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。     

Concise review: Deciphering the mechanism behind induced pluripotent stem cell generation

Regenerative medicine using spluripotent/multipotent stem cells holds a great promise in developing therapies for treating developmental abnormalities, degenerative disorders, and aging-related illness. However, supply and safety of the stem cells are two major problems with today's regenerative medicine. Recent development of induced pluripotent stem cells (iPSCs) has overcome the supply shortages by allowing the reprogramming of patients' body cells to embryonic stem cell (ESC)-like pluripotent cells. Still, the potential tumorigenicity of iPSCs remains as an obstacle. During early embryogenesis ESCs can be generated without tumor formation; therefore, understanding the mechanisms underlying ESC generation may help us to prevent iPSC tumorigenicity. Previous studies have shown that an ESC-enriched noncoding RNA, miR-302, induces somatic cell reprogramming (SCR) to form iPSCs, suggesting its pivotal role in stem cell generation. Recent research further revealed that miR-302-induced SCR involves an epigenetic reprogramming mechanism similar to the natural zygotic reprogramming process in the two- to eight-cell-stage embryos. These findings indicate that miR-302, as a cytoplasmic gene silencer, inhibits the translation of multiple key epigenetic regulators, including AOF1/2, methyl-CpG binding proteins 1 and 2, and DNA (cytosine-5-)-methyltransferase 1, to induce global DNA demethylation, which subsequently triggers the activation of the previously defined factors Oct4, Sox2, and Nanog to complete the reprogramming process. The same mechanism was also found in the event of somatic cell nuclear transfer. Based on these advanced understandings, this review describes the currently established SCR mechanism--as compared to the natural process of early ESC formation--and demonstrates how stem cell researchers may use this mechanism to improve iPSC generation.     

多能干细胞制备方法的研究进展及问题

多能干细胞为研究疾病的分子机制、药物筛选及再生医学应用提供理想的细胞来源,然而,当前多能干细胞的来源或制备方法面临众多瓶颈和挑战,本文回顾了多能干细胞制备方法的最新研究进展,为多能干细胞的后续研究提供思路。     

诱导多能性干细胞技术的进展

将人类体细胞诱导成为多能性干细胞(induced pluripotent stem cells,iPS cells)的研究成果为生物医学研究提供了广泛的前景,建立了一种全新的体细胞核重编程的方法,这种方法相对容易操作,而且比较稳定、安全,点燃了再生医学应用的新希望,本文从体细胞重编程的研究历程、iPS细胞技术的诱导因子、转录因子、载体及其应用价值方面进行了综述.     

SHH信号通路调控hiPSCs定向分化为腹侧底板类神经前体细胞

目的激活Sonic Hedgehog(SHH)信号通路促进人诱导性多能干细胞(hiPSCs)定向分化为腹侧底板类细胞。方法将hiPSCs设3个实验组:LDN193189+SB431542组(LSB组)、SHH C24-II+Purmorphamine组(SHH组)和Cyclopamine组(CYC组)。采用RT-qPCR检测诱导第7天各组细胞背侧前脑标志物PAX6、腹侧底板标志物FOXA2和SHH信号通路关键因子SHH、Ptc-1、Smo、Gli-1及诱导第11天多巴胺能神经前体细胞LMX1A、EN1的mRNA表达。免疫细胞化学(ICC)检测诱导第7天FOXA2、PAX6及分化第25天多巴胺能神经元标志物TH的蛋白表达。结果 RT-qPCR结果显示分化第7天,SHH组FOXA2的mRNA表达明显上调(P<0.01),PAX6的表达明显下调(P<0.01);SHH信号通路相关因子SHH、Ptc-1及Gli-1的表达明显上调(P<0.01),Smo的表达无明显变化;ICC结果显示LSB组、CYC组以PAX6+细胞为主;SHH组以FOXA2~+细胞细胞为主;分化第11天,SHH组多巴胺能神经前体标志物LMX1A及EN1的mRNA表达量较LSB组高(P<0.05);分化第25天ICC结果显示SHH组TH+细胞数量较LSB、CYC组多。结论分化早期激活SHH信号可诱导hiPSCs定向分化为FOXA2~+的腹侧底板细胞,该类细胞能进一步分化为多巴胺能神经前体细胞及多巴胺能神经元。     

人诱导性多潜能干细胞系的建立

背景：诱导性多潜能干细胞与肿瘤干细胞的发生过程极其相似,而且具有的干细胞特性极其接近人胚胎干细胞。因此,研究诱导性多潜能干细胞有利于人们进一步认识并了解人类发育以及肿瘤的发生过程。 目的：掌握建立人诱导性多潜能干细胞系的技术,以便为特异性疾病细胞的重编程建立技术平台,从而利用重编程技术研究疾病的发病机制。 方法：将含有Oct4、Sox2、Klf-4和c-Myc 4个转录因子的反转录病毒感染人皮肤成纤维细胞(HS27细胞),在人胚胎干细胞培养条件下诱导产生人胚胎干细胞样的克隆。挑取并进一步扩增,通过克隆形态、碱性磷酸酶活性、免疫荧光检测是否有人胚胎干细胞标记物Oct4、Sox2、c-Myc、Klf-4的表达,悬滴法检测HS27细胞来源的克隆形成畸胎瘤的能力和验证向3个胚层的分化能力。 结果与结论：经病毒感染诱导产生的胚胎干细胞样克隆呈绿色荧光蛋白阴性,克隆在细胞形态方面与人胚胎干细胞克隆相似,进一步扩增经碱性磷酸酶检测克隆呈阳性,免疫荧光检测克隆表达Oct4、Sox2、c-Myc、Klf-4,并且HS27细胞来源的克隆注入免疫缺陷小鼠体内可以形成畸胎瘤并经苏木精-伊红染色显示具有向三胚层分化能力。实验成功构建了人诱导性多潜能干细胞系,为下一步开展疾病细胞特异性重编程研究奠定了良好的实验基础。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：     

Crucial role of c-Myc in the generation of induced pluripotent stem cells

c-Myc transduction has been considered previously to be nonessential for induced pluripotent stem cell (iPSC) generation. In this study, we investigated the effects of c-Myc transduction on the generation of iPSCs from an inbred mouse strain using a genome integration-free vector to exclude the effects of the genetic background and the genomic integration of exogenous genes. Our findings reveal a clear difference between iPSCs generated using the four defined factors including c-Myc (4F-iPSCs) and those produced without c-Myc (3F-iPSCs). Molecular and cellular analyses did not reveal any differences between 3F-iPSCs and 4F-iPSCs, as reported previously. However, a chimeric mice formation test indicated clear differences, whereby few highly chimeric mice and no germline transmission was observed using 3F-iPSCs. Similar differences were also observed in the mouse line that has been widely used in iPSC studies. Furthermore, the defect in 3F-iPSCs was considerably improved by trichostatin A, a histone deacetyl transferase inhibitor, indicating that c-Myc plays a crucial role in iPSC generation through the control of histone acetylation. Indeed, low levels of histone acetylation were observed in 3F-iPSCs. Our results shed new light on iPSC generation mechanisms and strongly recommend c-Myc transduction for preparing high-quality iPSCs.     

Discovery of nonsteroidal anti-inflammatory drug and anticancer drug enhancing reprogramming and induced pluripotent stem cell generation

Recent breakthroughs in creating induced pluripotent stem cells (iPSCs) provide alternative means to obtain embryonic stem-like cells without destroying embryos by introducing four reprogramming factors (Oct3/4, Sox2, and Klf4/c-Myc or Nanog/Lin28) into somatic cells. iPSCs are versatile tools for investigating early developmental processes and could become sources of tissues or cells for regenerative therapies. Here, for the first time, we describe a strategy to analyze genomics datasets of mouse embryonic fibroblasts (MEFs) and embryonic stem cells to identify genes constituting barriers to iPSC reprogramming. We further show that computational chemical biology combined with genomics analysis can be used to identify small molecules regulating reprogramming. Specific downregulation by small interfering RNAs (siRNAs) of several key MEF-specific genes encoding proteins with catalytic or regulatory functions, including WISP1, PRRX1, HMGA2, NFIX, PRKG2, COX2, and TGFβ3, greatly increased reprogramming efficiency. Based on this rationale, we screened only 17 small molecules in reprogramming assays and discovered that the nonsteroidal anti-inflammatory drug Nabumetone and the anticancer drug 4-hydroxytamoxifen can generate iPSCs without Sox2. Nabumetone could also produce iPSCs in the absence of c-Myc or Sox2 without compromising self-renewal and pluripotency of derived iPSCs. In summary, we report a new concept of combining genomics and computational chemical biology to identify new drugs useful for iPSC generation. This hypothesis-driven approach provides an alternative to shot-gun screening and accelerates understanding of molecular mechanisms underlying iPSC induction.     

Induced pluripotent stem cell consensus genes: implication for the risk of tumorigenesis and cancers in induced pluripotent stem cell therapy

Induced pluripotent stem cells (iPSCs) have recently boomed enthusiasm in stem cell therapy, whereas high potential tumorigenesis of iPSCs has become the biggest obstacle for clinic application and the tumorigenic genes in iPSCs have not been well documented. In this investigation, using tools of bioinformatics, we analyzed the all available datasets regarded to iPSCs from 11 differentiated cell lines and revealed 593 iPSC consensus genes. Notably, of the 593 genes, 209 were expressed in human tumor cell lines and cancer tissues, and some of them were expressed in the iPSC-differentiated hepatocytes; remarkably, 5 oncogenes were overexpressed in the iPSCs and an oncogene RAB25 in the iPSC-differentiated cells, suggesting that these iPSC consensus genes are implicated with the risk of tumorigenesis and cancers. This investigation provides useful information for designing new strategies and methods to curtail the expression of oncogenic genes in iPSCs and produce safe iPSC derivatives for stem cell therapy.     

miR-148a促进人诱导性多能干细胞向心肌样细胞的分化

文题释义：人诱导性多能干细胞来源的心肌样细胞：在体外直接将人诱导性多能干细胞定向诱导分化为心肌样细胞具有供者个体基因的特异性,不仅可以进行发病机制的深入研究,还可以进行个体化药物治疗测试,为精准个性化治疗带来希望。来自于患者自身体细胞的心肌细胞扩增速度快,没有免疫排斥和伦理问题,但目前的主要问题是来自于人诱导性多能干细胞的心肌样细胞成熟程度不佳,分化的细胞不具备成人心肌细胞的电生理活动,且纯化有一定困难。 微小RNA的调节作用：微小RNA是一类内源性的非编码单链RNA,通常含有19-22个核苷酸,可通过靶向结合mRNA而使其降解或抑制其翻译,发挥对靶基因的转录后调控作用。近年来,基于芯片分析技术发现有多个miRNA在心肌分化发育或干细胞向心肌样细胞定向分化过程中发挥调控作用,有正向调控也有负向调控。 背景：研究发现,miR-148a可以促进人骨髓间充质干细胞定向分化为成熟心肌样细胞,但关于miR-148a对人诱导性多能干细胞向心肌样细胞分化的影响则未见报道。 目的：探讨miR-148a对人诱导性多能干细胞向心肌样细胞分化的影响。 方法：将人诱导性多能干细胞分为3组向心肌样细胞诱导分化,对照组细胞不做处理,低表达组细胞用miR-148a抑制物处理28 d,高表达组细胞用miR-148a模拟物处理28 d;另取常规培养28 d的人诱导性多能干细胞为空白对照组。CCK-8检测细胞增殖活力,qRT-PCR检测miR-148a mRNA的表达,免疫荧光染色和Western blot检测MHC和cTnT蛋白的表达。 结果与结论：①低表达组细胞内miR-148a mRNA表达、细胞增殖活力低于空白对照组和对照组,高表达组高于其他3组(P < 0.01);②空白对照组无MHC和cTnT的阳性表达,对照组、低表达组和高表达组均有MHC和cTnT的阳性表达;③低表达组细胞MHC和cTnT蛋白表达低于对照组,高表达组高于其他3组(P < 0.01);④以上结果提示,miR-148a可促进人诱导性多能干细胞向心肌样细胞分化。 ORCID: 0000-0003-1698-4393(毛庆) 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     

Application of induced pluripotent stem cells in generation of a tissue-engineered tooth-like structure

Stem cells, such as adult stem cells or embryonic stem cells, are the most important seed cells employed in tooth tissue engineering. Even though dental-derived stem cells are a good source of seed cells for such procedures, they are not often used in clinical applications because of the limited supply. Induced pluripotent stem (iPS) cells, with their high proliferation and differentiation ability, are now considered a promising alternative. The objectives of this study were to assess the role of iPS cells in tooth tissue engineering. We used real-time polymerase chain reaction to confirm that mouse iPS (miPS) cells can be induced to express both odontogenic and osteogenic gene profiles. We then established a tooth germ model and transplanted the recombinant tooth germ into a mouse subrenal capsule for 4 weeks to reproduce early-tooth organogenesis. After 4 weeks, hematoxylin and eosin staining results showed newly formed bone-like and dental pulp-like areas. Further immunohistochemical staining confirmed that osteopontin was present in the apical part of the tooth-like structure. These results demonstrate that miPS cells have the potential to differentiate into odontogenic cells, confirming that they could be a new source of seed cells for use in tooth tissue engineering.     

MicroRNAs control the apoptotic threshold in primed pluripotent stem cells through regulation of BIM

Mammalian primed pluripotent stem cells have been shown to be highly susceptible to cell death stimuli due to their low apoptotic threshold, but how this threshold is regulated remains largely unknown. Here we identify microRNA (miRNA)-mediated regulation as a key mechanism controlling apoptosis in the post-implantation epiblast. Moreover, we found that three miRNA families, miR-20, miR-92, and miR-302, control the mitochondrial apoptotic machinery by fine-tuning the levels of expression of the proapoptotic protein BIM. These families therefore represent an essential buffer needed to maintain cell survival in stem cells that are primed for not only differentiation but also cell death.     

Two-phase analysis of molecular pathways underlying induced pluripotent stem cell induction

Induced pluripotent stem cells (iPSCs) can be reprogrammed from adult somatic cells by transduction with Oct4, Sox2, Klf4, and c-Myc, but the molecular cascades initiated by these factors remain poorly understood. Impeding their elucidation is the stochastic nature of the iPS induction process, which results in heterogeneous cell populations. Here we have synchronized the reprogramming process by a two-phase induction: an initial stable intermediate phase following transduction with Oct4, Klf4, and c-Myc, and a final iPS phase following overexpression of Sox2. This approach has enabled us to examine temporal gene expression profiles, permitting the identification of Sox2 downstream genes critical for induction. Furthermore, we have validated the feasibility of our new approach by using it to confirm that downregulation of transforming growth factor β signaling by Sox2 proves essential to the reprogramming process. Thus, we present a novel means for dissecting the details underlying the induction of iPSCs, an approach with significant utility in this arena and the potential for wide-ranging implications in the study of other reprogramming mechanisms.     

MiR-1228 promotes breast cancer cell growth and metastasis through targeting SCAI protein

Breast cancer is the most common cancer in women around the world. However, the molecular mechanisms underlying breast cancer pathogenesis are only partially understood. Here, in this study, we found that miR-1228 was up-regulated in breast cancer cell lines and tissues. Ectopic expression of miR-1228 mimics leads to promoted cell growth, invasion and migration. Using bioinfomatic analysis and 3’UTR luciferase reporter assay, we determined SCAI can be directly targeted by miR-1228, which can down-regulate endogenous SCAI protein level. Furthermore, our findings demonstrate that SCAI was down-regulated in breast cancer cell lines and tissues. Rescue experiment demonstrated that miR-1228 promoted cell growth is attenuated by over-expression of MOAP1 and miR-1228 promoted cell invasion and migration can be attenuated by over-expression of SCAI. Taken together, this study provides evidences that miR-1228 serves as an oncogene to promote breast cancer proliferation, invasion and migration, which may become a critical therapeutic target for breast cancer treatment.     

PMA体外诱导小鼠多能干细胞向心肌细胞的分化

目的研究丙二醇甲醚醋酸酯(PMA)对小鼠诱导多能干细胞体外分化为心肌细胞的影响,以建立一种高效安全的体外诱导i PSC分化为心肌细胞的实验方法。方法用悬滴法形成拟胚体(EBs),PMA诱导其向心肌细胞定向分化。免疫细胞学标记检测心肌肌钙蛋白T(c Tn T)和α横纹肌辅肌动蛋白(α-actinin)的表达;RT-PCR和q-PCR检测Brachyury,c Tn T,MLC2a,NKX2.5和GATA4等mRNA的表达。以添加相应DMSO作为对照组,观察各组出现搏动拟胚体的数量,计算分化比率。结果 PMA诱导小鼠诱导多能干细胞分化为心肌细胞的最佳浓度为100 nmol/L,此时拟胚体搏动率可达53%,显著高于对照组(12%),且PMA诱导产生的心肌细胞表达多种心肌蛋白及基因,具有心肌细胞的结构特征。结论 PMA能够促进mi PSC在体外定向分化为心肌样细胞。