诱导多潜能干细胞

通过病毒载体导入4个外源转录因子Oct4、Sox2、c-Myc、Klf或者Oct4、Sox2、Nanog、Lin28入体细胞,可以诱导产生具有胚胎干细胞特性相似的诱导多潜能干细胞(induced pluripotent stem cells,iPS).iPS在疾病治疗和药物研究等领域具有非常重要的应用前景,但是目前存在诱导效率低以及致肿瘤性等缺点,采用改良方法诱导产生iPS是将来研究的重点.     

不同干细胞来源外泌体在心血管疾病治疗中的应用、作用及问题

文题释义：外泌体：是一种直径为30-100 nm,由细胞内多泡体与细胞膜融合后释放到细胞外基质中的膜囊泡。外泌体存在于多种体液中,如血清、唾液、羊水、母乳和尿液,同时在细胞培养基中也有外泌体的释放。外泌体含有DNA片段、mRNA、小RNA、功能蛋白及转录因子等多种具有生物活性的物质,其膜结构还表达多种抗原、抗体分子,能产生各种生物学效应,从而在临床治疗疾病中具有广泛的应用前景。 干细胞来源外泌体在心血管疾病方面的应用价值：干细胞来源外泌体有效地规避了干细胞移植带来的缺陷,如免疫排斥反应、致瘤性和输注毒性,这些局限性使干细胞在心血管疾病治疗中黯然失色。干细胞来源外泌体可通过移植到受体心脏来调节各种细胞过程,如增殖、凋亡、应激反应以及分化和血管生成等。因此,干细胞来源外泌体可以作为替代干细胞治疗心血管疾病安全有效的手段。 背景：外泌体含有DNA片段、mRNA、小RNA、功能蛋白及转录因子等多种具有生物活性的物质,同时其膜结构还表达多种抗原、抗体分子,从而能产生各种生物学效应。近年来的研究结果表明其具有类似于干细胞移植的治疗作用,可用于代替干细胞移植治疗心血管疾病。 目的：总结不同干细胞来源外泌体在心血管疾病治疗中的应用,为外泌体治疗心血管疾病提供参考及依据。 方法：通过检索 PubMed数据库2005至 2019年期间的相关文章,检索词为“exosomes,cardiovascular disease,embryonic stem cells,induced pluripotent stem cells,mesenchymal stem cells”,同时检索中国知网、维普数据库2014 至 2019年期间的相关文章,检索词为“外泌体,心血管疾病,胚胎干细胞,诱导多能干细胞,间充质干细胞”。对文献资料及参考文献进行逐一查阅。 结果与结论：干细胞来源外泌体比干细胞移植更安全、有效,在心血管疾病的治疗方面具有巨大潜力,但关于外泌体功能及用途的研究仍处于起步阶段,此外外泌体含量低,提取过程繁琐等问题限制了其临床应用。 ORCID: 0000-0001-8179-1040(刘卒) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

心脏组织工程中细胞和生物支架的研究热点

文题释义：心脏组织工程：是基于组织工程的技术和原理,利用合适来源的细胞和生物支架制造心脏移植物,用于代替受损心脏组织或促进心肌细胞增殖,以恢复或改善心脏功能的技术。 生物支架：是组织工程技术中用于对细胞成分起支撑作用的移植物,其构成成分类似于细胞外基质成分,部分支架具有孔隙等允许血液中氧气及营养物质通过。 背景：心脏组织工程技术的出现和发展,为心血管疾病尤其是心肌梗死的治疗提供了新的选择。 目的：通过对心脏组织工程的2个核心要素即细胞和生物支架的研究进展进行综述,以期为心脏组织工程技术应用于心血管疾病治疗提供参考及依据。 方法：通过检索PubMed 数据库及中国知网数据库2010至2019年期间心脏组织工程相关文章,以“cardiac tissue engineering,cardiomyocytes differentiation,cardiac tissue engineering,cardiomyocytes differentiation,bone marrow derived stem cells,human embryonic stem cells,induced pluripotent stem cells,menstrual blood stem cells,biological scaffolds”为英文检索词,以“心脏组织工程,心肌细胞分化,干细胞,生物支架”等为中文检索词,最终选择78篇英文文献纳入研究。 结果与结论：多种来源的细胞(包括心肌细胞、骨骼肌细胞、心脏成纤维细胞、骨髓来源干细胞、胚胎干细胞、诱导多能干细胞、月经血干细胞)和生物支架(包括水凝胶、脱细胞支架、细胞片及心脏芯片)都可应用于心脏组织工程,但心脏组织工程仍然存在诸多需要解决的问题,如合适的细胞来源、新型支架材料的研发、诱导分化技术的优化,植入时机及途径的优化。 ORCID:0000-0003-2763-5535(王萍) 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders

BACKGROUND: Human embryonic stem (hES) cells are pluripotent cells usually derived from the inner cell mass (ICM) of blastocysts. Because of their ability to differentiate into all three embryonic germ layers, hES cells represent an important material for studying developmental biology and cell replacement therapy. hES cell lines derived from blastocysts diagnosed as carrying a genetic disorder after PGD represent in vitro disease models. METHODS: ICMs isolated by immunosurgery from human blastocysts donated for research after IVF cycles and after PGD were plated in serum-free medium (except VUB01) on mouse feeder layers. RESULTS: Five hES cell lines were isolated, two from IVF embryos and three from PGD embryos. All lines behave similarly in culture and present a normal karyotype. The lines express all the markers considered characteristic of undifferentiated hES cells and were proven to be pluripotent both in vitro and in vivo (ongoing for VUB05_HD). CONCLUSIONS: We report here on the derivation of two hES cell lines presumed to be genetically normal (VUB01 and VUB02) and three hES cell lines carrying mutations for myotonic dystrophy type 1 (VUB03_DM1), cystic fibrosis (VUB04_CF) and Huntington disease (VUB05_HD).     

Screening of human pluripotent stem cells using CGH and FISH reveals low-grade mosaic aneuploidy and a recurrent amplification of chromosome 1q

Pluripotency and proliferative capacity of human embryonic stem cells (hESCs) make them a promising source for basic and applied research as well as in therapeutic medicine. The introduction of human induced pluripotent cells (hiPSCs) holds great promise for patient-tailored regenerative medicine therapies. However, for hESCs and hiPSCs to be applied for therapeutic purposes, long-term genomic stability in culture must be maintained. Until recently, G-banding analysis was considered as the default approach for detecting chromosomal abnormalities in stem cells. Our goal in this study was to apply fluorescence in-situ hybridization (FISH) and comparative genomic hybridization (CGH) for the screening of pluripotent stem cells, which will enable us identifying chromosomal abnormalities in stem cells genome with a better resolution. We studied three hESC lines and two hiPSC lines over long-term culture. Aneuploidy rates were evaluated at different passages, using FISH probes (12,13,16,17,18,21,X,Y). Genomic integrity was shown to be maintained at early passages of hESCs and hiPSCs but, at late passages, we observed low rates mosaiciam in hESCs, which implies a direct correlation between number of passages and increased aneuploidy rate. In addition, CGH analysis revealed a recurrent genomic instability, involving the gain of chromosome 1q. This finding was detected in two unrelated cell lines of different origin and implies that gains of chromosome 1q may endow a clonal advantage in culture. These findings, which could only partially be detected by conventional cytogenetic methods, emphasize the importance of using molecular cytogenetic methods for tracking genomic instability in stem cells.     

诱导多能干细胞建系及体外造血祖细胞定向分化体系的建立

背景：目前,大量文献报道了诱导多能性干细胞系的建立,但大规模体外诱导分化造血祖细胞的研究还缺乏深入的探讨。 目的：建立诱导多能性干细胞体外定向诱导形成造血祖细胞的方法。 方法：采用慢病毒感染的方法将含有Oct4、Sox2、Nanog和Lin28全能性基因的慢病毒颗粒转导人皮肤成纤维细胞,获得了诱导多能性干细胞;在诱导分化体系中添加了Y-27632,克服干细胞扩增中的凋亡现象;运用OP9细胞产生的条件培养液建立诱导多能性干细胞体外定向分化形成造血祖细胞的分化体系。 结果与结论：①前3代细胞克隆传代时,诱导多能性干细胞发生凋亡的现象很多,很难大规模扩增培养。培养基中添加阻断ROCK活化的抑制剂,能够明显抑制胚胎干细胞的凋亡。②诱导多能性干细胞在OP9细胞条件培养液作用下,经过体外诱导分化,形成CD34+造血祖细胞。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

人多能干细胞的冷冻与保存

背景：人多能干细胞的出现与发展是近年来生物医学研究领域的重大突破。但其在基础/临床研究中的广泛应用还有诸多限制,建立安全有效标准化的冷冻保存方案是人多能干细胞广泛应用面临的重大挑战。 目的：回顾人多能干细胞冷冻领域的研究进展,探索造成冷冻损伤的原因和机制及改进方式,致力于促进新的更有效的冷冻方案形成。 方法：以“人多能干细胞、人胚胎干细胞、人诱导多能干细胞、玻璃化、程序化冷冻、慢冻法、冷冻保存”为中文检索词,以“human pluripotent stem cells,human embryonic stem cell,human introduced pluripotent stem cell,vitrification,programmed cryopreservation,slow-freezing,cryopreservation”为英文检索词,应用计算机检索中国知网全文数据库、万方全文数据库、维普(VIP)期刊全文数据库、PubMed数据库有关人多能干细胞冷冻保存技术的文献,排除与研究目的无关及重复文献,保留58篇文献进一步总结分析。 结果与结论：了解人多能干细胞冷冻过程中造成冷冻损伤的原因和机制,是寻找高效的冻存方案的关键。需要更清晰的了解冷冻过程中损伤的原理,改进和创新低温生物技术来避免各种冷冻损伤的发生并致力于探讨可重复的,高效的,符合GMP要求的,能大规模冻人多能干细胞的方案。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Stem cell originated red blood cell cultures: status quo and outlook

The present paper reports the various obstacles cleared during the past decade with the view of generating red blood cells in vitro from various sources of stem cells, for transfusion purposes. We also consider the next developments to be performed for achieving this goal. Starting from the natural source, the haematopoietic stem cells, the major advance resides in the establishment of the proof of principle for transfusion in human, by showing a normal life span of cultured Red blood cells compared to their native counterpart. The best available source of highly proliferative adult stem cells is cord blood, with the capacity of generating the equivalent of 100 units of packed RBC from one average unit. It is, however, a limited source in terms of haematopoietic stem cells and remains dependent on donations as observed from conventional blood supply. Critical advances have allowed the in vitro production of functional RBC from pluripotent human stem cells, embryonic and induced pluripotent stem cells, in the past 5 years. Because induced pluripotent stem cells (iPS) can proliferate indefinitely and be selected for a phenotype of interest, they appear the most favourable source of stem cells. Proof of concept of the generation of RBC from iPS has been made, but still needs to be optimized. We also discuss the key points that remain to be resolved to achieve an application for clinical transfusion. Several crucial points remain to be resolved notably to ensure the safety of iPS of clinical grade, the optimization of the erythrocyte differentiation and cellular amplification, and finally, the definition of GMP conditions for industrial production.     

人胚胎干细胞建系培养及体外诱导分化的研究进展

人胚胎干细胞具有发育全能性,在特定条件下能分化成多种类型的细胞.人胚胎干细胞的研究对人胚胎发育机制、人基因功能研究和治疗性克隆有着重大的意义.本文从人胚胎干细胞建系、培养及体外诱导分化等方面作一综述.     

Identification and characterisation of mRif1: a mouse telomere-associated protein highly expressed in germ cells and embryo-derived pluripotent stem cells.

We have identified a mouse ortholog of the yeast Rif1 family of telomere-associated proteins on the basis of its high expression in primordial germ cells and embryo-derived pluripotent stem cell lines. mRif1 is also highly expressed in totipotent and pluripotent cells during early mouse development, and in male and female germ cells in adult mice. mRif1 expression is induced during derivation of embryonic stem cells and is rapidly down-regulated upon differentiation of embryonic stem cells in vitro. Furthermore, we show that mRif1 physically interacts with the telomere-associated protein mTrf2 and can be cross-linked to telomeric repeat DNA in mouse embryonic stem cells. mRif1 may be involved in the maintenance of telomere length or pluripotency in the germline and during early mouse development.     

诱导多能干细胞与p53基因的研究

背景：诱导多能干细胞可以绕过胚胎干细胞存在的伦理学问题,已成为目前干细胞研究的热点问题。 目的：探讨诱导多能干细胞的研究进展以及需要解决的问题。 方法：回顾分析诱导多能干细胞的发现,近年来的研究过程,目前存在的问题。检索汤森路透Web of Science数据库关于诱导多能干细胞和p53基因研究相关的文献进行分析。 结果与结论：近年来国内外学者对诱导多能干细胞进行大量研究。例如,日本正在筹备建立干细胞库,将为视网膜等疾病治疗提供基础。但是,在诱导多能干细胞能广泛应用之前,还面临许多安全问题,其中p53相关的功能性研究是必不可少的紧迫问题,p53基因中的其他成员p73也参与诱导多能干细胞的产生和分化也需要进一步深入。关于p53功能的发现将伴随着干细胞研究,促进再生医学的深入和发展。     

干细胞移植治疗糖尿病肾病

背景：糖尿病肾病所致的终末期肾病已成为慢性肾功能衰竭的主要原因,传统的血液透析和肾脏移植已无法满足临床治疗的需求,以干细胞为基础的再生医学研究有可能为糖尿病肾病的治疗带来新的希望。 目的：综合分析不同来源干细胞治疗糖尿病肾病的机制及临床研究进展。 方法：应用计算机检索CNKI和PubMed数据库中2006年1月至2011年8月关于干细胞治疗糖尿病肾病的文章,在标题和摘要中以“胚胎干细胞;骨髓间充质干细胞;诱导性多能干细胞;糖尿病肾病”或Embryonic stem cells; Bone marrow mesenchymal stem cells;induced poluripotent stem cells;induced multipotent stem cells; diabetic nephropathy”为检索词进行检索。最终选择26篇文章进行综述。 结果与结论：胚胎干细胞、骨髓间充质干细胞、诱导性多能干细胞均具有向肾脏组织细胞分化的潜能,大量研究表明干细胞移植对肾脏损伤和修复具有积极的作用。通过对干细胞相关特性的描述和相关机制的研究,干细胞移植有可能为糖尿病肾病提供一种新型的治疗方案。     

Primer and interviews: Promises and realities of induced pluripotent stem cells

In 2006, Yamanaka's group announced they had discovered the proverbial “fountain of youth” for human cells, forever changing the field of stem cell research. After misexpressing within them a cocktail of four genes, adult somatic cells revert into an embryonic stem cell (ESC)‐like state. These so‐called induced pluripotent stem cells (iPSCs) can differentiate into a wide variety of cell types, seemingly bypassing the need for politically charged ESCs. However, iPSCs differ from ESCs in potentially deleterious ways, precluding their use in regenerative medicine. In this primer and adjoining discussion with iPSC biologists William Lowry, PhD, and Clive Svendsen, PhD, we explore these issues as well how iPSCs promise to contribute to the understanding of developmental biology and the etiology, and treatment, of human diseases. Developmental Dynamics 240:2034–2041, 2011. © 2011 Wiley‐Liss, Inc.     

Evaluating human embryonic germ cells: concord and conflict as pluripotent stem cells

The realization of cell replacement therapy derived from human pluripotent stem cells requires full knowledge of the starting cell types as well as their differentiated progeny. Alongside embryonic stem cells, embryonic germ cells (EGCs) are an alternative source of pluripotent stem cell. Since 1998, four groups have described the derivation of human EGCs. This review analyzes the progress on derivation, culture, and differentiation, drawing comparison with other pluripotent stem cell populations.     

MT1-MMP 基因敲除细胞的诱导多能干细胞系的构建

目的: 观察野生型鼠和膜1型基质金属蛋白酶( MT1-MMP )基因敲除鼠的成纤维细胞重新编程为诱导多能干细胞(iPSCs)后,其细胞特性是否具有胚胎干细胞(ESCs)相似的潜能。方法: 利用逆转录病毒介导 Oct3/4、Sox2、c-Myc和Klf4 四种基因,转染到野生型鼠和 MT1-MMP 基因敲除鼠的成纤维细胞中。iPSCs克隆形成后,用免疫细胞化学检测ESCs特异性标志的表达情况。体外将iPSCs通过"悬滴法"向内皮细胞和心肌细胞分化,以检测其分化能力。结果: 转染野生型鼠和 MT1-MMP 基因敲除鼠的成纤维细胞2周后,可见ESCs样克隆开始形成。iPSCs明显表达ESCs特异性标志物碱性磷酸酶(AP)、阶段特异性胚胎抗原-1(SSEA-1)和八聚体结合转录因子3/4(OCT3/4)。诱导分化的内皮细胞表达早期内皮标志物Flk-1/KDR;诱导分化的心肌细胞出现跳动,表达心肌标志物肌钙蛋白I。结论: 野生型鼠和 MT1-MMP 基因敲除鼠的成纤维细胞可被重新编程为iPSCs,iPSCs具有ESCs的特征及增殖分化能力,因此可能为再生医学的研究和临床上进行细胞移植治疗提供理想的种子细胞来源。     

多潜能干细胞的研究进展

多潜能干细胞是一类具有向外胚层、中胚层以及内胚层细胞分化的干细胞,从胚胎发育早期分离获得的胚胎干细胞、胚胎生殖细胞到成体组织骨髓、睾丸中获得的干细胞,都具有三胚层分化能力.近年来,研究证实,可以将成体分化细胞经基因操作逆转为多能干细胞.就各种来源多潜能干细胞的特点予以综述,并对细胞间可能存在的关系加以探讨.     

Isolation and characterization of pluripotent human spermatogonial stem cell-derived cells

Several reports have documented the derivation of pluripotent cells (multipotent germline stem cells) from spermatogonial stem cells obtained from the adult mouse testis. These spermatogonia-derived stem cells express embryonic stem cell markers and differentiate to the three primary germ layers, as well as the germline. Data indicate that derivation may involve reprogramming of endogenous spermatogonia in culture. Here, we report the derivation of human multipotent germline stem cells (hMGSCs) from a testis biopsy. The cells express distinct markers of pluripotency, form embryoid bodies that contain derivatives of all three germ layers, maintain a normal XY karyotype, are hypomethylated at the H19 locus, and express high levels of telomerase. Teratoma assays indicate the presence of human cells 8 weeks post-transplantation but limited teratoma formation. Thus, these data suggest the potential to derive pluripotent cells from human testis biopsies but indicate a need for novel strategies to optimize hMGSC culture conditions and reprogramming.