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。     

用非整合重编程技术构建人外周血单个核细胞来源的诱导多能干细胞

目的用非整合重编程技术构建人外周血单个核细胞(PBMC)来源诱导多能干细胞(i PSC)。方法收集健康人外周血标本,分离PBMC,用含4个重编程转录因子(OCT3/4、SOX2、KLF4和c-MYC)的仙台病毒感染PBMC,并用无饲养层细胞的培养体系培育获得i PSC。通过细胞形态学、碱性磷酸酶(AP)染色、免疫荧光染色、拟胚体形成和分化实验对i PSC细胞进行多能性验证;用核型分析和仙台病毒基因组RNA检测对其进行安全性验证。结果在仙台病毒感染后16 d后,PBMC出现i PSC样克隆。i PSC可进行传代培养,且经多能性验证实验证明其具有多能性;安全性验证结果表明,i PSC核型正常,外源性病毒基因组RNA可被完全清除。结论建立了对PBMC进行非整合重编程的诱导体系,并获得了具有多能性、核型正常且不含外源基因的i PSC。     

外周血红系祖细胞来源诱导多能干细胞的建立

目的建立和鉴定外周血中红系祖细胞来源的诱导多能干细胞(induced pluripotent stem cells,i PSCs)。方法从健康人外周血标本中分选红系祖细胞,将表达Oct4、Sox2、Lin28、L-Myc和Klf4转录因子的ori P/EBNAl附着体电转染红系祖细胞使其重编程获得i PSCs,并通过核型鉴定、碱性磷酸酶染色、RT-PCR反应、畸胎瘤形成实验及类胚体形成实验检测其干细胞多能性特性。结果获得的i PSCs核型正常,碱性磷酸酶染色呈阳性,表达干细胞多能性基因Sox2、Oct4、Nanog和Klf4和Lin28,体内畸胎瘤形成实验可分化为内、中、外三胚层细胞,体外可形成类胚体。结论成功建立外周血红系祖细胞来源具有多向分化潜能的i PSCs。     

以4种基因诱导产前诊断绒毛细胞建立诱导性多能干细胞

背景:诱导性多能干细胞因具有多能性特征,可以诱导分化为特定的细胞,包括神经细胞、造血细胞等。目的:建立产前诊断绒毛细胞来源的诱导性多能干细胞。方法:运用反转录病毒介导4种基因hOct4、hSox2、hc-Myc、hKlf4诱导产前诊断绒毛细胞,对建立的诱导性多能干细胞进行多能性、体内外分化能力、核型等鉴定。结果与结论:建立的诱导性多能干细胞能维持自我更新状态,在蛋白和mRNA水平上高表达全能性的标志基因,具有体内、外分化潜能;在体外长期培养能维持正常核型。说明4种全能性基因转入绒毛细胞可获得具有多能性的诱导性多能干细胞,这为胎儿的细胞自体移植治疗提供理想来源,为产前诊断疾病机制研究提供很好的细胞模型。     

以4种基因诱导产前诊断绒毛细胞建立诱导性多能干细胞

背景：诱导性多能干细胞因具有多能性特征,可以诱导分化为特定的细胞,包括神经细胞、造血细胞等。目的：建立产前诊断绒毛细胞来源的诱导性多能干细胞。方法：运用反转录病毒介导4种基因hOct4、hSox2、hc-Myc、hKlf4诱导产前诊断绒毛细胞,对建立的诱导性多能干细胞进行多能性、体内外分化能力、核型等鉴定。结果与结论：建立的诱导性多能干细胞能维持自我更新状态,在蛋白和mRNA水平上高表达全能性的标志基因,具有体内、外分化潜能;在体外长期培养能维持正常核型。说明4种全能性基因转入绒毛细胞可获得具有多能性的诱导性多能干细胞,这为胎儿的细胞自体移植治疗提供理想来源,为产前诊断疾病机制研究提供很好的细胞模型。     

人诱导多能干细胞体外诱导产生红细胞的研究进展

诱导多能干细胞(iPSCs)不仅具有无限增殖的能力,还可以根据不同的需要筛选出特定的表型,iPSCs可定向诱导生成RBC,作为RBC输血的一个新来源。要将此技术应用到临床输血上,还需对一些关键程序进行优化,如用于生成iPSCs的细胞类型的选择、改进用于保障临床应用安全的重编程方法、红细胞分化过程的优化等。     

脆性X综合征遗传学诊断方法研究进展

脆性X综合征(FXS)是导致智力障碍和发育障碍的主要单基因病之一,呈X连锁不完全显性遗传。FXS的病因是FMR1基因内(CGG)n重复序列的不稳定扩展及其上游CpG岛的异常甲基化,进而导致脆性X智力低下蛋白(FMRP)减少或缺乏,FMRP的水平直接关系到临床表型的严重程度。临床表现和基因检测是诊断FXS的主要依据。然而,FMR1基因分子结构和遗传模式的特殊性使得FXS的分子诊断和遗传咨询面临挑战。因此,如何简便而准确地进行FMR1基因检测一直是临床关注的焦点。文章针对FXS遗传学诊断方法的研究进展进行综述,旨在促进FXS的规范诊断,为临床提供帮助。     

卵巢早衰与脆性X智力障碍基因1CGG异常重复扩增相关性的研究进展

脆性X综合征(FXS)常表现为遗传性智力障碍和自闭症等多系统疾病。脆性X智力障碍基因1(FMR1)为卵巢早衰(POF)发病相关的重要遗传学因素。患POF的女性仍有一定受孕概率,且FMR1基因CGG重复异常的携带者可能性较高,导致其生殖功能改变和不明原因复发性流产发生率增高。本文对POF与FMR1基因CGG异常重复相关研究进行综述,以期为遗传咨询和生育指导提供理论基础,进而降低生育缺陷。     

细胞重编程前后基因组的动态稳定性

背景：研究表明,人类早代诱导多能性干细胞发生拷贝数变异多于晚代、体细胞及人类胚胎干细胞。目的：分析重编程过程是否危及基因组稳定性,进一步探讨诱导多能性干细胞建系的有效性。 方法：利用高分辨率的Affymetrix Cytoscan HD芯片检测遗传性癫痫先证者的体细胞及早代诱导多能性干细胞,分析重编程后基因组拷贝数变异及杂合性缺失的变化。 结果与结论：与遗传性癫痫患者体细胞相比,其早代诱导多能性干细胞的杂合性缺失未发现明显差异,而存在更多的拷贝数变异,且均为微重复,涉及致癌基因。结果证明重编程过程中基因组稳定性动态变化,需要动态监测诱导多能性干细胞保证其基因组稳定性及临床安全性。     

建立非基因整合原发性骨髓纤维化诱导性多能干细胞株

目的:利用诱导多能性干细胞(iPSC)技术建立原发性骨髓纤维化(PMF)诱导多能性干细胞系,为研究血液病的发生发展提供一个实验模型。方法:采用非基因整合型质粒重编程携带JAK2V617F突变的PMF患者骨髓来源的单个核细胞,诱导生成该患者特异的iPS细胞株。结果:采用此方法建立的iPS细胞株,在体外能够稳定传代,无外源性基因整合,多能性基因表达水平与人胚胎干细胞类似,在体内具有形成三胚层结构的能力。测序结果显示,所建立的患者的iPS细胞株携带不同负荷的JAK2V617F突变。结论:成功地建立非基因整合的PMF患者特异的iPS细胞株,这为研究PMF的发病机制、化疗药物筛选及实现精准化治疗提供了一个重要的疾病模型。     

Modeling Human Bone Marrow Failure Syndromes Using Pluripotent Stem Cells and Genome Engineering

The combination of epigenetic reprogramming with advanced genome editing technologies opened a new avenue to study disease mechanisms, particularly of disorders with depleted target tissue. Bone marrow failure syndromes (BMFS) typically present with a marked reduction of peripheral blood cells due to a destroyed or dysfunctional bone marrow compartment. Somatic and germline mutations have been etiologically linked to many cases of BMFS. However, without the ability to study primary patient material, the exact pathogenesis for many entities remained fragmentary. Capturing the pathological genotype in induced pluripotent stem cells (iPSCs) allows studying potential developmental defects leading to a particular phenotype. The lack of hematopoietic stem and progenitor cells in these patients can also be overcome by differentiating patient-derived iPSCs into hematopoietic lineages. With fast growing genome editing techniques, such as CRISPR/Cas9, correction of disease-causing mutations in iPSCs or introduction of mutations in cells from healthy individuals enable comparative studies that may identify other genetic or epigenetic events contributing to a specific disease phenotype. In this review, we present recent progresses in disease modeling of inherited and acquired BMFS using reprogramming and genome editing techniques. We also discuss the challenges and potential shortcomings of iPSC-based models for hematological diseases.     

诱导多功能干细胞的研究进展

背景：由于胚胎干细胞移植存在致瘤性和伦理学争议,有关胚胎干细胞的研究及临床应用存在较大的限制。2006年Yamanaka实验室利用Oct3/4、Sox2、Klf4、c-Myc4种因子将鼠成纤维细胞重编程为诱导多功能干细胞,标志着一种新型类胚胎干细胞的问世。目的：了解诱导多功能干细胞的研究进展和应用前景。方法：由第一作者检索2006/2010PubMed数据库（http：//www.ncbi.nlm.nih.gov/PubMed）及万方数据库（http：//g.wanfangdata.com.cn/）有关诱导多功能干细胞的产生、细胞特征、产生技术的研究进展及应用前景等方面的文章,英文检索词为＂induced pluripotent stem cells,defined factors,reprogramming,vectors,disease＂,排除重复性研究,共保留其中的69篇进行归纳总结。结果与结论：诱导多功能干细胞研究在诱导因子种类,因子导入方式,重编程效率及应用研究等诸多方面取得进展。然而体细胞重编程为诱导多功能干细胞仍存在一定的风险,重编程效率还非常低。一旦解决诱导多功能干细胞的安全性和重编程效率问题,诱导多功能干细胞就可被广泛应用于疾病模型,药物测试,细胞移植及患者和疾病特异性多功能干细胞的建立等诸多方面。     

Generation and genetic modification of induced pluripotent stem cells

Importance of the field: The generation of induced pluripotent stem cells (iPSCs) enabled by exogenous expression of the canonical Oct4, Sox2, Klf4 and c-Myc reprogramming factors has opened new ways to create patient- or disease-specific pluripotent cells. iPSCs represent an almost inexhaustible source of cells for targeted differentiation into somatic effector cells and hence are likely to be invaluable for therapeutic applications and disease-related research.

Areas covered in this review: After an introduction on the biology of reprogramming we cover emerging technological advances, including new reprogramming approaches, small-molecule compounds and tailored genetic modification, and give an outlook towards potential clinical applications of iPSCs.

What the reader will gain: Although this field is progressing rapidly, reprogramming is still an inefficient process. The reader will learn about innovative tools to generate patient-specific iPSCs and how to modify these established lines in a safe way. Ideally, the disease-causing mutation is edited directly in the genome using novel technologies based on artificial nucleases, such as zinc-finger nucleases.

Take home message: Human iPSCs create fascinating options with regard to disease modeling, drug testing, developmental studies and therapeutic applications. However, important hurdles have to be taken and more efficient protocols to be established to achieve the ambitious goal of bringing iPSCs into clinical use.     

Recent progress of national banking project on homozygous HLA‐typed induced pluripotent stem cells in South Korea

Induced pluripotent stem cells (iPSCs) can be generated by introducing several factors into mature somatic cells. Banking of iPSCs can lead to wider application for treatment and research. In an economical view, it is important to store cells that can cover a high percentage of the population. Therefore, the use of homozygous human leukocyte antigen‐iPSCs (HLA‐iPSCs) is thought as a potential candidate for effective iPSC banking system for further clinical use. We screened the database stored in the Catholic Hematopoietic Stem Cell Bank of Korea and sorted the most frequent homozygous HLA types of the South Korean population. Blood cells with the selected homozygous HLA types were obtained and transferred to the GMP facility in the Catholic Institute of Cell Therapy. Cells were reprogrammed to iPSCs inside the facility and went through several quality controls. As a result, a total of 13 homozygous GMP‐grade iPSC lines were obtained in the facility. The generated iPSCs showed high pluripotency and normal karyotype after reprogramming. Five HLA‐homozygous iPSCs had the type that was included in the top five most frequent HLA types. Homozygous HLA‐iPSCs can open a new opportunity for further application of iPSCs in clinical research and therapy.     

Induced pluripotent stem cells: progress towards a biomedical application

Evaluation of: Burridge PW, Thompson S, Millrod MA et al. A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PLoS One 6(4), e18293 (2011).

Some 5 years ago, Takahashi and Yamanaka first obtained induced pluripotent stem cells (iPSCs) by genetically ‘reprogramming’ adult somatic cells (fibroblasts). This breakthrough opened a new frontier in regenerative medicine, in which iPSCs might effectively replace embryonic stem cells (ESCs), with the additional advantage of permitting autologous transplant. Unfortunately, the risk of aberrant reprogramming and of the complications related to the use of transgenes in the process still hinders iPSC clinical application. Nevertheless, differentiation of iPSCs derived from patients may already provide a formidable platform for the in vitro analysis of human disease mechanisms and their modulation by drugs. Such an approach has already been validated by the finding that iPSCs obtained from patients with a specific genetic syndrome can be differentiated into cardiomyocytes retaining the gene abnormality and recapitulating, at the cell level, the syndrome functional phenotype. Unlike the use of iPSCs in regenerative therapy, their development as disease models is unencumbered by safety constraints. Whatever the intended use, the availability of reliable and reproducible methods for somatic cell reprogramming, iPSC expansion and differentiation is pivotal and remains a major challenge to date. The article by Burridge and coworkers describes a process encompassing all the phases in the preparation of precursor-derived cardiomyocytes, characterized by unprecedented efficiency and, most notably, applicable to different human precursors, including ESCs and iPSCs derived from multiple somatic cell types. Provided that the process will prove reproducible when applied by different laboratories, the contribution of Burridge and coworkers may represent a genuine leap in the development of precursor-derived technologies.     

Modified Lentiviral LTRs Allow Flp Recombinase–mediated Cassette Exchange and In Vivo Tracing of “Factor-free” Induced Pluripotent Stem Cells

Methods for generating induced pluripotent stem cells (iPSCs) for disease modeling and cell therapies have progressed from integrating vectors to transient delivery of reprogramming factors, avoiding permanent genomic modification. A major limitation of unmodified iPSCs is the assessment of their distribution and contribution to adverse reactions in autologous cell therapy. Here, we report that polycistronic lentiviral vectors with single Flp recombinase (Flp) recognition target (FRT) sites can be used to generate murine iPSCs that are devoid of the reprogramming cassette but carry an intergenic 300-bp long terminal repeat sequence. Performing quantitative polymerase chain reaction on this marker, we could determine genetic identity and tissue contribution of iPSC-derived teratomas in mice. Moreover, we generated iPSCs carrying heterospecific FRT twin sites, enabling excision and recombinase-mediated cassette exchange (RMCE) of the reprogramming cassette for another expression unit of choice. Following screening of iPSCs for “safe harbor” integration sites, expression cassettes were introduced by RMCE into various previously silenced loci of selected single-copy iPSCs. Analysis of DNA methylation showed that RMCE reverted the local epigenetic signature, which allowed transgene expression in undifferentiated iPSCs and in differentiated progeny. These findings support the concept of creating clonotypically defined exchangeable and traceable pluripotent stem cells for disease research and cell therapy.     

Generation of HIV-1 Resistant and Functional Macrophages From Hematopoietic Stem Cell�Cderived Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from adult individuals. By developing iPSCs to treat HIV, there is the potential for generating a continuous supply of therapeutic cells for transplantation into HIV-infected patients. In this study, we have used human hematopoietic stem cells (HSCs) to generate anti-HIV gene expressing iPSCs for HIV gene therapy. HSCs were dedifferentiated into continuously growing iPSC lines with four reprogramming factors and a combination anti-HIV lentiviral vector containing a CCR5 short hairpin RNA (shRNA) and a human/rhesus chimeric TRIM5α gene. Upon directed differentiation of the anti-HIV iPSCs toward the hematopoietic lineage, a robust quantity of colony-forming CD133⁺ HSCs were obtained. These cells were further differentiated into functional end-stage macrophages which displayed a normal phenotypic profile. Upon viral challenge, the anti-HIV iPSC-derived macrophages exhibited strong protection from HIV-1 infection. Here, we demonstrate the ability of iPSCs to develop into HIV-1 resistant immune cells and highlight the potential use of iPSCs for HIV gene and cellular therapies.     

Simplified molecular diagnosis of fragile X syndrome by fluorescent methylation-specific PCR and GeneScan analysis

BACKGROUND: Fragile X syndrome (FXS), the most common cause of inherited mental impairment, is most commonly related to hyperexpansion and hypermethylation of a polymorphic CGG trinucleotide repeat in the 5' untranslated region of the FMR1 gene. Southern blot analysis is the most commonly used method for molecular diagnosis of FXS. We describe a simplified strategy based on fluorescent methylation-specific PCR (ms-PCR) and GeneScan analysis for molecular diagnosis of fragile X syndrome. METHODS: We used sodium bisulfite treatment to selectively modify genomic DNA from fragile X and normal lymphoblastoid cell lines and from patients. We then performed ms-PCR amplification using fluorescently-labeled primers complementary to modified methylated or unmethylated DNA. Amplification products were resolved by capillary electrophoresis. FMR1 mutational status was determined by a combination of fluorescent peak sizes and patterns on the GeneScan electropherogram. RESULTS: DNA samples from male and female persons with known NL, PM, and FM FMR1 CGG repeats were analyzed. Each FMR1 genotype produced a unique GeneScan electropherogram pattern, thus providing a way to identify the various disease states. The number of CGG repeats in all NL and PM alleles were determined accurately. Analysis by both the new assay and Southern blot of a family segregating with FXS showed complete concordance between both methods. CONCLUSIONS: This simplified molecular diagnostic test, based on fluorescent methylation-specific PCR, may be a suitable alternative or complement to Southern blot analysis for the diagnosis of FXS.