应用小鼠早期胚胎活性因子诱导小鼠成纤维细胞再程序化为多能干细胞的实验研究

目的:研究并建立应用早期胚胎活性因子诱导成体细胞再程序化的实验体系。方法:通过体外受精方法大量收集小鼠3.5 d囊胚,从小鼠囊胚中提取早期胚胎蛋白,加入到小鼠成纤维细胞培养体系中,诱导其再程序化为多能干细胞。对得到的多能干细胞采用PCR鉴定Oct3/4基因表达;在诱导多能干细胞分化为脂肪细胞和成骨细胞后,采用油红O及茜素红染色确定其多向分化潜能。结果:诱导再程序化后获得的多能干细胞能够长期传代培养,表达干细胞特异性基因Oct3/4,采用油红O及茜素红染色确定其具有向脂肪和成骨诱导分化潜能。结论:本研究成功应用小鼠囊胚活性因子诱导小鼠成纤维细胞再程序化生成小鼠多能干细胞。     

人胚胎干细胞无饲养层培养体系的研究进展

人胚胎干细胞具有重要的基础研究价值和巨大的临床应用前景。建立一个理想的人胚胎干细胞培养体系是利用它的前提,但传统的饲养层培养体系中的动物源性物质和大量的未知成分影响了研究人员对人胚胎干细胞培养体系的研究,所以建立一个人胚胎干细胞无饲养层培养体系是非常有必要的。本文主要从细胞外基质、条件培养基、无血清培养基和成分确定的培养基等方面对人胚胎干细胞无饲养层培养体系的研究进展进行综述。     

建立植入前遗传学检测人胚胎来源的无动物源性干细胞系

【目的】在无动物源性的干细胞培养体系中建立植入前遗传学检测（PGT）胚胎来源的人胚胎干细胞系,为医学研究提供疾病模型。【方法】在我们的研究中,对无动物源性的人类包皮成纤维细胞饲养层（XF-HFF）与成分确定的培养基（CDM）构建的无动物源性培养体系进行了评估。在该培养体系中,利用染色体平衡易位患者PGT来源的废弃胚胎建立一个新的人胚干细胞（hESC）细胞系。【结果】新建立的人胚胎干细胞系在无动物源性培养系统中可长期培养传代（>45个代次）。核型分析显示,在传代45代次后,新建立的人胚胎干细胞仍保持一致的核型。XF-HFF/CDM中的hESC仍保持多能性。通过荧光免疫染色检测到SSEA-3、SSEA-4、SSEA-1、TRA-1-60、TRA-1-81等多能标志物的表达;RT-PCR分析显示,在XF-HFF/CDM培养体系上生长的hESC中存在干细胞标记。小鼠体内实验也证实了hESC在体内维持其多能性。【结论】本研究建立了PGT来源的人胚胎干细胞系,为进一步建立携带遗传疾病基因的hESC系并为临床研究和治疗提供疾病模型打下了基础。     

LncRNA在多能干细胞早期谱系分化中的作用与机制研究进展

多能干细胞,包括胚胎干细胞和诱导多能干细胞,均为未分化的细胞,具有自我更新和多向分化潜能,为细胞分化提供了无限的细胞来源,对基础研究和临床疾病治疗研究具有重要意义.然而,多能干细胞的早期谱系分化和调控机制尚不完全清楚.长链非编码RNA(lncRNA)能通过精确控制基因表达网络决定多能干细胞的分化方向,在多能干细胞的分化...     

诱导多能干细胞在肝脏疾病中的研究进展

干细胞,特别是诱导多能干细胞(iPS细胞)可分化为有功能的肝细胞,同时避开了胚胎干细胞的伦理问题,为终末期肝病的替代治疗提供了新的种子细胞.文章对iPS细胞在肝脏疾病研究中的实验方法、研究结果以及临床应用前景予以阐述,为iPS细胞在肝病治疗方面的实验和临床应用提供借鉴.     

人羊膜上皮细胞干细胞特性及向胰岛素分泌细胞分化的研究

目的建立人羊膜上皮细胞(hAECs)的分离及培养方法,探讨其向胰岛素分泌细胞分化的能力。方法观察不同培养方法对hAECs生长倍增时间的影响以优化其培养体系;通过细胞免疫荧光法检测不同培养代数细胞多能干细胞的表面标记;通过体外添加诱导因子的方法,探讨诱导hAECs向胰岛素分泌细胞分化的潜能。结果在10 ng表皮生长因子培养下,hAECs可长期传代。hAECs表达胚胎干细胞表面标记如阶段特异性胚胎抗原4等;采用悬浮培养法诱导分化,可检测到胰岛发育、胰岛功能基因如胰腺十二指肠同源盒、神经源素3、同源异形盒转录因子6、胰岛素、胰高糖素的表达,免疫组化检测细胞可以表达胰岛素,并且随着外界葡萄糖浓度升高,胰岛素分泌显著增加。结论建立了人羊膜上皮细胞的分离和培养方法(不添加动物血清);在体外可诱导分化为胰岛素分泌细胞。     

诱导多能干细胞向表皮干细胞分化的研究进展

皮肤来源是烧伤及慢性创面皮损修复中亟须解决的一大问题,近年来组织工程皮肤为烧伤创面的修复提供了新的方法,但仍未达到完全意义上的功能愈合.诱导性多能干细胞(iPSc)和胚胎干细胞(ESC)在形态及功能上几近相同,为解决这一难题提供了新的思路和可能手段,且iPSc的诞生克服了ESC在临床应用时涉及的移植免疫排斥与伦理道德问题,因此具有重要的临床应用价值.现就近年来iPSc向表皮干细胞转化的研究做一综述.     

干细胞神经定向分化研究与医学转化

作为一类具有自我更新和多向分化潜能的细胞,人类多能性干细胞,包括人胚干细胞(human embryonic stem cells,hESCs)及人诱导多能干细胞(induced pluripotent stem cells,iPSCs)等,能进一步分化成为多种类型的功能细胞,修复受损组织,有望为解决退行性疾病与组织损伤等难题提供新的途径。根据发育生物学理论,目前已建立有效的干细胞分化方案,可将人多能干细胞分化为特定类型的神经元和胶质细胞,包括GABA能、多巴胺能、脊髓运动神经元,以及星形胶质细胞和少突胶质细胞。本文综述了当前干细胞生物学中的与神经分化相关的几个热点问题,着重介绍人类与小鼠干细胞生物学方面的差异及干细胞研究向再生医学转化中的问题,特别是我国干细胞研究医学转化所面临的机遇与挑战。这些问题的解决将对提高我国的干细胞研究水平,推动干细胞研究向医学应用转化具有重要意义。     

携带α-地中海贫血基因人胚胎干细胞系的建立

【目的】 建立携带α-地中海贫血纯合子基因人胚胎干细胞系.【方法】 收集经胚胎种植前遗传学诊断技术诊断为携带α-地中海贫血纯合子基因的囊胚,机械法分离内细胞团,用无血清培养基进行人胚胎干细胞培养,建立携带α-地中海贫血纯合子基因的人胚胎干细胞系?对所得人胚胎干细胞进行α-地中海贫血基因的DNA序列分析与全能性鉴定. 【结果】 本研究共收集44枚PGD筛查后囊胚,分离12个囊胚内细胞团,建立两株人胚胎干细胞系.两株人胚胎干细胞系均携带α-地中海贫血纯合子基因.两株胚胎干细胞系均有正常的染色体核型.OCT-4基因与细胞表面抗原SSEA-3?SSEA-4?TRA-1-60与TRA-1-80的表达,能够向三胚层细胞分化.【结论】 携带α-地中海贫血纯合子基因的人胚胎干细胞具有向三胚层细胞分化的全能性,携带致病基因的胚胎可用于建立携带遗传疾病基因的人胚胎干细胞系.     

人脑类器官在神经发育疾病研究中的应用

人脑类器官是由人多能干细胞(hPSC)包括人胚胎干细胞(hESC)和人诱导多功能干细胞(hiPSC)衍生而来的三维组织,能模拟人大脑的结构和功能,作为神经发育疾病的体外研究模型独具优势.本文将对神经疾病体外模型建立和发展的历程、脑类器官在神经发育疾病研究中的应用、相关前沿技术和脑类器官技术的结合等进行综述和展望.     

玻璃化冷冻法保存人类胚胎干细胞

[目的]探讨玻璃化冷冻方法保存人类胚胎干细胞的效率.[方法]冷冻保存人类胚胎干细胞,按冷冻方法的不同分为玻璃化冷冻组和慢速冷冻组,比较两组解冻后人类胚胎干细胞的细胞复苏率、克隆生长与分化情况,并比较分析解冻后的与未经冷冻的胚胎干细胞的生长、分化情况,鉴定解冻后人类胚胎干细胞的全能性.[结果]玻璃化冷冻组与慢速冷冻组解冻后的细胞复苏率分别为81.9%和22.8%(P＜0.001).慢速冷冻组解冻后克隆生长较玻璃化冷冻组缓慢且更易分化.两组未分化的胚胎干细胞继续培养的生长与分化情况一致.玻璃化冷冻组解冻后第6代人类胚胎干细胞染色体核型正常,SSEA-4、SSEA-3阳性,表达OCT-4基因.人类胚胎干细胞来源的畸胎瘤包含了来源于3个胚层的组织细胞.[结论]玻璃化冷冻是保存人类胚胎干细胞的有效方法,解冻后的干细胞在继续培养过程中仍可保持其正常核型和全能性.     

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.

     

提升上清液Wnt3a蛋白含量的L-Wnt3a细胞培养体系及其促进多能干细胞诱导黑素细胞分化的效应研究

目的: 明确L-Wnt3a条件培养基的收集方案并标定Wnt3a蛋白浓度,验证其作为多能干细胞向黑素细胞分化体系中Wnt3a来源的作用。方法: 通过观察L-Wnt3a细胞的生长特性及其分泌Wnt3a蛋白量特点,设定收集条件培养基的参数,使用ELISA法对收集到的条件培养基进行Wnt3a蛋白浓度的标定,配制出明确Wnt3a浓度的黑素细胞分化培养基,对胚胎干细胞株WA09及诱导多能干细胞株WTC-11细胞进行黑素细胞的悬浮诱导分化,对获得的诱导黑素细胞进行黑素相关基因表达检测及Masson-Fontana染色等相关验证。结果: 优化后的L-Wnt3a 条件培养基收集体系能够稳定高效获取Wnt3a蛋白,明确Wnt3a浓度的分化体系能够使WA09和WTC-11成功诱导黑素细胞,使用相应浓度的Wnt3a重组蛋白也能够达到相似的效果。结论: 通过优化收集方案能够高效获得明确浓度的L-Wnt3a 条件培养基,将其应用于明确Wnt3a浓度的培养体系能够成功诱导多能干细胞分化为功能性黑素细胞。     

Establishment of human embryonic stem cell line from gamete donors

Background Human embryonic stem (HES) cell derived from human blastocyst can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent. It has exciting potential in human developmental biology, drug discovery, and transplantation medicine. But there are insufficient HES cell lines for further study. Methods Three oocyte donors were studied, and 3 in vitro fertilization (IVF) cycles were carried out to get blastocysts for the establishment of HES cell line. Isolated from blastocysts immunosurgically, inner cell mass (ICM) was cultured and propagated on mouse embryonic fibroblasts (MEFs). Once established, morphology, cell surface markers, karyotype and differentiating ability of the cell line were thoroughly analyzed. Results Four ICMs from 7 blastocysts were cultured on MEFs. After culture, one cell line ( criES-1 ) was established and met the criteria for defining human pluripotent stem cells including a series of markers used to identify pluripotent stem cells, morphological similarity to primate embryonic stem cells and HES reported else where. Normal and stable karyotype maintained over 60 passages, and demonstrated ability to differentiate into a wide variety of cell types. Conclusions HES cell lines can be established from gamete donors at a relatively highly efficient rate. The establishment will exert a widesoread impact on biomedical research.     

Neural stem cells: Brain building blocks and beyond

Abstract

Neural stem cells are the origins of neurons and glia and generate all the differentiated neural cells of the mammalian central nervous system via the formation of intermediate precursors. Although less frequent, neural stem cells persevere in the postnatal brain where they generate neurons and glia. Adult neurogenesis occurs throughout life in a few limited brain regions. Regulation of neural stem cell number during central nervous system development and in adult life is associated with rigorous control. Failure in this regulation may lead to e.g. brain malformation, impaired learning and memory, or tumor development. Signaling pathways that are perturbed in glioma are the same that are important for neural stem cell self-renewal, differentiation, survival, and migration. The heterogeneity of human gliomas has impeded efficient treatment, but detailed molecular characterization together with novel stem cell-like glioma cell models that reflect the original tumor gives opportunities for research into new therapies. The observation that neural stem cells can be isolated and expanded in vitro has opened new avenues for medical research, with the hope that they could be used to compensate the loss of cells that features in several severe neurological diseases. Multipotent neural stem cells can be isolated from the embryonic and adult brain and maintained in culture in a defined medium. In addition, neural stem cells can be derived from embryonic stem cells and induced pluripotent stem cells by in vitro differentiation, thus adding to available models to study stem cells in health and disease.     

Generation of patient-specific pluripotent stem cells and directed differentiation of embryonic stem cells for regenerative medicine

Embryonic stem(ES) cells are pluripotent cells that can give rise to derivatives of all three embryonic germ layers. Due to its characteristics, the patient-specific ES cells are of great potential for transplantation therapies. Several strategies can reprogramme somatic cells back to pluripotent stem cells: nuclear transfer, fusion with ES cells, treatment with cell extract and induction by specific factors. Considering the future clinical use, the differentiation from ES to neurons, cardiomyocytes and many other types of cells currently provide basic cognition and experience to regenerative medicine. This article will review two courses, the reprogramming of differentiated cells and the differentiation of ES cells to specific cell types.     

人体胚胎干细胞蛋白质组学的研究进展

人体胚胎干细胞是一类具有自我更新能力的多潜能细胞,在一定条件下,可分化成超过200种人体细胞类型,它在发育生物学和再生医学中具有重要的研究价值。其多潜能性使得一系列疾病,包括癌症、阿尔茨海默病和帕金森病的治疗看到了希望。而胚胎干细胞蛋白质组学的研究对揭示胚胎干细胞增殖和分化的机制以及其多潜能的维持具有重大意义。在此,总结在过去几年中已报道的部分关于人体胚胎干细胞蛋白质组学研究取得的进步及其对人体胚胎干细胞研究的促进作用。     

胚胎干细胞诱导分化机制及方法

胚胎干细胞是来源于附植前胚胎的内细胞团或附植后胎儿的原始生殖细胞具有发育全能性的细胞。近年来在胚胎干细胞分化机制研究和定向诱导分化方面都有长足进展，但其分化机制和定向分化的方法一直是人们需要解决的难点问题，本文主要就胚胎干细胞的定向分化机制、方法及目前存在的问题等方面进行概括性论述。