诱导性多能干细胞技术的应用

诱导性多能干细胞在形态与增殖能力方面与胚胎干细胞极其类似,是将特定的转录因子导入细胞中,诱导其重编程而得。诱导性多能干细胞与胚胎干细胞高度相似,避开了细胞来源、伦理、法律和宗教等的限制,且解决了免疫排斥问题,又因其来源广泛、容易获得,在疾病治疗、药物研发等领域的研究中有着广泛的应用前景。目前,诱导性多能干细胞领域依然存在着诱导效率低、遗传安全性不明确等问题。     

诱导性多能干细胞技术的研究进展及其应用前景

在分化的体细胞中表达转录因子可以诱导体细胞重编程,获得诱导性多能干细胞（induced pluripotent stem cells,iPS cells）.这些细胞具有不断的自我更新能力和多向分化潜能,这些细胞重编程领域的突破性研究进展,为细胞重编程机制、人类疾病发病机制的研究及发展新的治疗方法提供了一种强有力的工具.iPS细胞技术是当前干细胞研究领域的热点之一,近年来取得了迅猛的发展.最初,研究者利用逆转录病毒作为载体将4种转录因子导入小鼠成纤维细胞诱导其重编程.近年来,iPS细胞的诱导方法不断改进,包括使用不整合入宿主细胞基因组的病毒载体、非病毒载体或者用基因敲除的方法切除导入的外源基因,从而产生了更为安全的iPS细胞系,许多小分子化合物也被证实能显著提高重编程效率.iPS细胞在再生医学、疾病模型的建立及药物筛选等领域正逐渐显现出它巨大的应用价值.本文回顾过去几年iPS细胞技术的研究进展,包括诱导方法的改进、iPS细胞诱导效率的提高和安全性的提高,并探讨iPS细胞的临床应用前景及当前研究存在的问题.     

一种人诱导多能干细胞分化生成巨核细胞的优化方案

目的 优化现有spin-EB培养法,促进人诱导多能干细胞(human induced pluripotent stem cells, hiPSCs)分化生成巨核细胞(megakaryocytes, MKs)。方法 本研究通过将hiPSCs初始接种量从3 500个细胞/孔提高至8 000个细胞/孔,增加了形成EB的大小。通过观察分化过程中EB-造血细胞的产生时间,检测不同阶段CD34⁺造血干祖细胞、CD41⁺MKs的增殖情况,研究该优化方案是否促进hiPSCs向造血祖细胞(hematopoietic progenitor cells, HPCs)和MKs分化。结果 通过提高hiPSCs初始接种量,增加形成EB的大小可促进hiPSCs向HPCs和MKs分化的进程和细胞产生效率。结论 我们研究描述了一种优化的和可重复的分化方法,在相对短的时间内可以更高的产量从hiPSCs产生造血干祖细胞和成熟的巨核细胞。不断优化体外hiPSCs分化生成MKs和血小板的培养方案,并促进输血医学中大规模的体外血小板生成,具有重要的临床意义和广阔的科研前景。     

诱导性多能干细胞技术用于研究人类疾病

<正>获得遗传学上与患者相配的大量细胞是再生医学的梦想。诱导性多能干细胞技术通过在培养皿中培养细胞来研究发病机理,建立疾病模型,观察疾病的分子机制,并发现新细胞基础的药物。诱导多能干(iPS)细胞从非多能细胞诱导产生,但拥有相似于胚胎干(ES)细胞的多能性,Takahashi等[1-4]首先实现了这个突破,在体外成功表达4个转录因子,即Oct4、Sox2、Klf4和c-Myc,这个步骤不需要卵母细胞。自从发现iPS细     

诱导多能干细胞的研究进展及其瓶颈

背景：随着研究的深入,诱导多能干细胞被应用于疾病模型建立、药物筛选和再生医学等多个研究领域,尤其是应用于神经系统疾病的研究。 目的：总结诱导多能干细胞目前的进展并结合相关领域的最新研究提出问题和展望。 方法：第一作者检索2006年1月至2014年9月PubMed数据库中有关诱导多能干细胞的相关文献,检索词为“iPS,induced pluripotent stem cel”,包括综述、临床研究和基础研究,最终纳入60篇文献。 结果与结论：2006年Yamanaka团队的诱导多能干细胞问世至2012年诺贝尔奖的颁发,短短6年间诱导多能干细胞相关的研究不断取得突破。诱导多能干细胞研究在疾病模型、药物筛选和再生医学等方面拥有广阔的前景,目前存在包括重编程方式、细胞稳定性、临床转化等一系列问题亟待解决,仍需进一步研究深化。     

体外诱导人类诱导性多能干细胞分化为造血干 / 祖细胞的研究简

本研究探讨体外诱导人诱导性多能干细胞（induced pluripotent stem cell,iPSC）分化为造血干/祖细胞的能力.在体外用小鼠骨髓基质细胞OP9与人类iPSC共培养的方法,将iPSC诱导分化为造血干/祖细胞;用流式细胞术检测造血干/祖细胞表面标志物的表达水平;用实时定量PCR检测分化过程中iPSC及造血干/祖细胞的相关基因mRNA表达水平的变化;用免疫磁珠法分离CD34＋造血干/祖细胞并进行半固体集落形成实验检测细胞的集落形成能力.结果表明,iPSC与OP9细胞共培养诱导造血分化的第4天即可观察到iPSC形态变化;流式细胞术检测显示,分化得到的细胞表达已知的造血干/祖细胞相关表面标志物CD34和CD43分子.在体外分化过程中多能性的标志基因Oct4的表达逐渐下降,造血相关转录因子Gata-2的表达逐渐升高,而Runx-1的表达量则呈波浪式变化,CD34表达量逐渐升高.集落培养14 d能够得到红系集落（CFU-E）,粒系集落（CFU-G）,巨核系集落（CFU-M）,粒-巨核系集落（CFU-GM）和混合系集落（CFU-GEMM）.结论：iPSC细胞能够在体外通过与OP9细胞共培养分化为造血干/祖细胞.     

构建脆性X染色体综合征患者来源的诱导多能干细胞系

目的构建脆性X综合征(FXS)患者来源的诱导多能干细胞(iPSCs)。方法采集2个FXS家系患者外周血并分离外周血单个核细胞(PBMC),用含4个重编程转录因子(OCT4、SOX2、KLF4和c-MYC)的仙台病毒感染PBMC,获得患者来源的iPSCs;采用细胞形态学、免疫荧光染色等技术对iPSCs进行多能性鉴定;核型分析验证其基因组完整性;利用PCR扩增结合微流控毛细管电泳技术检测重编程前后CGG重复数。结果成功对2个脆性X家系患者的PBMC细胞进行了重编程。患者来源的iPSCs经过多能性验证试验证实,其表达多能性标志基因并具有向三胚层分化潜能;核型分析结果证实iPSCs核型正常;2例先证者细胞系重编程前后的CGG重复数差异无统计学意义。结论通过非整合重编程技术成功构建2个FXS患者来源iPSCs细胞系,为进一步研究FXS疾病提供多能干细胞资源。     

诱导多能干细胞在血液学方面的研究进展

诱导多能干细胞(iPSCs)是通过基因转染技术将某些转录因子导人到动物或人的体细胞中,使体细胞直接重构成为胚胎样干细胞样的多潜能细胞,因其广泛的科研价值及应用前景而迅速成为科学领域的研究热点.近年来,iPSCs在医学再生领域以及自身技术改进方面取得了令人瞩目的成绩,为其应用于临床提供了有力的理论保障.     

构建阵挛性癫痫伴破碎红纤维综合征患者来源的诱导多能干细胞系

目的构建阵挛性癫痫伴破碎红纤维综合征(MERRF)患者来源的诱导多能干细胞(i PSCs)。方法采集MERRF患者外周血并分离PBMC,用含有4个重编程转录因子(OCT4、SOX2、KLF4和c-MYC)的仙台病毒感染PBMC,获得患者来源的i PSCs;用细胞形态学、免疫荧光染色、拟胚体形成分化和RT-PCR对i PSCs的多能性进行鉴定;核型分析验证其安全性;焦磷酸测序检测患者来源的i PSCs中线粒体DNA(mt DNA)第8344位的突变比例。结果患者来源的i PSCs经过多能性验证实验证明其具有多能性;核型分析表明,i PSCs核型正常;建立的6个i PSCs细胞系中,mt DNA 8344位点的突变比例分别为68.24%、60.51%、45.95%、24.06%、62.11%和0。结论通过非整合重编程技术成功构建MERRF患者来源i PSCs细胞系,为进一步研究MERRF疾病提供多能干细胞资源。     

白细胞蛋白酶抑制因子在真皮多能干细胞中的表达

目的研究白细胞蛋白酶抑制因子(secretory leukocyte protease inhibitor,SLPI)在真皮多能干细胞中的表达以及同伤口液刺激的关系。方法采用前期建立的技术分离、纯化并扩增真皮多能干细胞,收集伤后1d伤口液,比较伤口液刺激前后真皮多能干细胞和创伤前后大鼠皮肤组织中SLPI mRNA表达的变化。通过Western blot了解伤口液刺激前后真皮多能干细胞中SLPI蛋白表达的变化。结果分离、纯化的真皮多能干细胞形态较为均一,增殖能力强,RT-PCR和Western blot提示,SLPI在真皮多能干细胞中表达,伤口液刺激后表达增强。同时检测到SLPI在创伤后1d大鼠皮肤组织中表达升高。结论SLPI高表达可能是真皮多能干细胞参与创伤修复的重要途径之一。     

Generation of Engraftable Hematopoietic Stem Cells From Induced Pluripotent Stem Cells by Way of Teratoma Formation

In vitro generation of hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs) has the potential to provide novel therapeutic approaches for replacing bone marrow (BM) transplantation without rejection or graft versus host disease. Hitherto, however, it has proved difficult to generate truly functional HSCs transplantable to adult host mice. Here, we demonstrate a unique in vivo differentiation system yielding engraftable HSCs from mouse and human iPSCs in teratoma-bearing animals in combination with a maneuver to facilitate hematopoiesis. In mice, we found that iPSC-derived HSCs migrate from teratomas into the BM and their intravenous injection into irradiated recipients resulted in multilineage and long-term reconstitution of the hematolymphopoietic system in serial transfers. Using this in vivo generation system, we could demonstrate that X-linked severe combined immunodeficiency (X-SCID) mice can be treated by HSCs derived from gene-corrected clonal iPSCs. It should also be noted that neither leukemia nor tumors were observed in recipients after transplantation of iPSC-derived HSCs. Taken our findings together, our system presented in this report should provide a useful tool not only for the study of HSCs, but also for practical application of iPSCs in the treatment of hematologic and immunologic diseases.     

Induced Pluripotent Stem Cells: The Most Versatile Source for Stem Cell Therapy

Cell therapy has existed since the first bone marrow transplant in the 1950s involving identical twins. The blood-forming stem cells were used to restore healthy blood cells for the twin with leukemia. It was not until 1968 that genetic matching (known as human leukocyte antigen matching) was known to be important, and not until 1973 that bone marrow transplants were performed from non–twin-related and nonrelated donors. The most important application of human stem cells is for the generation of cells and tissues for cell-based therapies. Currently, donated organs and tissues are often the only option to replace diseased, injured, or destroyed tissue. The availability for these transplantable tissues and organs is very limited, however. To satisfy the demand for a source for these cells and tissues, induced pluripotent stem cells that have been differentiated into specific cell types can serve as a renewable source of replacement cells and tissues. A bank of suitable human leukocyte antigen–matched cells will be an important source providing immediate availability of cells that are readily scalable, economical, and well characterized. Areas of active pursuit with stem cell therapy is being investigated for treating diseases such as macular degeneration, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis, rheumatoid arthritis, and neurodegenerative diseases. This article describes the advantages and hurdles for the use of induced pluripotent cells as the starting material for a source of replacement cells for regenerative medicine.     

非基因整合建立人诱导多能性干细胞方法的优化研究

诱导多能性干细胞（iPSC）技术具有临床应用前景,但iPSC的遗传稳定性和成瘤性阻碍了其可能的临床应用。非整合质粒（Episomal）方法无外源基因整合到宿主基因组上并且方法简单,适宜推广,是目前保证iPSC遗传安全性的最佳方案之一,但其诱导效率偏低,严重阻碍了其应用。本研究旨在优化Episomal方法,将脐血单个核细胞（CBMNC）重编程为诱导多能性干细胞（iPSC）,建立无基因整合的iPSC的高效生成技术体系,为以后建立疾病iPSC奠定基础。利用CBMNC,通过比较不同氧含量,诱导质粒,MNC培养方法和预刺激时间等条件对Episo—mal方法进行优化。结果表明：CBMNC采用红系培养液,培养8d,使用启动子为sFFV（spleen focus forming virus）的Episomal载体,在低氧（3％）条件下诱导,CBMNC重编程效率最高,可达到0．12％。通过分析最佳条件下供体细胞成分发现,表型为CD36＋CD71＋CD235alow的有核红细胞是重编程最主要的供体细胞来源。结论：本研究成功建立并优化出一种可推广的高效安全的,可以用于临床应用研究的iPSC诱导技术。     

Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes by Soluble Factors from Human Mesenchymal Stem Cells

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Application of Epigenome‐Modifying Small Molecules in Induced Pluripotent Stem Cells

Recent breakthroughs in generating induced pluripotent stem cells (iPSCs) using four defined factors have revealed the potential utility of stem cells in biological research and clinical applications. However, the low efficiency and slow kinetics of reprogramming related to producing these cells and underlying safety issues, such as viral integration and genetic and epigenetic abnormalities of iPSCs, hamper the further application of iPSCs in laboratory and clinical settings. Previous studies have suggested that reprogramming efficiency can be enhanced and that reprogramming kinetics can be accelerated by manipulating epigenetic status. Herein, we review recent studies on the application of epigenome‐modifying small molecules in enhancing reprogramming and functionally replacing some reprogramming factors. We mainly focus on studies that have used small molecules to interfere with epigenome‐modifying enzymes, such as DNA methyltransferase, histone acetyltransferase, and histone methyltransferase. The potential use of these small molecules in inducing iPSCs and new ways to identify small molecules of higher potency and fewer side effects are also discussed.     

Inhibition of PTEN Tumor Suppressor Promotes the Generation of Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) can be generated from patients with specific diseases by the transduction of reprogramming factors and can be useful as a cell source for cell transplantation therapy for various diseases with impaired organs. However, the low efficiency of iPSC derived from somatic cells (0.01–0.1%) is one of the major problems in the field. The phosphoinositide 3-kinase (PI3K) pathway is thought to be important for self-renewal, proliferation, and maintenance of embryonic stem cells (ESCs), but the contribution of this pathway or its well-known negative regulator, phosphatase, and tensin homolog deleted on chromosome ten (Pten), to somatic cell reprogramming remains largely unknown. Here, we show that activation of the PI3K pathway by the Pten inhibitor, dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate, improves the efficiency of germline-competent iPSC derivation from mouse somatic cells. This simple method provides a new approach for efficient generation of iPSCs.     

人参皂苷Rg1参与诱导小鼠多潜能干细胞的研究

目的:观察人参皂苷Rg1对鼠胚成纤维细胞(MEF)向诱导多潜能干细胞(iPSCs)诱导转化效率的影响。方法:采用经典四因子的逆转录病毒载体感染,在细胞感染后3 d内在MEF培养基里加入人参皂苷Rg1(0、0.3、1、3、10μg/mL),进行iPSCs诱导及鉴定。结果:人参皂苷Rg1(1μg/mL)组可明显提高iPSCs的诱导效率,诱导效率为(0.0450±0.0019)%,人参皂苷Rg1(0μg/mL)组为(0.0100±0.0033)%,所获iPSCs经鉴定为阳性。结论:人参皂苷Rg1可能在提高小鼠iPSCs的诱导效率方面发挥一定作用。