iPSCs遗传稳定性与重编程机制的研究进展

诱导性多能干细胞(Induced pluripotent stem cells, iPSCs)是采用特定转录因子,将体细胞重编程为具有多能性的干细胞。iPSCs已成功由多种体细胞诱导出来,不仅具有发育多能性还能避免胚胎干细胞(Embryonic stem cells, ESCs)的伦理道德问题,已成为生命科学领域不可或缺的研究工具,具有广阔的应用前景。但获得高质量、遗传稳定的iPSCs是当前亟须解决的问题。文章对iPSCs重编程机制和遗传稳定性的研究进展进行了综述,以期为提高iPSCs的诱导效率、降低诱导成本、掌握iPSCs质量控制的关键点提供参考,从而推进多能性干细胞临床应用的发展。     

诱导多潜能干细胞（iPSCs）：安全高效的诱导策略

在分化的体细胞中,导入特定的转录因子能诱导得到诱导多潜能干细胞(induced pluripotent stem cells,iPSCs).iPSCs在细胞形态、生长特性、表面标志物以及畸胎瘤形成等方面与胚胎干细胞(embryonic stem cells,ESCs)非常相似,而且跟ESCs相比, iPSCs具有避免免疫排斥和不涉及伦理问题的优势,因此iPSCs的临床应用潜力巨大.然而,iPSCs具有成瘤性,而且其诱导效率极低,此二者严重阻碍了iPSCs的临床应用.为解决这两大难题,本综述将主要探讨安全高效的iPS细胞诱导策略,以期为促进其临床应用提供借鉴.     

诱导多能干细胞在医学方面的应用

2006年,首次报道在体外简单的转录因子就可以使体细胞重编程为多能性细胞。自从这项技术诞生以来,人们为改善诱导多能干细胞(iPSCs)技术做出了巨大努力,发展各种方法用于将重编程因子导入体细胞制备诱导多能干细胞(iPSCs)。诱导多能干细胞(iPSCs)技术彻底改变了人类对疾病发病机制的探索和药物开发的进程。本文简述了诱导多能干细胞的来源及诱导策略、近年来iPSCs在疾病建模、药物研发、再生医学等方面的应用,同时探讨了该技术当前存在的问题,并对未来进行了展望。     

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.     

人诱导多能干细胞与人胚胎干细胞分化过程中Oct4/Nanog基因表达的比较

目的：比较通过慢病毒方法获得的人诱导多能性干细胞（iPSCs）与人胚胎干细胞（hESCs）分化过程中全能性基因Oct4、Nanog的表达变化。方法：收集分化不同时间点的拟胚体（EBs）,检测三胚层分化基因以及全能性基因Oct4/Nanog的表达,并通过畸胎瘤组织切片的荧光染色分析Oct4的表达。结果：iPSCs获得的EB中内外三胚层分化基因表达的出现明显晚于hESCs来源的EB。不同于hESCs,iPSCs悬浮培养获得的EBs在体外培养18天未见内源性Oct4、Nanog基因表达的下调。未分化的iPSCs注射严重联合免疫缺陷（SCID）小鼠培养10周后获得的畸胎瘤中仍存在Oct4阳性的细胞,但iPS-#2中明显少于iPS-#5。结论：通过慢病毒方法获得的iPSCs虽然具有向三胚层分化的能力,但在分化过程中仍维持较高水平的全能性基因Oct4、Nanog的表达。     

Nanofiber-based polyethersulfone scaffold and efficient differentiation of human induced pluripotent stem cells into osteoblastic lineage

Human induced pluripotent stem cells (iPSCs) have been shown to have promising potential for regenerative medicine and tissue engineering applications. In the present study, osteogenic differentiation of human iPSCs was evaluated on polyethersulfone (PES) nanofibrous scaffold. According to the results, higher significant expressions of common osteogenic-related genes such as runx2, collagen type I, osteocalcin and osteonectin was observed in PES seeded human iPSCs compared with control. Alizarin red staining and alkaline phosphatase activity of differentiated iPSCs demonstrated significant osteoblastic differentiation potential of these cells. In this study biocompatibility of PES nanofibrous scaffold confirmed by flattened and spreading morphology of iPSCs under osteoblastic differentiation inductive culture. Taking together, nanofiber-based PES scaffold seeded iPSCs showed the highest capacity for differentiation into osteoblasts-like cells. These cells and PES scaffold were demonstrated to have great efficiency for treatment of bone damages and lesions.     

RNAs介导的干细胞诱导技术研究进展

诱导性多能干细胞(i PSCs)技术可重编程体细胞为胚胎干细胞(ESCs)样的多能性细胞,在药物筛选、再生医学等领域具有巨大的应用潜力。i PSCs技术自2006年首次报道用逆转录病毒转导一组转录因子,将小鼠(Mus musculus)成纤维细胞成功重编程为i PSCs以来,便不断改进和完善。近年来,不引起任何基因组改变的RNAs介导的i PSCs技术成为新兴的研究热点,主要包括修饰m RNAs法、mi RNAs法、si RNAs法和lnc RNAs法等。本文综述了RNAs介导的各种i PSCs技术的研究进展,分析了这些技术的优势、存在的不足及改进的方向等,为i PSCs技术的发展与应用提供参考。     

Efficient feeder-free episomal reprogramming with small molecules

Genetic reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) could offer replenishable cell sources for transplantation therapies. To fulfill their promises, human iPSCs will ideally be free of exogenous DNA (footprint-free), and be derived and cultured in chemically defined media free of feeder cells. Currently, methods are available to enable efficient derivation of footprint-free human iPSCs. However, each of these methods has its limitations. We have previously derived footprint-free human iPSCs by employing episomal vectors for transgene delivery, but the process was inefficient and required feeder cells. Here, we have greatly improved the episomal reprogramming efficiency using a cocktail containing MEK inhibitor PD0325901, GSK3β inhibitor CHIR99021, TGF-β/Activin/Nodal receptor inhibitor A-83-01, ROCK inhibitor HA-100 and human leukemia inhibitory factor. Moreover, we have successfully established a feeder-free reprogramming condition using chemically defined medium with bFGF and N2B27 supplements and chemically defined human ESC medium mTeSR1 for the derivation of footprint-free human iPSCs. These improvements enabled the routine derivation of footprint-free human iPSCs from skin fibroblasts, adipose tissue-derived cells and cord blood cells. This technology will likely be valuable for the production of clinical-grade human iPSCs.     

Simple Derivation of Transgene-Free iPS Cells by a Dual Recombinase Approach

Mammalian cells can be reprogrammed into induced pluripotent stem cells (iPSCs), a valuable tool for in vitro disease modeling and regenerative medicine. These applications demand for iPSCs devoid of reprogramming factor transgenes, but current procedures for the derivation of transgene-free iPSCs are inefficient and cumbersome. Here, we describe a new approach for the simple derivation of transgene-free iPSCs by the sequential use of two DNA recombinases, C31 Integrase and Cre, to control the genomic insertion and excision of a single, non-viral reprogramming vector. We show that such transgene-free iPSCs exhibit gene expression profiles and pluripotent developmental potential comparable to genuine, blastocyst-derived embryonic stem cells. As shown by a reporter iPSC line for the differentiation into midbrain dopaminergic neurons, the dual recombinase approach offers a simple and efficient way to derive transgene-free iPSCs for studying disease mechanisms and cell replacement therapies.     

iPSCs的潜在致瘤性及降低致瘤性方法的研究进展

自从2006年山中伸弥成功地将小鼠皮肤成纤维细胞重编程为诱导性多能干细胞(Induced pluripotent stem cells,i PSCs),i PSCs已成为科学家们研究的热点之一。而i PSCs在为基础研究和再生医学提供了无限可能的同时,相关争论焦点也随之产生,如i PSCs有可能导致肿瘤已成为其在临床应用前需要进一步证实、面对和解决的问题。目前已经有相关研究人员对i PSCs是否具有潜在致瘤性进行了探索。研究结果表明,i PSCs基因表达谱与癌细胞基因表达谱具有交集,重编程过程中积累了基因损伤,以及在培养过程中的基因突变都是其致瘤性产生的原因之一。研究人员目前已经找到很多减少i PSCs致瘤性的方法,比如优化促重编程因子、筛选表达载体、筛选细胞株等。文中对i PSCs致瘤可能性的原因和如何消除其致瘤性进行了综述。     

The genomic stability of induced pluripotent stem cells

With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells of human patients with defined factors, hold promise for regenerative medicine because they can provide a renewable source of autologous cells for cell therapy without the concern for immune rejection. In addition, iPSCs provide a unique opportunity to model human diseases with complex genetic traits, and a panel of human diseases have been successfully modeled in vitro by patient-specific iPSCs. Despite these progresses, recent studies have raised the concern for genetic and epigenetic abnormalities of iPSCs that could contribute to the immunogenicity of some cells differentiated from iPSCs. The oncogenic potential of iPSCs is further underscored by the findings that the critical tumor suppressor p53, known as the guardian of the genome, suppresses induced pluripotency. Therefore, the clinic application of iPSCs will require the optimization of the reprogramming technology to minimize the genetic and epigenetic abnormalities associated with induced pluripotency.     

Generation of Induced Pluripotent Stem Cells with High Efficiency from Human Umbilical Cord Blood Mononuclear Cells

Human induced pluripotent stem cells （iPSCs） hold great promise for regenerative med- icine. Generating iPSCs from immunologically immature newborn umbilical cord blood mononu- clear cells （UCBMCs） is of great significance. Here we report generation of human iPSCs with great efficiency from UCBMCs using a dox-inducible lentiviral system carrying four Yamanaka factors. We generated these cells by optimizing the existing iPSC induction protocol. The UCBMC-derived iPSCs （UCB-iPSCs） have characteristics that are identical to pluripotent human embryonic stem cells （hESCs）. This study highlights the use of UCBMCs to generate highly functional human iPSCs that could accelerate the development of cell-based regenerative therapy for patients suffering from various diseases.     

Derivation of male germ cells from induced pluripotent stem cells by inducers: A review

Induced pluripotent stem cells (iPSCs) refer to stem cells that are artificially produced using a new technology known as cellular reprogramming, which can use gene transduction in somatic cells. There are numerous potential applications for iPSCs in the field of stem cell biology becauase they are able to give rise to several different cell features of lineages such as three-germ layers. Primordial germ cells, generated via in vitro differentiation of iPSCs, have been demonstrated to produce functional gametes. Therefore, in this review we discussed past and recent advances in the in vitro differentiation of germ cells using pluripotent stem cells with an emphasis on iPSCs. Although this domain of research is still in its infancy, exploring development mechanisms of germ cells is promising, especially in humans, to promote future reproductive and developmental engineering technologies. While few studies have evaluated the ability and efficiency of iPSCs to differentiate toward male germ cells in vitro by different inducers, the given effect was investigated in this review.     

DNA methylation dynamics in human induced pluripotent stem cells over time

Epigenetic reprogramming is a critical event in the generation of induced pluripotent stem cells (iPSCs). Here, we determined the DNA methylation profiles of 22 human iPSC lines derived from five different cell types (human endometrium, placental artery endothelium, amnion, fetal lung fibroblast, and menstrual blood cell) and five human embryonic stem cell (ESC) lines, and we followed the aberrant methylation sites in iPSCs for up to 42 weeks. The iPSCs exhibited distinct epigenetic differences from ESCs, which were caused by aberrant methylation at early passages. Multiple appearances and then disappearances of random aberrant methylation were detected throughout iPSC reprogramming. Continuous passaging of the iPSCs diminished the differences between iPSCs and ESCs, implying that iPSCs lose the characteristics inherited from the parent cells and adapt to very closely resemble ESCs over time. Human iPSCs were gradually reprogrammed through the "convergence" of aberrant hyper-methylation events that continuously appeared in a de novo manner. This iPS reprogramming consisted of stochastic de novo methylation and selection/fixation of methylation in an environment suitable for ESCs. Taken together, random methylation and convergence are driving forces for long-term reprogramming of iPSCs to ESCs.     

诱导多潜能干细胞(iPSCs)的研究与应用进展

诱导多潜能干细胞(induced pluripotent stem cells,iPSCs)是体细胞在外源因子作用下,经直接细胞核程序重整而重新获得多潜能的干细胞.iPSCs在疾病的模型建立与机理研究、细胞治疗、药物的发现与评价等方面有着巨大的潜在应用价值.在过去几年中,科学家们致力于改进体细胞重编程技术并取得许多突破.然而,为实现其在临床上的应用,必须克服体细胞重编程效率低和iPSCs成瘤风险两大挑战,而且重编程机制有待进一步阐明.结合iPSCs最新研究成果,评述了有关领域国内外研究进展,重点讨论当前存在问题,并展望未来研究方向.