potentia generandi of treated and control rats were then examined. HUMSCs injected into the ovary only distributed to the ovary and uterus, while HUMSCs injected via tail vein were detected in the ovary, uterus, kidney, liver and lung. The estrous cycle, levels of sex hormones and potentia generandi of the treated rats were also recovered to a certain degree. Moreover, in some transplanted rats, fertility was restored and their offspring developed normally. While ovary injection could recover ovarian function faster, both methods produced similar results in the later stages of observation. Therefore, our results suggest that transplantation of HUMSCs by tail vein injection represents a minimally invasive and effective treatment method for ovarian injury.     

人脐带间充质干细胞静脉输注小鼠的安全性

目的探讨C57／BL6J小鼠重复多次尾静脉输注人脐带间充质干细胞后的免疫反应和毒性。方法将SPF级别的32只C57／BL6J小鼠随机分为阴性对照组、细胞移植组,每组16只,雌雄各半,细胞移植组小鼠尾静脉注射分离培养的第5代人脐带间充质干细胞,一次5×10。／只,每周注射一次,连续注射4周;阴性对照组每次注射相同容积的PBS。注射后后观察小鼠的一般症状,末次注射后i周、4周进行血细胞计数、血生化、免疫反应指标、脏器质量测定和组织病理学检查。结果细胞移植组小鼠血细胞计数、血生化、脏器重量和脏器系数与对照组无显著性差异（P〉0．05）,脏器组织病理学在光镜下检查结果与对照组无形态学差别,以及免疫结果测定（T细胞亚群CD3＋、CD4＋、CD8＋及CD4＋／CD8＋）与对照组无显著性差异（P〉0．05）。结论人脐带间充质干细胞重复多次尾静脉输注C57／BL6J小鼠是安全可行的,对受者无明显免疫反应和毒副作用。     

bFGF promotes adipocyte differentiation in human mesenchymal stem cells derived from embryonic stem cells

In this work we describe the establishment of mesenchymal stem cells (MSCs) derived from embryonic stem cells (ESCs) and the role of bFGF in adipocyte differentiation. The totipotency of ESCs and MSCs was assessed by immunofluorescence staining and RT-PCR of totipotency factors. MSCs were successfully used to induce osteoblasts, chondrocytes and adipocytes. MSCs that differentiated into adipocytes were stimulated with and without bFGF. The OD/DNA (optical density/content of total DNA) and expression levels of the specific adipocyte genes PPARγ2 (peroxisome proliferator activated receptor γ2) and C/EBPs were higher in bFGF cells. Embryonic bodies had a higher adipocyte level compared with cells cultured in plates. These findings indicate that bFGF promotes adipocyte differentiation. MSCs may be useful cells for seeding in tissue engineering and have enormous therapeutic potential for adipose tissue engineering.     

In vitro differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocyte-like cells

In addition to long-term self-renewal capability, human mesenchymal stem cells (MSCs) possess versatile differentiation potential ranging from mesenchyme-related multipotency to neuroectodermal and endodermal competency. Of particular concern is hepatogenic potential that can be used for liver-directed stem cell therapy and transplantation. In this study, we have investigated whether human umbilical cord blood (UCB)-derived MSCs are also able to differentiate into hepatocyte-like cells. MSCs isolated from UCB were cultured under the pro-hepatogenic condition similar to that for bone marrow (BM)-derived MSCs. Expression of a variety of hepatic lineage markers was analyzed by flow cytometry, RT-PCR, Western blot, and immunofluorescence. The functionality of differentiated cells was assessed by their ability to incorporate DiI-acetylated low-density lipoprotein (DiI-Ac-LDL). As the cells were morphologically transformed into hepatocyte-like cells, they expressed Thy-1, c-Kit, and Flt-3 at the cell surface, as well as albumin, alpha-fetoprotein, and cytokeratin-18 and 19 in the interior. Moreover, about a half of the cells were found to acquire the capability to transport DiI-Ac-LDL. Based on these observations, and taking into account immense advantages of UCB over other stem cell sources, we conclude that UCB-derived MSCs retain hepatogenic potential suitable for cell therapy and transplantation against intractable liver diseases.     

Mouse embryonic stem cell-derived feeder cells support the growth of their own mouse embryonic stem cells

Feeder cells are usually used in culturing embryonic stem cells (ESCs) to maintain their undifferentiated and pluripotent status. To test whether mouse embryonic stem cells (mESCs) may be a source of feeder cells to support their own growth, 48 fibroblast-like cell lines were isolated from the same mouse embryoid bodies (mEBs) at three phases (10th day, 15th day, 20th day), and five of them, mostly derived from 15th day mEBs, were capable of maintaining mESCs in an undifferentiated and pluripotent state over 10 passages, even up to passage 20. mESCs cultured on the feeder system derived from these five cell lines expressed alkaline phosphatase and specific mESCs markers, including SSEA-1, Oct-4, Nanog, and formed mEBs in vitro and teratomas in vivo. These results suggest that mEB-derived fibroblasts (mEB-dFs) could serve as feeder cells that could sustain the undifferentiated growth and pluripotency of their own mESCs in culture. This study not only provides a novel feeder system for mESCs culture, avoiding a lot of disadvantages of commonly used mouse embryonic fibroblasts as feeder cells, but also indicates that fibroblast-like cells derived from mESCs take on different functions. Investigating the molecular mechanisms of these different functional fibroblast-like cells to act on mESCs will contribute to the understanding of the mechanisms of mESCs self-renewal.     

The immunologic and hematopoietic profiles of mesenchymal stem cells derived from different sections of human umbilical cord

Mesenchymal stem cells （MSCs） have been widely used in allogeneic stem cell transplantation. We compared im- munologic and hematopoietic characteristics of MSCs derived from whole human umbilical cord （UC）, as well as from different sections of UCs, including the amniotic membrane （AM）, Wharton＇s jelly （WJ）, and umbilical vessel （UV）. Cell phenotypes were examined by flow cytometry. Lymphocyte transformation test and mixed lymphocyte reaction were performed to evaluate the immuno-modulatory activity of MSCs derived from UCs. The mRNA expression of cytokines was detected by real- time polymerase chain reaction. Hematopoietic function was studied by co-culturing MSCs with CD34＋ cells iso- lated from cord blood. Our results showed that MSCs separated from these four different sections including UC, W J, UV, and AM had similar biological characteristics. All of the MSCs had multi-lineage differentiation ability and were able to differentiate into osteoblasts, adipocytes, and chondrocytes. The MSCs also inhibited the proliferation of allogeneic T cells in a dose-dependent manner. The relative mRNA expression of cytokines was examined, and the results showed that UCMSCs had higher interleukin-6 （IL6）, ILll, stem cell factor, and FLT3 expression than MSCs derived from specific sections of UCs. CD34＋ cells had high propagation efficiencies when co-cultured with MSCs derived from different sections of UCs, among which UCMSCs are the most efficient feeding layer. Our study demonstrated that MSCs could be isolated from whole UC or specific sections of UC with similar immuno- modulation and hematopoiesis supporting characteristics.     

人脐带间充质干细胞对1型糖尿病鼠肝脏损伤的保护作用

目的探讨人脐带间充质干细胞（hUCMSCs）对初发1型非肥胖型糖尿病（NOD）小鼠肝脏损伤的保护作用。 方法雌性NOD小鼠共33只,饲养9周后,将成模的21只小鼠随机分为糖尿病组和干细胞组,每组10只,其中干细胞（MSCs）组发病后第3天尾静脉注射hUCMSCs 1?次;另取10只未发病小鼠为正常对照组。各组小鼠每周检测随机血糖（GLU）水平,8周后处死小鼠,取肝脏,HE染色后观察肝脏结构改变,ELISA法检测糖基化终末产物（AGEs）水平,Real-time PCR法检测糖基化终末产物受体（RAGE）、NF-κB P65、白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α） mRNA的表达水平。采用单因素方差分析和SNK-q检验进行统计学分析。 结果MSCs治疗8周后,MSCs组小鼠随机血糖（8.46±1.37）mmol/L比T1DM组（32.82±0.59）?mmol/L降低,差异具有统计学意义（P < 0.05）。同时T1DM组肝脏细胞形态异常,炎症细胞浸润,而MSCs组的较T1DM组明显改善。MSCs组小鼠肝脏组织的AGEs浓度（0.72±0.10）μg/ml低于T1DM组（1.35±0.22）μg/ml;同时MSCs组的NF-κB P65、IL-6、TNF-α、RAGE mRNA水平（分别为10.08±1.94、9.31±1.67、11.92±1.82、3.87±0.27）,均低于T1DM组（分别为15.46±3.09、18.04±1.69、22.12±3.23、5.12±0.26）,差异具有统计学意义（P < 0.05）。 结论hUCMSCs可以降低糖尿病小鼠血糖水平,改善肝脏微观病理状态,降低AGEs浓度及某些炎性因子的水平以减轻肝脏损伤。