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干细胞移植治疗心脏病的最新研究进展心脏病是导致全球许多人死亡的主要原因之一，世界卫生组织估计全球每年有1700万人死于各种心脏病。
干细胞疗法作为一种新兴的治疗方法，近年来受到了广泛的关注。
干细胞具有自我更新和分化为各种细胞类型的能力，因此在心脏病的治疗中具有巨大的潜力。
本文将探讨干细胞移植治疗心脏病的最新研究进展。
干细胞移植治疗心脏病的基础是采用干细胞的自我更新和分化能力来修复受损的心脏组织。
现在已知有很多种来源的干细胞可以用于心脏病的治疗，如自体干细胞、胚胎干细胞、多能干细胞等。
其中，自体干细胞是指从患者自身身体组织中提取的干细胞，具有避免免疫排斥的优势。
目前的研究表明，干细胞移植治疗心脏病是安全且有效的。
近年来，一项最新的研究发现了一种新型干细胞，被称为诱导多能干细胞(induced pluripotent stem cells, iPSCs)。
iPSCs是通过将成熟的体细胞转化为干细胞的过程中获得的。
这一发现极大地推进了干细胞移植治疗心脏病的研究。
iPSCs具有多能性，能够分化为心细胞并进行心脏组织的修复。
研究人员发现，iPSCs移植后可以促进心肌细胞的生成和血管再生，有效恢复了受损心脏的功能。
除了iPSCs外，研究人员还发现胚胎干细胞在心脏病治疗中具有巨大的潜力。
胚胎干细胞来源于胚胎早期，具有极强的分化潜能。
研究显示，胚胎干细胞可以分化为心肌细胞、心内膜细胞等心脏组织，从而实现心脏组织的修复和再生。
然而，胚胎干细胞的使用还面临着伦理和法律等诸多限制，因此其应用范围受到了一定的限制。
另外一项最新的研究表明，干细胞可以通过释放细胞外囊泡(extracellular vesicles, EVs)来治疗心脏病。
EVs是一种包裹着细胞分泌物的微小膜囊，包含有多种活性因子和生物活性分子。
研究发现，干细胞释放的EVs可以通过转运生物活性物质，促进心脏细胞的增殖和血管生成，从而发挥修复心脏组织的作用。
与传统的干细胞移植治疗相比，使用EVs的治疗方法具有更低的免疫排斥风险和更高的安全性。
诱导多能干细胞名词解释概述诱导多能干细胞（induced pluripotent stem cells，简称iPSCs）是一种在实验室中通过重新编程成熟细胞而获得的一类多能干细胞。
与胚胎干细胞相比，iPSCs具有相似的多能性和自我更新能力，但不涉及胚胎的形成过程，从而避免了伦理和法律争议。
iPSCs的发现被认为是一项重大突破，为疾病治疗、组织再生和药物筛选等领域带来了新的可能。
历史背景iPSCs的发现可以追溯到2006年，由日本科学家山中伸弥和他的团队首次成功地通过将成熟的人体皮肤细胞转录因子的表达方式，将其重新编程成具有胚胎干细胞样特征的细胞。
这项研究是在老鼠细胞中进行的，但随后的研究证实了在人体细胞中也可以实现这一转化。
山中伸弥因此成为首位获得诺贝尔生理学或医学奖的日本科学家。
iPSCs的制备方法iPSCs的制备方法通常包括以下几个关键步骤：1.选择原始细胞：可以使用多种类型的成熟细胞作为起始细胞，如皮肤细胞、血液细胞等。
起始细胞的选择在一定程度上会影响后续iPSCs的性质和应用。
2.重编程方法：通过引入转录因子或使用其他技术，将起始细胞中特定转录因子的表达重新激活，使其回到未分化状态。
这些转录因子通常包括Oct4、Sox2、Klf4和c-Myc等。
3.细胞培养和扩增：将重编程后的细胞进行培养和扩增，使其成为一个细胞系。
这个细胞系中的细胞具有类似于胚胎干细胞的特征，包括多能性和自我更新能力。
4.鉴定和纯化：通过特定的标志物或性状，对iPSCs进行鉴定和纯化。
这些标志物包括Oct4、Nanog、Sox2等。
纯化后的iPSCs可以用于进一步的应用研究。
iPSCs的特性iPSCs具有以下几个重要的特性：1.多能性：iPSCs具有向三个胚胎发层（内胚层、中胚层和外胚层）分化的潜能，从而有能力分化成各种类型的成熟细胞，如神经细胞、心肌细胞、肝细胞等。
这种多能性使得iPSCs在疾病治疗和组织再生方面具有巨大潜力。
干细胞科研领域的领军人物第一位、山中伸弥（Shinya Yamanaka）简介：山中伸弥（Shinya Yamanaka），1962年出生于日本大阪府，日本医学家，京都大学再生医科研究所干细胞生物系教授，大阪市立大学医学博士（1993年），美国加利福尼亚州旧金山心血管疾病研究所高级研究员。
所获奖项：2008年邵逸夫生命科学与医学奖2009年拉斯克基础医学奖2011年获得国际最高学术大奖之一的沃尔夫医学奖，与其一起获奖的还有美国怀特黑德研究所的Rudolf Jaenisch。
2012年山中伸弥与美国软件工程师利努斯〃托瓦兹获得芬兰“千年技术奖”，二人分别获得60万欧元的奖金。
[5]2012年10月与英国发育生物学家约翰〃格登（John Gurdon）因在细胞核重新编程研究领域的杰出贡献而获得诺贝尔生理学或医学奖。
研究方向主要有两个：1、建立符合临床应用标准的人的iPS细胞库；2、研究iPS 细胞产生的分子机制。
代表作：1. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell.2007 Nov 30;131(5):861-72.2. Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent inducedpluripotent stem cells. Nature. 2007 Jul 19;448(7151):313-7.3. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006 Aug 25;126(4):663-76. Epub 2006 Aug 10.第二位、约翰〃格登（John B. Gurdon）简介：约翰〃伯特兰〃格登（John Bertrand Gurdon）是一位英国发育生物学家，1933年10月2日出生于英国，中学毕业于伊顿公学。
2种诱导 iPSC 向神经干细胞分化方法的比较杨坦;刘华;汪运山【摘要】目的：整体比较2种促进诱导性多能干细胞（ induced pluripotent stem cells ， iPSC）向神经干细胞（neural stem cells，NSC）分化的方法，确定一种稳定、高效的获得NSC的方法，并对NSC进行系统鉴定。
方法：方法A：SB431542和drosomophorin的浓度均为5μmmol／L，诱导初始密度100％；方法B：SB431542的浓度为5 mmol／L， drosomophorin的浓度为1 mmol／L，诱导初始密度为40％。
比较及鉴定方法：镜下观察诱导获得NSC的状态；real-time PCR比较神经干细胞相关基因Pax6、nestin、Sox1、Sox2等表达量；流式细胞术分析诱导第16天Pax6阳性率；免疫荧光定性分析神经干细胞相关蛋白的表达及其自发分化的能力。
结果：方法A获得的NSC悬起后成球趋势明显，圆形，透明；方法B诱导获得NSC形状不规则，色灰暗。
Real-time PCR结果证明方法A诱导获得的细胞神经干细胞相关基因的表达量高于方法B。
流式细胞术分析证明第16天，PAX6的阳性率，方法A高于方法B。
经鉴定，方法A获得的神经干细胞高表达Pax6、nestin、Sox2等基因自发分化30 d，形成明显的神经纤维束，表达TUJ-1、MAP2及GFAP等神经元和胶质细胞的特异性标志物。
结论：方法A整体优于方法B，我们推荐方法A作为诱导iPSC向神经干细胞分化的方法。
%AIM:To select an efficient way of promoting induced pluripotent stem cells ( iPSC) to differentiate into neural stem cells (NSC) by comparing 2 methods.METHODS:The culture system in method A contained SB431542 (5 mmol/L) and drosomophorin (5 mmol/L) with 100%initial cell density, while that in method B contained SB431542 (5 mmol/L) and drosomophorin (1 mmol/L) with 30%~50% initial cell density.Forcomparison and identification of the 2 methods, the growth state was observed under microscope , and the expression of Pax6, nestin, Sox1 and Sox2 was quantitatively detected by real-time PCR and flow cytometry .The related protein expression and the ability of spontaneous differentiation were determined by immunofluorescence analysis .RESULTS: The cells derived from method A with 5 mmol/L of SB431542 and drosomophorin and 100% initial cell density achieved the higher expression of Pax 6, nestin, Sox1 and Sox2.The growth state was better and the cells differentiated into neurons and astrocytes normally .CONCLU-SION:The method A was superior to method B , and we recommend the method A with 5 mmol/L of SB431542 and droso-mophorin and 100%initial cell density as the method for differentiating NSC .【期刊名称】《中国病理生理杂志》【年(卷),期】2015(000)001【总页数】5页(P188-192)【关键词】诱导性多能干细胞;分化;神经干细胞【作者】杨坦;刘华;汪运山【作者单位】山东省医学科学院基础医学研究所，济南大学-山东省医学科学院医学与生命科学学院; 山东大学附属济南市中心医院中心实验室;山东大学附属济南市中心医院中心实验室; 山东省移植与组织工程研究中心，山东济南250013;山东大学附属济南市中心医院中心实验室; 山东省移植与组织工程研究中心，山东济南250013【正文语种】中文【中图分类】R33-3神经干细胞(neural stem cells, NSC)可用于建立神经系统疾病的细胞模型，因此在神经系统疾病的机制及治疗的研究中日趋重要[1]。
诱导性多潜能干细胞现状及前景展望一、本文概述随着生物科技的飞速发展，诱导性多潜能干细胞（Induced Pluripotent Stem Cells，简称iPSCs）的研究已经成为当代生物医学领域的一个热点。
作为一种具有自我更新能力和多向分化潜能的细胞类型，iPSCs的出现在很大程度上颠覆了我们对细胞命运的认知，为疾病治疗、药物筛选、再生医学等领域提供了新的可能性。
本文旨在全面概述诱导性多潜能干细胞的研究现状，深入剖析其潜在的应用价值，并展望未来的发展前景。
我们将从iPSCs的诱导技术、分化机制、临床应用等方面展开讨论，以期为读者提供一个清晰、深入的iPSCs研究全貌。
我们也将关注当前面临的挑战与问题，以期推动iPSCs技术的进一步发展。
二、诱导性多潜能干细胞的研究现状诱导性多潜能干细胞（Induced Pluripotent Stem Cells, iPSCs）是近年来生物医学领域的一个重大突破。
自从2006年日本科学家山中伸弥首次成功地将成体细胞诱导为具有类似胚胎干细胞特性的多潜能干细胞以来，iPSCs的研究已经取得了长足的进展。
在研究现状方面，iPSCs的制备方法已经从最初的病毒载体法发展到现在的非整合型质粒法、mRNA法以及蛋白法，显著提高了诱导过程的安全性和效率。
同时，关于iPSCs的分化机制也取得了重要突破，研究人员已经能够控制iPSCs向特定细胞类型分化，如心肌细胞、神经细胞、胰岛细胞等，这为再生医学和疾病治疗提供了可能。
在疾病模型方面，iPSCs技术的出现使得研究者能够利用患者自身的细胞诱导出iPSCs，进而分化为疾病相关的细胞类型，为研究疾病的发生机制和开发新的治疗方法提供了独特的工具。
例如，利用iPSCs技术，研究人员已经成功模拟了多种遗传性疾病和退行性疾病，如帕金森病、阿尔茨海默症等。
在临床应用方面，虽然目前iPSCs技术还面临诸多挑战，如分化效率、安全性等问题，但已经有一些初步的临床试验在进行。
“线粒体转移”：干细胞发挥作用的新靶点来自中国广东省人民医院的李欣教授团队最新研究发现，诱导多能干细胞来源的间充质干细胞可以通过细胞间的隧道纳米管结构，转移自身“健康”的线粒体到缺氧损伤的PC12细胞中，并且显著改善了PC12细胞的存活率，从而模拟了心跳骤停后干细胞发挥抗缺氧损伤作用，并从“线粒体转移”的角度揭示了干细胞发挥作用的机制。
心跳骤停后引发的缺血缺氧脑损伤是全球患者死亡的重要原因之一，尤其是在发展中国家。
越来越多的研究也表明，神经元中的线粒体功能障碍是缺血缺氧诱导的脑损伤的重要机制，缺血缺氧诱导的线粒体功能障碍导致过量的反应性氧化物质产生，引发了神经元的凋亡。
因此，如何恢复神经元线粒体能量代谢功能、保护线粒体膜电位，以抑制后续损伤成为了治疗的关键所在。
干细胞是一类具有自我更新及分化能力的细胞，以干细胞为基础的再生医学也是未来治疗缺血缺氧性脑损伤的最有前景的手段之一。
目前认为干细胞发挥作用的主要机制为分化替代、旁分泌、抗炎等，也有报道骨髓间充质干细胞骨髓间充质干细胞可以通过转移线粒体到受损细胞从而发挥治疗作用。
那么，对于以线粒体损伤为特点的缺血缺氧性脑损伤，干细胞是否也可以通过这一方式来发挥治疗作用呢？值得我们进一步深入探讨。
此次李欣等发现，在模拟细胞缺氧状态时，CoCl2呈浓度依赖性地降低PC12细胞的存活率，而且PC12细胞的线粒体结构出现显著性退变。
而当诱导多能干细胞来源的间充质干细胞与PC12细胞共培养时，发现PC12细胞均能与诱导多能干细胞来源的间充质干细胞间形成隧道纳米管结构，并且通过隧道纳米管这一特殊通道，诱导多能干细胞来源的间充质干细胞可以将自身“健康”的线粒体转移到PC12细胞中，从而对抗CoCl2对PC12细胞线粒体结构和功能的损伤，减轻氧化应激，改善能量代谢，对缺氧诱导的细胞凋亡发挥抑制作用。
该研究在证明诱导多能干细胞来源的间充质干细胞对缺氧损伤有治疗作用的基础上，从线粒体转移替代的角度首次探讨了其中的具体机制，将有助于更大程度上发挥干细胞治疗的作用，为其临床使用提供理论基础和科学依据。
排名第一：ShinyaYamanaka和JamesA.Thomson博士是建立了可诱导的万能干细胞，在干细胞再生和分化重排机理上做出了最具突破性的进展性工作；这毫无疑问是诺贝尔奖级的工作，其他几位平时工作很杰出，可是没有这种级别的工作，只好屈居次位；排名第二：RudolfJaenisch博士长期从事于干细胞核的替代重组和干细胞的表观遗传修饰工作，卓有成绩，这也是培养诱导干细胞的核心工作，重要性无人能代替；排名第三：Rebortlanza博士领导和指挥着全球最领先的干细胞生物技术公司，独创和建立了分离和培养单个胚胎干细胞的方法和技术。
主编了所有重要的干细胞参考书籍。
每一相重大干细胞技术的出现，美欧主流媒体都要听他的意见，可谓干细胞领域的大腕人物；排名第四：AlanTrounson博士是国际免疫学和干细胞研究的先驱者，领导和指挥原澳大利亚Monash 大学免疫学和干细胞研究实验室，使Monash大学成为世界上最成功的大学之一。
手下的弟子MartinPerl博士出任南加州大学第一界干细胞和系统生物学所所长。
2007年成为美国眼下资金最多，实力最强的加州再生医学研究研究所所长，成为美国干细胞研究中最大的老板；排名第五：哈佛大学干细胞研究所所长DouglasA.Melton博士和斯坦福大学干细胞和再生医学研究所所长IrvingL.Weissman博士两人都是干细胞研究领域的顶尖高手，又各了带领着东西两岸这两所美国奈至全世界的顶尖学府的干细胞研究的竟赛。
排名第六：哈佛大学干细胞研究所共同所长DavidT.Scadden,博士和密西根大学干细胞中心主SeanMorrison博士两人是干细胞研究的中青年骨干，专长于干细胞分化再生的微环境调控机理的研究，.Scadden,博士是麻省总医院再生医学研究所所长，侧重于干细胞的临床应用。
Morrison博士则是休斯医学研究所研究员，是美国中西部大学中干细胞研究的顶级人物。
ReportsPluripotent stem cells, such as embryonic stem cells (ESCs), can self-renew and differentiate into all somatic cell types. Somatic cells can be reprogrammed to become pluripotent via nuclear transfer into oocytes or through the ectopic expression of defined factors (1–4). However, exogenous pluripotency-associated factors, especially Oct4, are indis-pensable for establishing pluripotency (5–7), and previous reprogram-ming strategies have raised concerns regarding the clinical applications (8, 9). Small molecules have advantages because they can be cell per-meable, non-immunogenic, more cost-effective, and can be more easily synthesized, preserved, and standardized. Moreover, their effects on inhibiting and activating the function of specific proteins are often re-versible and can be finely tuned by varying the concentrations. Here, we identified small-molecule combinations that were able to drive the re-programming of mouse somatic cells toward pluripotent cells.To identify small molecules that facilitate cell reprogramming, we searched for small molecules that enable reprogramming in the absence of Oct4 using Oct4 promoter-driven GFP expression (OG) mouse embryonic fibroblasts (MEFs), with viral expression of Sox2, Klf4, and c-Myc. After screening up to 10,000 small molecules (table S1A), we identified For-skolin (FSK), 2-Methyl-5-hydroxytryptamine (2-Me-5HT), and D4476 (table S1B) as chemical “substitutes” for Oct4 (Fig. 1, A and B, and figs. S1 and S2). Previously, we had developed a small-molecule combination “VC6T” [VPA, CHIR99021 (CHIR), 616452, Tranylcypromine], that enables reprogramming with a single gene, Oct4(6). We next treated OG-MEFs with VC6T plus the chemical substitutes of Oct4in the ab-sence of transgenes. We found VC6T plus FSK (VC6TF) induced some GFP-positive clusters expressing E-cadherin, a mesen-chyme-to-epithelium transition marker, reminiscent of early reprogram-ming by transcription factors (10, 11)(Fig. 1C and fig. S3). However, theexpression of Oct4 and Nanog was notdetectable and their promoters remainedhypermethylated, suggesting a repressedepigenetic state (fig. S3).To identify small molecules that fa-cilitate late reprogramming, we used adoxycycline (DOX)–inducible Oct4expression screening system, addingDOX only in the first 4-8 days (6).Small-molecule hits, including severalcAMP agonists (FSK, Prostaglandin E2,and Rolipram) and epigenetic modula-tors (3-deazaneplanocin A (DZNep),5-Azacytidine, Sodium butyrate, andRG108), were identified in this screen(fig. S4 and table S1B).To achieve complete chemical re-programming without theOct4-inducible system, these smallmolecules were further tested in thechemical reprogramming of OG-MEFswithout transgenes. When DZNep wasadded 16 days after treatment withVC6TF (VC6TFZ), GFP-positive cellswere obtained up to 65-fold more fre-quently than those treated with VC6TF,forming compact, epithelioid,GFP-positive colonies without clear-cutedges (Fig. 1, D and E, and fig. S5). Inthese cells, the expression levels of mostpluripotency marker genes were ele-vated but still lower than in ESCs, sug-gesting an incomplete reprogramming state (fig. S6). After switching to 2i-medium with dual inhibition (2i) of glycogen synthase kinase-3 and mitogen-activated protein kinase sig-naling after day 28 post-treatment, certain GFP-positive colonies devel-oped an ESC-like morphology (domed, phase-bright, homogeneous with clear-cut edges) (Fig. 1F) (12, 13). These colonies could be further cul-tured for more than 30 passages, maintaining an ESC-like morphology (Fig. 1, G and H). We refer to these 2i-competent, ESC-like, and GFP-positive cells as chemically induced pluripotent stem cells (CiPSCs).Next, we optimized the dosages and treatment duration of the small molecules, and were able to generate 1-20 CiPSC colonies from 50,000 initially plated MEFs (fig. S7). After an additional screen, we identified some small-molecule boosters of chemical reprogramming, among which, a synthetic retinoic acid receptor ligand, TTNPB, enhanced chemical reprogramming efficiency up to 40-fold, to a frequency com-parable to transcription factor-induced reprogramming (up to 0.2%) (fig.S8 and table S1B). Furthermore, using the small-molecule combinationVC6TFZ, we obtained CiPSC lines from mouse neonatal fibroblasts (MNFs), mouse adult fibroblasts (MAFs), and adipose-derived stem cells (ADSCs) with OG cassettes by an efficiency approximately 10 times lower than that obtained from MEFs (fig. S9 and table S3). Moreover, we induced CiPSCs from wild-type MEFs without OG cassettes or any other genetic modifications by a comparable efficiency to that achieved from MEFs with OG cassettes (fig. S9). The CiPSCs were also confirmed to beviral-vector free by genomic PCR and Southern blot analysis (fig. S10).The established CiPSC lines were then further characterized. They grew with a doubling time (14.1-15.1 hours) similar to that of ESCs (14.7 hours), maintained alkaline phosphatase activity, and expressed pluripo-tency markers, as detected by immunofluorescence and RT-PCR (Fig. 2,Pluripotent Stem Cells Induced from Mouse Somatic Cells bySmall-Molecule CompoundsPingping Hou,1* Yanqin Li,1* Xu Zhang,1,2* Chun Liu,1,2* Jingyang Guan,1* Honggang Li,1* Ting Zhao,1† Junqing Ye,1,2† Weifeng Yang,3†Kang Liu,1† Jian Ge,1,2† Jun Xu,1† Qiang Zhang,1,2† Yang Zhao,1‡ Hongkui Deng1,2‡1College of Life Sciences and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China. 2School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China. 3Beijing Vitalstar Biotechnology Co., Ltd., Beijing 100012, China.*These authors contributed equally to this work.†These authors contributed equally to this work.‡Corresponding author. E-mail: hongkui_deng@ (H.D.); yangzhao@ (Y.Z.).Pluripotent stem cells can be induced from somatic cells, providing an unlimited cell resource, with potential for studying disease and use in regenerative medicine. However, genetic manipulation and technically challenging strategies such as nuclear transfer used in reprogramming limit their clinical applications. Here, weshow that pluripotent stem cells can be generated from mouse somatic cells at a frequency up to 0.2% using a combination of seven small-molecule compounds. The chemically induced pluripotent stem cells (CiPSCs) resemble embryonic stem cells (ESCs) in terms of their gene expression profiles, epigenetic status, and potential for differentiation and germline transmission. By using small molecules, exogenous “master genes” are dispensable for cell fate reprogramming. This chemical reprogramming strategy has potential use in generating functional desirable cell types for clinical applications. o n J u l y 1 8 , 2 0 1 3 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o mA and B, and fig. S11). The gene expression profiles were similar in CiPSCs, ESCs, and OSKM-iPSCs (iPSCs induced by Oct4, Sox2, Klf4 and c-Myc) (Fig. 2C and fig. S12). DNA methylation state and histone modifications at Oct4 and Nanog promoters in CiPSCs were similar to that in ESCs (Fig. 2D and fig. S13). In addition, CiPSCs maintained a normal karyotype and genetic integrity for up to 13 passages (fig. S14 and table S2).To characterize their differentiation potential, we injected CiPSCs into immunodeficient (SCID) mice. The cells were able to differentiate into tissues of all three germ layers (Fig. 3A and fig. S15). When injected into eight-cell embryos or blastocysts, CiPSCs were capable of integra-tion into organs of all three germ layers including gonads and transmis-sion to subsequent generations (Fig. 3, B to E, and fig. S16). Unlike chimeric mice generated from iPSCs induced by transcription factors including c-Myc(14), the chimeric mice generated from CiPSCs were 100% viable and apparently healthy for up to 6 months (Fig. 3F). These observations suggest that the CiPSCs were fully reprogrammed into pluripotency (table S3).We next determined which of these small molecules were critical in inducing CiPSCs. We found four essential small molecules whose indi-vidual withdrawal from the cocktails generated significantly reduced GFP-positive colonies and no CiPSCs (Fig. 4, A to C). These small molecules (C6FZ) include: CHIR (C), a glycogen synthase kinase 3 inhibitor (15); 616452 (6), a transforming growth factor-beta inhibitor (16); FSK (F), a cAMP agonist (fig. S17) (17); and DZNep (Z), an S-adenosylhomocysteine (SAH) hydrolase inhibitor (figs. S18 and S19) (18, 19). Moreover, C6FZ was able to induce CiPSCs from both MEFs and MAFs, albeit by a 10 times lower efficiency than that induced by VC6TFZ (fig. S20 and table S3).To better understand the pluripotency-inducing properties of these small molecules, we profiled the global gene expression during chemical reprogramming and observed the sequential activation of certain key pluripotency genes, which was validated by real-time PCR and immuno-fluorescence (fig. S21). The expression levels of two pluripotency-related genes, Sall4 and Sox2, were most significantly induced in the early phase in response to VC6TF, as was the expression of several extra-embryonic endoderm (XEN) markers Gata4, Gata6, and Sox17 (Fig. 4, D to F, and figs. S22 to S24). The expression of Sall4was enhanced most signifi-cantly as early as 12 hours after small-molecule treatment, suggesting that Sall4 may be involved in the first step toward pluripotency in chemical reprogramming (fig. S22B). We further examined the roles of the en-dogenous expression of these genes in chemical reprogramming, using gene overexpression and knockdown strategies. We found the concomi-tant overexpression of Sall4 and Sox2 was able to activate an Oct4 pro-moter-driven luciferase reporter (fig. S25) and was sufficient to replace C6F in inducing Oct4 expression and generating iPSCs (Fig. 4, G and H, and fig. S26). The endogenous expression of Sall4, but not Sox2, requires the activation of the XEN genes, and vice versa (fig. S27). This suggests a positive feedback network formed by Sall4, Gata4, Gata6, and Sox17, similar to which was previously described in mouse XEN formation (20). Moreover, knockdown of Sall4 or these XEN genes impaired Oct4 acti-vation and the subsequent establishment of pluripotency (fig. S28), in consistent with our previous finding that Gata4 and Gata6 can contribute to inducing pluripotency (21). Taken together, these findings revealed a Sall4-mediated molecular pathway that acts in the early phase of chemical reprogramming (Fig. 4L). This step resembles a Sall4-mediated dedif-ferentiation process in vivo during amphibian limb regeneration (22).We next investigated the role of DZNep, which was added in the late phase of chemical reprogramming. We found that Oct4 expression was enhanced significantly after the addition of DZNep in chemical repro-gramming (Fig. 4D), and DZNep was critical for stimulating the expres-sion of Oct4 but not the other pluripotency genes (Fig. 4I). As an SAH hydrolase inhibitor, DZNep elevates the concentration ratio of SAH to S-adenosylmethionine (SAM) and may thereby repress the SAM-dependent cellular methylation process (fig. S18) (18, 19). Con-sistently, DZNep significantly decreased DNA and H3K9 methylation at the Oct4 promoter, which may account for its role in Oct4 activation (Fig. 4, J and K) (23, 24). As master pluripotency genes, Oct4 and Sox2 may thereby activate other pluripotency-related genes, and fulfill the chemical reprogramming process, along with the activation of Nanog and silencing of Gata6, in the presence of 2i (12, 13, 25, 26) (Fig. 4F and fig. S29). In summary, as a master switch governing pluripotency, Oct4 expression, which is kept repressed in somatic cells by multiple epigenetic modifica-tions, is unlocked in chemical reprogramming by the epigenetic modu-lator DZNep, and stimulated by C6F-induced expression of Sox2and Sall4 (Fig. 4L).Our proof-of-principle study demonstrates that somatic reprogram-ming toward pluripotency can be manipulated using only small-molecule compounds (fig. S30). It reveals that the endogenous pluripotency pro-gram can be established by the modulation of molecular pathways non-specific to pluripotency via small molecules rather than by exogenously provided “master genes.” These findings increase our understanding about the establishment of cell identities and open up the possibility of generating functionally desirable cell types in regenerative medicine by cell fate reprogramming using specific chemicals or drugs, instead of genetic manipulation and difficult-to-manufacture biologics. To date, the complete chemical reprogramming approach remains to be further im-proved to reprogram human somatic cells and ultimately meet the needs of regenerative medicine.References and Notes1. I. Wilmut, A. E. Schnieke, J. McWhir, A. J. Kind, K. H. Campbell,Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810–813 (1997). doi:10.1038/385810a0 Medline2. K. Takahashi, S. Yamanaka, Induction of pluripotent stem cells frommouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006). doi:10.1016/j.cell.2006.07.024 Medline3. S. Yamanaka, H. M. Blau, Nuclear reprogramming to a pluripotentstate by three approaches. Nature465, 704–712 (2010).doi:10.1038/nature09229 Medline4. M. Stadtfeld, K. Hochedlinger, Induced pluripotency: History,mechanisms, and applications. Genes Dev.24, 2239–2263 (2010).doi:10.1101/gad.1963910 Medline5. S. Zhu, W. Wei, S. Ding, Chemical strategies for stem cell biology andregenerative medicine. Annu. Rev. 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This work was supported by grants from the National 973 Basic Research Program of China (2012CD966401, 2010CB945204), the Key New Drug Creation and Manufacturing Program (2011ZX09102-010-03), the National Natural Science Foundation of China (90919031), the Ministry of Science and Technology (2011DFA30730, 2013DFG30680), the Beijing Science and Technology Plan (Z121100005212001), the Ministry of Education of China (111 project), and a Postdoctoral Fellowship of Peking-Tsinghua Center for Life Sciences. Microarray and RNA-seq data are deposited in the Gene Expression Omnibus (GEO) database (accession number GSE48243). The authors have filed a patent for the small-molecule combinations used in the chemical reprogramming reported in this paper. Supplementary Materials/cgi/content/full/science.1239278/DC1 Materials and Methods Figs. S1 to S30 Tables S1 to S5 References (27–37) o n J u l y 18, 2013w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o m17 April 2013; accepted 11 July 2013 Published online 18 July 2013 10.1126/science.1239278o n J u l y 18, 2013w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mFig. 1. Generation of CiPSCs by small-molecule compounds. (A and B) Numbers of iPSC colonies induced from MEFs infected by SKM (A) or SK (B) plus chemicals or Oct4. Error bars indicate standard deviation (s.d.) (n = 3). (C) Morphology of MEFs for chemical reprogramming on day 0 (D0) and a GFP-positive cluster generated using VC6TF on day 20 (D20) after chemical treatment. (D) Numbers of GFP-positive colonies induced after DZNep treatment on day 36. Error bars indicate the s.d. (n = 2). (E to G) Morphology of a compact, epithelioid, GFP-positive colony on day 32 (D32) after treatment (E), a primary CiPSC colony on day 40 (D40) after treatment (F), and passaged CiPSC colonies (G). (H) Schematic diagram illustrating the process of CiPSC generation. For (C) and (E to G), scale bars, 100 μm. For (D), cells fo r reprogramming were replated on day 12.o n J u l y 1 8 , 2 0 1 3 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o mFig. 2. Characterization of CiPSCs. (A and B) Pluripotency marker expression as illustrated by immunofluorescence (A, clone CiPS-25) and RT-PCR (B). Scale bars,100 μm. (C) Hierarchical clustering of global transcriptional profiles. 1-PCC, Pearson correlation coefficient. (D) Bisulfite genomic sequencing of the Oct4 and Nanog promoter regions. MNF-CiPS-7, MNF-derived CiPSC line #7; MAF-CiPS-1, MAF-derived CiPSC line #1. o n J u l y 1 8 , 2 0 1 3 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o mFig. 3. Pluripotency of CiPSCs.(A) Hematoxylin and eosin staining of CiPSC-derived teratoma (clone CiPS-30). (B to D) Chimeric mice (B, clone CiPS-34), germline contribution of CiPSCs in testis, (C, clone CiPS-45) and F2 offspring (D, clone CiPS-34). Scale bars, 100 μm. (E) Genomic PCR analyzing pOct4-GFP cassettes in the tissues of chimeras. (F) Survival curves of chimeras. n, total numbers of chimeras studied. o n J u l y 1 8 , 2 0 1 3 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o mFig. 4. Step-wise establishment of the pluripotency circuitry during chemical reprogramming. (A and B) Numbers of GFP-positive (A) and CiPSC (B) colonies induced by removing individual chemicals from VC6TFZ. The results of three independent experiments are shown with different colors (white, gray and black).(C) Structures of the essential chemicals. (D and E) The expression of pluripotency-related genes (D) and Gata6, Gata4 and Sox17 (E) as measured by real-time PCR. (F) Gene expression heat map at the single colony level. The value indicates the log2-transformed fold change (relative to Gapdh and normalized to the highest value). (G and H) Oct4 activation (G) and numbers of GFP-positive and iPSC colonies (H) induced by the overexpression of Sall4 and Sox2, with removing C6F from VC6TFZ. (I to K) The expression of pluripotency-related genes (I), DNA methylation (J) and H3K9 dimethylation (K) states of the Oct4promoter in the presence and absence of DZNep on day 32. (L) Schematic diagram illustrating the step-wise establishment of the pluripotency circuitry during chemical reprogramming. Error bars indicate the s.d. (n≥ 2).o n J u l y 1 8 , 2 0 1 3 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o m。
