Volume: 11, Issue: 5

Preface

Induced Pluripotent Stem Cells: Current Progress and Future Perspectives

Qi Zhou

Page 257-258

Review

Lineage Specifiers: New Players in the Induction of Pluripotency

Jian Shu, Hongkui Deng

Pluripotency-associated factors and their rivals, lineage specifiers, have long been considered the determining factors for the identity of pluripotent and differentiated cells, respectively. Therefore, factors that are employed for cellular reprogramming in order to induce pluripotency have been identified mainly from embryonic stem cell (ESC)-enriched and pluripotency-associated factors. Recently, lineage specifiers have been identified to play important roles in orchestrating the process of restoring pluripotency. In this review, we summarize the latest discoveries regarding cell fate conversion using pluripotency-associated factors and lineage specifiers. We highlight the value of the “seesaw” model in defining cellular identity, opening up a novel scenario to consider pluripotency and lineage specification.

Page 259-263

Review

Cellular Reprogramming of Human Peripheral Blood Cells

Xiao-Bing Zhang

Breakthroughs in cell fate conversion have made it possible to generate large quantities of patient-specific cells for regenerative medicine. Due to multiple advantages of peripheral blood cells over fibroblasts from skin biopsy, the use of blood mononuclear cells (MNCs) instead of skin fibroblasts will expedite reprogramming research and broaden the application of reprogramming technology. This review discusses current progress and challenges of generating induced pluripotent stem cells (iPSCs) from peripheral blood MNCs and of in vitro and in vivo conversion of blood cells into cells of therapeutic value, such as mesenchymal stem cells, neural cells and hepatocytes. An optimized design of lentiviral vectors is necessary to achieve high reprogramming efficiency of peripheral blood cells. More recently, non-integrating vectors such as Sendai virus and episomal vectors have been successfully employed in generating integration-free iPSCs and somatic stem cells.
2006年，日本科学家山中伸弥及其学生，利用病毒载体把OCT4，SOX2，KLF4和MYC等四个基因转导入成纤维细胞后，细胞在培养皿中被逐渐重塑或重编程为诱导多能性干细胞（iPSC）。这一技术被认为是生命科学的一项革命性突破，为干细胞生物学的基础研究和临床细胞替代治疗带来了前所未有的机遇。 但是，从皮肤获得足够数量的成纤维细胞相当耗时费力，所以我们及其他研究小组着眼于人体中可快速获取大量细胞的外周血，然后从中分离出单个核细胞（MNC）用于重编程。经过优化的载体设计，我们能够利用慢病毒载体高效地重编程血细胞为iPSC。但利用慢病毒等整合型载体进行重编程难以用于临床治疗，于是很多实验室利用游离型载体（Episomal vectors）和仙台病毒（Sendai virus）等非整合技术。我们利用优化设计后的游离型载体实现了成人外周血细胞的高效重编程，尤其是我们发现，加入BCL-XL可以提高重编程效率10倍以上。我们尚未发表的结果显示，通过不断优化载体设计和因子组合，抽取1毫升外周血就可以得到数千个iPSC克隆。 我们还首次报道，只用OCT4一个因子在优化的培养条件下就能将人CD34+造血祖细胞转变为诱导间充质干细胞（iMSC）。加入小分子化合物组合后，还可以将细胞直接重编程为神经干细胞（NSC）。我们相信，通过不断地技术创新，包括载体设计，新因子组合，小分子化合物组合，培养条件优化等，成人外周血细胞可以直接在体外甚至在体内直接转变为其他有重要临床治疗价值的细胞类型。

Page 264-274

Review

The Functions of MicroRNAs and Long Non-coding RNAs in Embryonic and Induced Pluripotent Stem Cells

Wenwen Jia, Wen Chen, Jiuhong Kang

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) hold immense promise for regenerative medicine due to their abilities to self-renew and to differentiate into all cell types. This unique property is controlled by a complex interplay between transcriptional factors and epigenetic regulators. Recent research indicates that the epigenetic role of non-coding RNAs (ncRNAs) is an integral component of this regulatory network. This report will summarize findings that focus on two classes of regulatory ncRNAs, microRNAs (miRNAs) and long ncRNAs (lncRNAs), in the induction, maintenance and directed differentiation of ESCs and iPSCs. Manipulating these two important types of ncRNAs would be crucial to unlock the therapeutic and research potential of pluripotent stem cells.
胚胎干细胞和诱导多能干细胞在转化医学方面的应用前景巨大，这主要是因为它们具有自我更新的能力和分化成为所有类型细胞的能力。这种独一无二的特性受到来自于转录因子和表观遗传调控因子二者间复杂相互作用的调控。近年来研究表明，非编码RNA的表观遗传作用在该调控网络中扮演非常重要的角色。本篇报告总结了关于两类具有调控功能的非编码RNA即microRNA（miRNAs）和long ncRNAs(lncRNA)在胚胎干细胞和诱导多能干细胞诱导、维持、定向分化方面的发现。深入的探究这两类重要的非编码RNA对于开启多能干细胞治疗至关重要。

Page 275–283

Review

Integration-free Methods for Generating Induced Pluripotent Stem Cells

Yi-ye Zhou, Fanyi Zeng

Induced pluripotent stem (iPS) cells can be generated from mouse or human fibroblasts by exogenous expression of four factors, Oct4, Sox2, Klf4 and c-Myc, and hold great potential for transplantation therapies and regenerative medicine. However, use of retroviral vectors during iPS cell generation has limited the technique’s clinical application due to the potential risks resulting from genome integration of transgenes, including insertional mutations and altered differentiation potentials of the target cells, which may lead to pathologies such as tumorigenesis. Here we review recent progress in generating safer transgene-free or integration-free iPS cells, including the use of non-integrating vectors, excision of vectors after integration, DNA-free delivery of factors and chemical induction of pluripotency.

Page 284–287

Review

Induced Pluripotency for Translational Research

Menghua Wu, Guilai Chen, Baoyang Hu

The advent of induced pluripotent stem cells (iPSCs) has revolutionized the concept of cellular reprogramming and potentially will solve the immunological compatibility issues that have so far hindered the application of human pluripotent stem cells in regenerative medicine. Recent findings showed that pluripotency is defined by a state of balanced lineage potency, which can be artificially instated through various procedures, including the conventional Yamanaka strategy. As a type of pluripotent stem cell, iPSCs are subject to the usual concerns over purity of differentiated derivatives and risks of tumor formation when used for cell-based therapy, though they provide certain advantages in translational research, especially in the areas of personalized medicine, disease modeling and drug screening. iPSC-based technology, human embryonic stem cells (hESCs) and direct lineage conversion each will play distinct roles in specific aspects of translational medicine, and continue yielding surprises for scientists and the public.
IPSCs的出现革新了细胞重编程的概念，而多能性的定义由化学重编程iPSCs的出现受到冲击。多能性的维持需要不同细胞谱系特异分子力量之间的调整平衡。多能性相关因子可以作为细胞谱系特异性分子指导细胞命运的转变。而传统的“发育时钟”被认为是单向不可逆的，多能性相关因子和谱系特异决定因子分别能够确定多能细胞和分化细胞的身份。虽然iPSCs在转化应用研究上特别是个性化医疗、疾病模型和药物筛选上，具有一些优势，但是作为一种多能性细胞，iPSCs在细胞治疗上仍会遇到与ESCs类似的问题，包括分化得到的细胞的纯度问题，肿瘤形成的风险问题等。IPSCs、hESCs及直接谱系转换这三者均在转化医学的特定方面具有不同的优势，将会持续给科学家和大众带来不一样的精彩。在这篇综述中，我们着重归总了iPSCs和重编程领域的最新进展情况，特别是细胞命运决定及转变的新的概念性框架的提出和在转化研究中的潜在应用，也提出了iPSCs在转化应用中存在的问题和挑战。

Page 288–293

Review

Clinical Therapy Using iPSCs: Hopes and Challenges

Xiao Lu, Tongbiao Zhao

Induced pluripotent stem cells (iPSCs) are generated by ectopic expression of defined transcription factors in somatic cells. They can undergo unlimited self-renewal and maintain the embryonic stem cells (ESCs)-like ability to differentiate into all three germ layers. iPSCs can potentially provide unlimited autologous cells for therapy and therefore hold great promise for regenerative medicine. Here we reviewed the recent advances in iPSC studies on disease modeling and clinical treatment as well as challenges correlated with clinical development of iPSCs, like tumorigenicity, immunogenicity and genomic instability.
诱导多能干细胞（iPSC）的发现是干细胞研究领域的一项里程碑式突破。由体细胞重编程而来的iPSC细胞与胚胎干细胞类似，可以无限的自我更新并且可以分化为机体的各种组织器官。从理论上讲iPSC可以为再生医学提供无限的自体来源细胞，同时又避免了胚胎干细胞所免临的伦理问题，因此iPSC在临床应用方面有着巨大的前景。iPSC发现以来在基础理论研究与再生医学研究方面均取得重要进展。通过病人的体细胞重编程获得特定疾病的iPSC细胞，再定向分化成相应的细胞类型，一方面对于从发育的的角度探索疾病发生机理具有重要意义，另一方面iPSC疾病模型为针对性药物筛选及毒性毒理检测等研究提供了新的模型。目前，大量的研究报道了从多能干细胞（包括诱导多能干细胞）成功分化获得了神经、心脏、肝脏、肾脏、胰腺等组织器官的细胞，并建立了相应组织器官疾病的iPSC分化细胞模型。人iPSC分化的视网膜色素上皮细胞也已经应用于临床实验。然而，与此同时，由于目前定向分化技术的局限性及体内真正生理条件下发病的复杂性，体外多能干细胞的高效分化、精准疾病细胞模型及干细胞的替代治疗等仍面临着巨大的困难。此外iPSC重编程过程引起的基因组及表观基因组的不稳定性及其潜在的致瘤性与免疫原性等问题亦是诱导多能干细胞领域内所必须面对的挑战。

Page 294–298

Review

Pluripotency of Induced Pluripotent Stem Cells

Chunjing Feng, Yun-Dan Jia, Xiao-Yang Zhao

Induced pluripotent stem (iPS) cells can be generated by forced expression of four pluripotency factors in somatic cells. This has received much attention in recent years since it may offer us a promising donor cell source for cell transplantation therapy. There has been great progress in iPS cell research in the past few years. However, several issues need to be further addressed in the near future before the clinical application of iPS cells, like the immunogenicity of iPS cells, the variability of differentiation potential and most importantly tumor formation of the iPS derivative cells. Here, we review recent progress in research into the pluripotency of iPS cells.
过表达四种多能性因子，可以将体细胞重编程获得诱导多能干细胞（induced pluripotent stem cells, iPSCs）。由于iPSCs具有多潜能分化的能力，能够分化获得多种组织和细胞，是未来再生医学的重要种子细胞，因而受到极大关注。但目前iPSCs仍面临一些技术缺陷导致的应用瓶颈如iPSCs的免疫原性，分化潜能的可变性，以及iPSCs衍生产品的致瘤性问题。这里我们将过去几年iPSCs多能性方面的研究进展进行了总结。

Page 299–303

Original Research

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

Juan Wang, Qi Gu, Jie Hao, Donghui Bai, Lei Liu, Xiaoyang Zhao, Zhonghua Liu, Liu Wang, Qi Zhou

Human induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine. Generating iPSCs from immunologically immature newborn umbilical cord blood mononuclear 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.
人诱导多能性干细胞（induced pluripotent stem cells, iPSCs）在再生医学中有着广泛的应用前景。从免疫系统未成熟的新生儿脐带血单个核细胞（umbilical cord blood mononuclear cells, UCBMCs）中获得多能性干细胞具有十分重要的意义。在本文中，我们运用Dox-诱导的携带Yamanaka四因子的慢病毒系统，通过改善现有iPSCs诱导流程，成功地将新生儿脐带血单个核细胞高效诱导成为人iPSCs。所获得的iPSCs具有人胚胎干细胞的特性，包括：集落边缘光滑，细胞之间连接紧密，呈扁平样，碱性磷酸酶阳性，核型正常，表达多能性细胞的标志基因，在体外和体内均可以随机分化成三胚层的细胞。该研究为将UCBMCs重编程为iPSCs提供了一种新方法，有助于加速人iPSCs在再生医学中的应用步伐。

Page 304–311

Original Research

Induced Neural Stem Cells Generated from Rat Fibroblasts

Guangjun Xi, Pingfang Hu, Cunye Qu, Shenfeng Qiu, Chang Tong, Qi-Long Ying

The generation of induced tissue-specific stem cells has been hampered by the lack of well-established methods for the maintenance of pure tissue-specific stem cells like the ones we have for embryonic stem (ES) cell cultures. Using a cocktail of cytokines and small molecules, we demonstrate that primitive neural stem (NS) cells derived from mouse ES cells and rat embryos can be maintained. Furthermore, using the same set of cytokines and small molecules, we show that induced NS (iNS) cells can be generated from rat fibroblasts by forced expression of the transcriptional factors Oct4, Sox2 and c-Myc. The generation and long-term maintenance of iNS cells could have wide and momentous implications.
由于缺乏像胚胎干细胞一样能够长期在体外稳定维持的可靠培养条件，可诱导的组织特异性干细胞的研究工作受到很大的阻碍。我们的研究发现，采用细胞因子和小分子化合物相组合的培养体系，可以很好将小鼠胚胎干细胞分化的神经干细胞及大鼠胚胎发育的神经干细胞在体外长期稳定的培养维持。在这种培养体系中，我们发现通过转入Oct4、Sox2和c-Myc三个基因可以将大鼠成纤维细胞重编程为神经干细胞。同时，可诱导的神经干细胞在这种特定的培养体系下可以长久的保存，为神经干细胞的广泛应用打下坚实的基础。

Page 312–319

Original Research

Induced Pluripotent Stem Cells Are Sensitive to DNA Damage

Minjie Zhang, Caiyun Yang, Huixian Liu, Yingli Sun

Induced pluripotent stem cells (iPSCs) resemble embryonic stem cells (ESCs) in morphology, gene expression and in vitro differentiation, raising new hope for personalized clinical therapy. While many efforts have been made to improve reprogramming efficiency, significant problems such as genomic instability of iPSCs need to be addressed before clinical therapy. In this study, we try to figure out the real genomic state of iPSCs and their DNA damage response to ionizing radiation (IR). We found that iPSC line 3FB4-1 had lower DNA damage repair ability than mouse embryonic fibroblast (MEF) cells, from which 3FB4-1line was derived. After the introduction of DNA damage by IR, the number of γ-H2AX foci in 3FB4-1 increased modestly compared to a large increase seen in MEF, albeit both significantly (P < 0.01). In addition, whole-genome sequencing analysis showed that after IR, 3FB4-1 possessed more point mutations than MEF and the point mutations spread all over chromosomes. These observations provide evidence that iPSCs are more sensitive to ionizing radiation and their relatively low DNA damage repair capacity may account for their high radiosensitivity. The compromised DNA damage repair capacity of iPSCs should be considered when used in clinical therapy.
诱导多功能干细胞（induced pluripotent stem cells, iPSCs）是体细胞经重编程而得到了类似于胚胎干细胞的一种细胞类型。其在疾病治疗中具有巨大的应用价值，进一步拉近了干细胞和临床治疗的距离。然而iPSCs在体内的成瘤性限制了其广大应用。本研究过主要通过全基因组测序分析方法，发现iPSCs在经电离照射后基因组上点突变总数上升率明显大于体细胞MEF，且照射引起的突变并没有聚集在某条染色体上，而是基本平均分布于整套染色体。此外通过分别检测两种细胞系的DNA损伤修复能力发现，iPSCs在电离照射后γH2AX的上升幅度明显低于体细胞MEF，揭示其具有较低的DNA损伤修复能力，进而表明iPSCs较低的DNA损伤修复能力是其基因组不稳定性的一个重要原因。本研究阐明了iPSCs基因组不稳定性是iPSCs在临床应用中致瘤性的一个重要因素，并在机制层面揭示了DNA损伤修复能力在其中发挥着重要的作用。为进一步降低iPSCs的成瘤性，提高iPSCs临床应用提供了一定的理论依据。

Page 320–326

Original Research

Generation and Developmental Characteristics of Porcine Tetraploid Embryos and Tetraploid/diploid Chimeric Embryos

Wenteng He, Qingran Kong, Yongqian Shi, Bingteng Xie, Mingxia Jiao, Tianqing Huang, Shimeng Guo, Kui Hu, Zhonghua Liu

The aim of this study was to optimize electrofusion conditions for generating porcine tetraploid (4n) embryos and produce tetraploid/diploid (4n/2n) chimeric embryos. Different electric field intensities were tested and 2 direct current (DC) pulses of 0.9 kV/cm for 30 μs was selected as the optimum condition for electrofusion of 2-cell embryos to produce 4n embryos. The fusion rate of 2-cell embryos and the development rate to blastocyst of presumably 4n embryos, reached 85.4% and 28.5%, respectively. 68.18% of the fused embryos were found to be 4n as demonstrated by fluorescent in situ hybridization (FISH). Although the number of blastomeres in 4n blastocysts was significantly lower than in 2n blastocysts (P < 0.05), there was no significant difference in developmental rates of blastocysts between 2n and 4n embryos (P > 0.05), suggesting that the blastocyst forming capacity in 4n embryos is similar to those in 2n embryos. Moreover, 4n/2n chimeric embryos were obtained by aggregation of 4n and 2n embryos. We found that the developmental rate and cell number of blastocysts of 4-cell (4n)/4-cell (2n) chimeric embryos were significantly higher than those of 2-cell (4n)/4-cell (2n), 4-cell (4n)/8-cell (2n), 4-cell (4n)/2-cell (2n) chimeric embryos (P < 0.05). Consistent with mouse chimeras, the majority of 4n cells contribute to the trophectoderm (TE), while the 2n cells are mainly present in the inner cell mass (ICM) of porcine 4n/2n chimeric embryos. Our study established a feasible and efficient approach to produce porcine 4n embryos and 4n/2n chimeric embryos.
嵌合体是由不同遗传组成的细胞或者组织构成的嵌合生物个体。通过电融合制作小鼠四倍体胚胎与二倍体胚胎或者ES细胞嵌合后，四倍体胚胎来源的细胞贡献到胚外谱系中，二倍体细胞可以贡献到胚内和胚外谱系中，胚胎发育到期后能够得到完全来自二倍体胚胎或者ES的个体，这种方法称为四倍体补偿技术。因此，四倍体补偿技术可以绕过传统二倍体嵌合小鼠阶段，直接得到基因修饰的小鼠。除此之外，嵌合体的研究有助于胚胎细胞发育分化的研究和特殊物种基因保存，在动物育种、生产转基因动物研究、医学、产业化应用等众多领域也都具有广泛应用价值。目前，关于猪嵌合体的研究鲜有报道，如何优化猪四倍体胚胎的制作方法，二倍体胚胎与四倍体胚胎嵌合最佳时期的确定，以及嵌合后不同倍性来源的细胞在嵌合囊胚中时如何分布等，都没有相关的报道，有待于研究。据此，本研究优化了猪四倍体胚胎制作体系，探索了四倍体与二倍体嵌合胚胎的发育模式，为建立与猪相关的疾病模型和转基因动物制作方法等奠定基础。

Page 327–333