搅拌式生物反应器培养促进拟胚体的形成及其向心肌细胞分化

目的：摸索搅拌式生物反应器培养小鼠胚胎干细胞（mESC）的最佳条件,建立一种批量制备拟胚体（EB）的方法。方法：研究mESC不同接种密度及生物反应器初始搅拌速度对EB形成的数量和质量的影响,以细菌培养皿中形成的EB为对照,用抗坏血酸诱导其向心肌细胞分化,比较两种培养体系对EB心肌细胞分化潜能的影响,通过免疫荧光染色及RT PCR对ESC来源的心肌细胞进行鉴定。结果：当mESC接种密度为1×105~3×105个/ml,搅拌速度设定为15~30r/min时,搅拌式生物反应器能高效制备出大量相对均一的EB,EB中几乎没有坏死细胞。与细菌培养皿制备的EB相比,生物反应器培养的EB向心肌细胞分化的效率更高,并表达心肌特异性基因。结论：搅拌式生物反应器培养促进EB的形成及其向心肌细胞分化,是一种更为理想的EB培养系统。     

利用胚胎干细胞分化的拟胚体研究小鼠早期胚胎发育过程

小鼠胚胎干细胞(ES细胞)具有分化的全能性已经得到广泛共识。ES细胞在体外分化所形成的拟胚体在结构上能够模仿早期胚胎发育过程,包括在内细胞团表面形成内胚层、柱状上皮细胞的分化,以及中央空腔的形成。本文介绍利用拟胚体研究小鼠早期胚胎发育过程中各个胚胎阶段的发育、细胞程序性死亡的发生及TGF-β信号在胚胎发育过程中的作用。     

应用旋转生物反应器批量制备拟胚体的研究

以小鼠胚胎干细胞(ES-D3)为模型,应用新型细胞培养系统——STLV型旋转生物反应器(rotarycellculturesystem,RCCS)建立一种批量制备拟胚体(embryoidbodies,EBs)的新方法,研究不同细胞接种密度及培养时间对RCCS内EBs产生效率的影响。为了进一步研究该制备方法是否对EBs的分化潜能产生影响,对照传统方法制备的EBs,利用形态学及RT-PCR方法测定经旋转生物反应器制备的EBs在自发性或诱导条件下(1%DMSO)向心肌细胞的分化能力。结果表明:ES-D3在RCCS内能够高效形成EBs,与传统的直接悬浮法比较,其EBs的形成效率可达到后者的2倍。1×104个/ml为最佳细胞接种密度,培养时间也是在RCCS制备EBs过程中的重要因素之一,培养第4~5天为最佳收获EBs的时间。与悬滴法制备的EBs比较,该方法制备的EBs分化为心肌细胞的潜能未改变。由此,应用旋转生物反应器可以高效制备EBs,该方法制备的EBs可以用于发育生物学等基础及应用领域的相关研究。     

长期培养小鼠胚胎干细胞拟胚体(EB)的观察

胚胎干细胞在体外培养条件下能够维持自我更新,并具有向多种细胞类型分化的能力,因此被广泛用于研究细胞分化的分子机理以及药物筛选.形成拟胚体(Embryoid body,EB)是胚胎干细胞分化常用的技术手段.为了便于今后利用EB做进一步的药物筛选及分化研究,严格规范了形成EB的条件,得到了分化状态均一性很高的EB.利用这一条件,观察到在分化条件下长期培养(长达60 d)的EB中仍有表达各项多能性指标的细胞集落.有关这一现象的进一步分析工作正在进行中.     

人类胚胎干细胞体外诱导分化为神经干细胞

人类胚胎干细胞是替代治疗充满希望的细胞来源. 描述了从人胚胎干细胞诱导分化出神经干细胞的方法. 将人胚胎干细胞系PKU1, PKU2在细菌培养皿中悬浮培养, 分化形成囊性拟胚体. 拟胚体接种至组织培养皿, 加入N2培养液和生长因子bFGF培养2周, 拟胚体贴壁、展开,中心出现灶状增生, 有突起的小细胞. 用机械方法取下此种细胞, 重新接种, 则细胞团悬浮生长,形成神经球. 培养10天后, 将神经球打散成单细胞接种, 该细胞贴壁生长旺盛. 免疫荧光检测显示为几乎100% 纯净的nestin阳性细胞. 将培养液中的生长因子撤除, 继续培养7~10天, 细胞分化为神经元, 该细胞呈现β-tubulin isotype_Ⅲ 阳性、GABA阳性、serotonin阳性、synaptophysin阳性. 在生长因子PDGF-AA诱导下, 细胞分化为星形胶质细胞, 其GFAP阳性; 或少突胶质细胞, 其O4阳性. 可见, 人类胚胎干细胞经上述方法培养可分化为典型神经干细胞, 表达神经干细胞特异的标志分子nestin、能自我更新、具有分化为神经系统三类主要细胞的能力.     

人诱导多能干细胞与人胚胎干细胞分化过程中Oct4/Nanog基因表达的比较

目的：比较通过慢病毒方法获得的人诱导多能性干细胞（iPSCs）与人胚胎干细胞（hESCs）分化过程中全能性基因Oct4、Nanog的表达变化。方法：收集分化不同时间点的拟胚体（EBs）,检测三胚层分化基因以及全能性基因Oct4/Nanog的表达,并通过畸胎瘤组织切片的荧光染色分析Oct4的表达。结果：iPSCs获得的EB中内外三胚层分化基因表达的出现明显晚于hESCs来源的EB。不同于hESCs,iPSCs悬浮培养获得的EBs在体外培养18天未见内源性Oct4、Nanog基因表达的下调。未分化的iPSCs注射严重联合免疫缺陷（SCID）小鼠培养10周后获得的畸胎瘤中仍存在Oct4阳性的细胞,但iPS-#2中明显少于iPS-#5。结论：通过慢病毒方法获得的iPSCs虽然具有向三胚层分化的能力,但在分化过程中仍维持较高水平的全能性基因Oct4、Nanog的表达。     

人诱导多能干细胞与人胚胎干细胞分化过程中Oct4/Nanog 基因 表达的比较

目的:比较通过慢病毒方法获得的人诱导多能性干细胞(iPSCs)与人胚胎干细胞(hESCs)分化过程中全能性基因Oct4、Nanog的表达变化。方法:收集分化不同时间点的拟胚体(EBs),检测三胚层分化基因以及全能性基因Oct4/Nanog的表达,并通过畸胎瘤组织切片的荧光染色分析Oct4的表达。结果:iPSCs获得的EB中内外三胚层分化基因表达的出现明显晚于hESCs来源的EB。不同于hESCs,iPSCs悬浮培养获得的EBs在体外培养18天未见内源性Oct4、Nanog基因表达的下调。未分化的iPSCs注射严重联合免疫缺陷(SCID)小鼠培养10周后获得的畸胎瘤中仍存在Oct4阳性的细胞,但iPS-#2中明显少于iPS-#5。结论:通过慢病毒方法获得的iPSCs虽然具有向三胚层分化的能力,但在分化过程中仍维持较高水平的全能性基因Oct4、Nanog的表达。     

诱导胚胎干细胞心肌定向分化的方法及其机制

胚胎干细胞是具有分化为各种类型组织细胞潜能的全能干细胞,可在体外大量扩增,细胞因子、激素、诱导剂和细胞内转录因子等可诱导和调控胚胎干细胞进行心肌细胞定向分化．这将使干细胞移植治疗心肌损伤性疾病成为可能。该文介绍胚胎干细胞定向心肌分化的诱导因素及其机制的研究进展。     

人胚胎干细胞传代培养及细胞化学染色特性

目的 体外建立人胚胎干细胞传代培养方法,研究人胚胎干细胞细胞化学染色特性.方法 以小鼠胚胎成纤维细胞作为饲养层传代培养人胚胎干细胞,检测人胚胎干细胞、自发分化克隆及拟胚体的细胞化学染色特性.结果 人胚胎干细胞在小鼠胚胎成纤维细胞饲养层上传30代以上其形态保持不变;人胚胎十细胞碱性磷酸酶、过碘酸-雪夫反应、α-醋酸萘酚酯酶染色阳性,自发分化克隆细胞阳性程度明显减弱;人胚胎干细胞形成的拟胚体碱性磷酸酶染色弱阳性,过碘酸-雪夫反应、α-醋酸萘酚酯酶染色阳性.结论 小鼠胚胎成纤维细胞能支持人胚胎干细胞传代培养,细胞化学染色结果能初步鉴别人胚胎干细胞未分化特性.     

小鼠胚胎干细胞分化形成拟胚体过程中的细胞程序性死亡

为了检测小鼠胚胎干细胞 (embryonicstemcell ,ES细胞 )体外分化的拟胚体 (embryoidbodies ,EBs)形成过程中细胞程序性死亡 (programmedcelldeath ,PCD)的发生 ,通过悬滴、悬浮培养技术定向诱导未分化的ES细胞分化为拟胚体 ,并用RT PCR检测原始内胚层、原始外胚层、中胚层、内脏内胚层 4种分子标记物在EBs中的表达 .通过TUNEL染色、电镜、激光共聚焦显微镜及Western印迹以确定凋亡发生 .结果表明 :ES细胞体外分化为拟胚体并且表达各胚层相应的分子标记物 ;在拟胚体的发育过程中出现明显的空腔化过程 ,TUNEL染色及电镜观察到凋亡生成 ,同时线粒体膜电位 (ΔΨm)在拟胚体发育过程中降低 ,通过Western印迹检测到caspase3、caspase8的激活 .表明小鼠ES细胞所分化的拟胚体可以作为研究早期胚胎发育的实验模型 ,线粒体在拟胚体的细胞程序性死亡过程中发挥重要的作用 .为进一步利用拟胚体研究细胞程序性死亡及相关信号分子在小鼠胚胎发育早期的作用奠定了基础     

Cluster characterization of mouse embryonic stem cell-derived pluripotent embryoid bodies in four distinct developmental stages

The formation of embryoid bodies (EBs) is the principal step in the differentiation of embryonic stem (ES) cells. In this study, the morphological characteristics and gene expression patterns of EBs related to the sequential stages of embryonic development were well defined in four distinct developmental groups over 112 days of culture: early-stage EBs groups (1–7 days of differentiation), mid-stage EBs groups (9–15 days of differentiation), maturing EBs groups (17–45 days of differentiation) and matured EBs groups (50 days of differentiation). We first determined definite histological location of apoptosis within EBs and the sequential expression of molecular markers representing stem cells (Oct4, SSEA-1, Sox-2 and AKP), germ cells (Fragilis, Dazl, c-kit, StellaR, Mvh and Stra8), ectoderm (Neurod, Nestin and Neurofilament), mesoderm (Gata-1, Flk-1 and Hbb) and endoderm (AFP and Transthyretin). Our results revealed that developing EBs possess either pluripotent stem cell or germ cell states and that three-dimensional aggregates of EBs initiate mES cell differentiation during prolonged culture in vitro. Therefore, we suggest that this EB system to some extent recapitulates the early developmental processes occurring in vivo.     

The reversal of hyperglycemia after transplantation of mouse embryonic stem cells induced into early hepatocyte-like cells in streptozotocin-induced diabetic mice

Cellular replacement therapy is a potential therapeutic strategy for diabetes. In this study, we investigated the effect of transplantation of induced mouse embryonic stem cells (mESCs) into endoderm and early hepatocyte-like cells in streptozotocin (STZ)-diabetic mice. After embryoid body (EB) formation from mESC, the EBs were cultured in the presence of dexamethasone (DEX) and insulin for 4 days then was added acidic fibroblast growth factor (aFGF), hepatocyte growth factor (HGF) and oncostatin M (OSM) for 10 days, respectively. Blood glucose levels, intraperitoneal glucose tolerance (IGT) test and islet histology were assessed. The result revealed that transplantation of induced mESCs into early hepatocyte-like cells could repair pancreatic islets of control group. Blood glucose levels and intraperitoneal glucose tolerance test were significantly improved in test group compared to control group. Furthermore, there was significant increase in the number of islets in test group compared to control group. The findings declare that induced mESCs into endoderm and early hepatocyte-like cells, are appropriate candidate for regenerative therapy of pancreatic islets in type I diabetes.     

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.     

Temporal changes in the carbohydrates expressed on BG01 human embryonic stem cells during differentiation as embryoid bodies

Cell surface carbohydrates present on BG01 human embryonic stem cells after 28 days of differentiation were examined using two classes of carbohydrate binding proteins: lectins and antibodies specific for carbohydrate epitopes. Specificity of lectin staining was verified using carbohydrate ligands to block lectin interaction, glycohydrolases to cleave specific sugar residues that are receptors for these proteins, and periodate oxidation to destroy susceptible sugar residues. Specific antibodies were used to identify various tissue types and germ layers present in the 12- and 28-day differentiating embryoid bodies. Results from 12 and 28-day differentiated embryoid bodies were compared to determine changes over time. A slight increase in the sialylation of α-GalNAc was seen between 12 and 28 days of differentiation due to the presence of sialyl Tn and/or other sialylated α-GalNAc residues. Increases were also observed in GalNAc, the T antigen (Gal β1,3 GalNAc), and difucosylated LacNAc residues during this time interval. Additionally, some distinct differences in the pattern of lectin staining between 12 and 28 days were observed. Not unexpectedly, the presence of most differentiated cell-types increased during this time period with the exception of neural progenitors, which decreased. Undifferentiated cells, which were prevalent in the 12-day EBs, were undetectable after 28 days. We conclude that several changes in glycosylation occurred during the differentiation of embryonic stem cells, and that these changes may play a role in embryonic development. Lectin abbreviations can be found in Table 1.     

Non-invasive label-free monitoring the cardiac differentiation of human embryonic stem cells in-vitro by Raman spectroscopy

Background

Online label-free monitoring of in-vitro differentiation of stem cells remains a major challenge in stem cell research. In this paper we report the use of Raman micro-spectroscopy (RMS) to measure time- and spatially-resolved molecular changes in intact embryoid bodies (EBs) during in-vitro cardiogenic differentiation.

Methods

EBs formed by aggregation of human embryonic stem cells (hESCs) were cultured in defined medium to induce differentiation towards cardiac phenotype and maintained in purpose-built micro-bioreactors on the Raman microscope for 5 days (between days 5 and 9 of differentiation) and spatially-resolved spectra were recorded at 24 h intervals.

Results

The Raman spectra showed that the onset of spontaneous beating of EBs at day 7 coincided with an increase in the intensity of the Raman bands at 1340 cm^− 1, 1083 cm^− 1, 937 cm^− 1, 858 cm^− 1, 577 cm^− 1 and 482 cm^− 1. The spectral maps corresponding to these bands had a high positive correlation with the expression of the cardiac-specific α-actinin obtained by immuno-fluorescence imaging of the same EBs. The spectral markers obtained here are also in agreement with previous studies performed on individual live hESC-derived CMs.

Conclusions

The intensity profile of these Raman bands can be used for label-free in-situ monitoring of EBs to estimate the efficacy of cardiogenic differentiation.

General significance

As the acquisition of the time-course Raman spectra did not affect the viability or the differentiation potential of the hESCs, this study demonstrates the feasibility of using RMS for on-line non-invasive continuous monitoring of such processes inside bioreactor culture systems.