Induced pluripotent stem cell (iPS) technology: promises and challenges

In 2006, an article published in Cell by Shinya Yamanaka took by surprise the stem cell research community. By performing systematic retroviral transduction of factors enriched in embryonic stem (ES) cells, the authors demonstrated the reprogramming of mouse fibroblasts into an ES cell-like state. These cells, baptized iPS (induced pluripotent stem) cells, were immediately recognized as a ground-breaking discovery. Subsequently, the same authors and other groups reported a similar achievement with human fibroblasts. Two years later, the number of top quality papers on iPS is astonishing, and interest in the scientific community has risen to a fever pitch. But although iPS has the potential to revolutionize Regenerative Medicine, important questions still remain unanswered. Work from multiple laboratories worldwide including ours is focused on deciphering the molecular mechanisms of iPS, and trying to improve the technique to make it suitable for the clinic. In this review article we briefly discuss the past, present and future of iPS, with emphasis on urgent issues to be solved. Supported by the National Nature Science Foundation of China (Grant Nos. 30725012, 30630039 and 90813033), Knowledge Innovation Project of Chinese Academy of Sciences (Grant No. KSCX2-YW-R-48), National Key Basic Research and Development Program of China (Grant Nos. 2006CB701504, 2006CB943600, 2007CB948002, 2007CB947804. 2007CB947900) and Guangzhou Science and Technology Development Funds (Grant No. 2008A1-E4011)     

<Emphasis Type="Italic">In vitro</Emphasis> induction of mouse meningeal-derived ips cells into neural-like cells

Previous research has shown that mouse embryonic stem (ES) cells can be induced to form neural cells in adherent monocultures. In this study, pluripotent stem (iPS) C5 cells derived from meningeal membranes were converted successfully into neural-like cells using the same protocol generally used for ES cells. Meningeal-iPS C5 cells were induced to express neural markers Sox1, Sox3, Pax6, Nestin and Tuj1 and to reduce the expression of ES markers Oct4 and Nanog during neural differentiation, and can be differentiated into Pax6 and Nestin positive neural progenitors, and further into neuronal, astrocytic, and oligodendrocytic cells. In vitro differentiation of iPS cells into patient-specific neural cells could serve as a model to study mechanisms of genetic diseases and develop promising candidates for therapeutic applications in dysfunctional or aging neural tissues. Meningeal cells express a high level of the embryonic master regulator Sox2, allowing them to be reprogrammed into iPS cells more easily than other somatic cells.     

Nuclear reprogramming by nuclear transplantation and defined transcription factors

In the past ten years, great breakthroughs have been achieved in the nuclear reprogramming area. It has been demonstrated that highly differentiated somatic cell genome could be reprogrammed to a pluripotent state, which indicates that differentiated cell fate is not irreversible. Nuclear transplantation and induced pluripotent stem (iPS) cell generation are the two major approaches to inducing reprogramming of differentiated somatic cell genome. In the present review, we will summarize the recent progress of nuclear reprogramming and further discuss the potential to generate patient specific pluripotent stem cells from differentiated somatic cells for therapeutic purpose. Supported by the National High Technology Research and Development Program of China (Grant No. 2005AA210930)     

On the “plasticity”of adult stem cells and its application in regenerative medicine

In recent years, with the increasingly further studies on embryonic stem cells and the recognition of the biologic characteristics of adult stem cells, it has been discovered that adult stem cells have another phenomenon of “plasticity” in addition to the characteristics of strong potential for self-renewal, proliferation and multi-differentiation, which brings us the hope for regenerative medicine—renewing new organ or tissue cells to replace those damaged by injury or diseases. Although the mechanism of “plasticity” and its application in the regenerative medicine are still in doubt, thorough exploration in these subjects would open up broad prospects for the use in cell and tissue engineering in the near future.     

Copy number variation and selection during reprogramming to pluripotency

The mechanisms underlying the low efficiency of reprogramming somatic cells into induced pluripotent stem (iPS) cells are poorly understood. There is a clear need to study whether the reprogramming process itself compromises genomic integrity and, through this, the efficiency of iPS cell establishment. Using a high-resolution single nucleotide polymorphism array, we compared copy number variations (CNVs) of different passages of human iPS cells with their fibroblast cell origins and with human embryonic stem (ES) cells. Here we show that significantly more CNVs are present in early-passage human iPS cells than intermediate passage human iPS cells, fibroblasts or human ES cells. Most CNVs are formed de novo and generate genetic mosaicism in early-passage human iPS cells. Most of these novel CNVs rendered the affected cells at a selective disadvantage. Remarkably, expansion of human iPS cells in culture selects rapidly against mutated cells, driving the lines towards a genetic state resembling human ES cells.     

干细胞的研究和应用

干细胞是一类具有自我更新和多向分化潜能的细胞群体,它能长期地自我更新,在特定的条件下具有分化形成多种终末细胞的能力,不同来源的干细胞分化潜能各异。近年来干细胞的研究与应用几乎涉及到所有生命科学和生物医学领域。本文概述了干细胞的生物学特性和它的可塑性,综述了分离培养技术及其在基础研究和临床上应用的研究进展,最后,提出了问题和展望。     

我国干细胞产业发展态势及其对策研究

干细胞研究是近年来生物医学领域的最热门的研究方向之一,近年来受到世界各国的高度重视,具有巨大的社会效益和市场前景。本文针对我国干细胞产业的发展现状和可持续发展要求,对干细胞产业发展的市场需求、产业目标、技术壁垒、研发需求等进行分析,并对该产业中的共性关键技术进行凝练,包括干细胞采集及存储业务、干细胞技术研发、干细胞移植及治疗、与其他产业结合等整个产业链,研究我国干细胞产业的技术发展现状,并提出干细胞产业发展的相关对策。     

干细胞研究国际发展态势分析

干细胞以其自我更新和多向分化的能力得到了国际社会的普遍关注,为干细胞疗法的临床应用带来了希望.该文结合文献计量方法,对干细胞以及诱导多能干细胞、胚胎干细胞和间充质干细胞等3个干细胞研究重点领域的科研现状进行了分析,总结了这些领域的发展趋势和重点研究方向,并根据分析结果,对我国干细胞领域采取的措施、今后发展的方向提出了建...     

干细胞技术进展

干细胞技术是人类后基因组计划的一部分。随着这一技术的科研和应用的快速发展,人们开始认识到这一技术的具大诱惑。本文对干细胞的概念、种类、技术原理、进行了概括,对干细胞技术应用领域、发展前景等进行了研究。     

大鼠胚胎神经干细胞体外培养及诱导分化

神经干细胞在理论研究和临床应用上有着广泛的前景.本文主要在体外分离培养SD大鼠胚胎前脑的神经干细胞,并分别用去除生长因子或添加全反式维甲酸(ATRA)两种方法诱导分化.免疫荧光染色技术分别检测细胞巢蛋白(Nestin)的表达及分化后β微管蛋白Ⅲ(β-Ⅲtubulin)、胶质纤维酸性蛋白(GFAP)的表达,计算分化率.结果显示:细胞生长状态良好,呈Nestin表达阳性.分化后可获得β-Ⅲtubulin及GFAP表达阳性的细胞,其中ATRA诱导方法获得β-Ⅲtubulin阳性细胞较多.     

Akt–Oct4 regulatory circuit in pluripotent stem cells

Oct4 is mainly expressed in embryonic stem cells(ESCs),germline stem cells,and embryonal carcinoma cells(ECCs)and plays an indispensable role in maintaining the pluripotency and self-renewal of these pluripotent stem cells.Akt serine/threonine kinase,a wellestablished anti-apoptosis and cell survival factor,has also been implicated as an important regulator of stemness.Emerging evidence indicated that Oct4 is reciprocally connected to Akt via a number of routes,and moreover,a direct interaction between Oct4 and Akt has recently been revealed.These components collectively form the Akt–Oct4 regulatory circuit.In this review,we summarize our current knowledge about the Akt–Oct4 regulatory circuit in ESCs and discuss its alterations in ECCs that may underlie the tumorigenesis of pluripotent stem cells.     

Neural stem cell transplantation in the repair of spinal cord injury

Neural stem cells are a pronising candidate for neural transplantation aimed at neural cell replacement and repair of the damaged host central nervous system (CNS). Recent studies using neural stem cells have shown that implanted neural stem cells can effectively incorporate into the damaged CNS and differentiate into neurons, astrocytes, and oligodendrocytes. The recent explosion in the field of neural stem cell research has provided insight into the inductive factors influencing neural stem cell differentiation and may yield potential therapies for several neurological disorders, including spinal cord injury. In this review, we summarize recent studies involving neural stem cell biology in both rodents and humans. We also discuss unique advantages and possible mechanisms of using neural stem cell trans plantation in the repair of spinal cord injury.     

人胎儿骨髓间充质干细胞的分离及生物学鉴定

通过原代细胞培养,从引产胎儿骨髓组织中分离干细胞,然后进行生物学鉴定,旨在体外建立培养胎儿骨髓干细胞的有效方法,为进一步研究干细胞奠定基础．本研究对四个月的引产胎儿骨髓组织进行原代细胞培养,采用贴壁筛选法,在含有15％胎牛血清的L-DMEM／IMDM(1:1)混和培养液中培养,7 d后细胞可长满瓶底．显微镜下观察,细胞形态均一,呈长梭形．传代后,在8代以内的细胞贴壁能力较强,生长速度较快．将其命名为BMMS-03．在第3代时,对培养的细胞进行了于细胞标志物的生物学鉴定,采用流式细胞仪对经免疫荧光染色的细胞进行检测．结果显示:97．2％的细胞呈CD105阳性反应,66．0％的细胞呈CD106阳性反应, 9．2％的细胞呈CD34阳性反应．阴性对照组阳性反应为0．5％．生物学鉴定的初步结果提示,从胎儿骨髓组织中分离培养成功的细胞为骨髓间充质干细胞,其细胞形态学特征、CD105 、CD106 和CD34-的检测结果均符合间充质干细胞的特征．本研究成功地建立了体外培养胎儿骨髓间充质干细胞的有效方法,所获得的间充质干细胞纯度较高,增殖较快,适用于干细胞生物学和组织工程学的研究．     

2007年国外重大科学进展

对2007年国外自然科学领域的重大进展进行回顾,遴选出"皮肤细胞转类胚胎干细胞"等10项,简要介绍并予以评述。     

造血干细胞的研究进展

干细胞是一类具有自我更新、高度增殖和多向分化潜能的原始细胞．造血干细胞在胚胎时期的发生、发育过程已得到进一步阐明．造血干细胞的移植已成功的应用于治疗多种血液系统和相关系统疾病．作者对造血干细胞的来源、细胞表面标志和其临床应用前景进行了综述．     

Generation of germline-competent induced pluripotent stem cells

We have previously shown that pluripotent stem cells can be induced from mouse fibroblasts by retroviral introduction of Oct3/4 (also called Pou5f1), Sox2, c-Myc and Klf4, and subsequent selection for Fbx15 (also called Fbxo15) expression. These induced pluripotent stem (iPS) cells (hereafter called Fbx15 iPS cells) are similar to embryonic stem (ES) cells in morphology, proliferation and teratoma formation; however, they are different with regards to gene expression and DNA methylation patterns, and fail to produce adult chimaeras. Here we show that selection for Nanog expression results in germline-competent iPS cells with increased ES-cell-like gene expression and DNA methylation patterns compared with Fbx15 iPS cells. The four transgenes (Oct3/4, Sox2, c-myc and Klf4) were strongly silenced in Nanog iPS cells. We obtained adult chimaeras from seven Nanog iPS cell clones, with one clone being transmitted through the germ line to the next generation. Approximately 20% of the offspring developed tumours attributable to reactivation of the c-myc transgene. Thus, iPS cells competent for germline chimaeras can be obtained from fibroblasts, but retroviral introduction of c-Myc should be avoided for clinical application.     

一种测定动物细胞平均沉降速率的新方法

通过构建细胞沉降过程的理想化模型，提出了一种简易的细胞沉降速率测定方法。标准颗粒沉降实验结果验证了这种方法的准确性。利用该方法测定杂交瘤细胞的沉降速率，定量描述了杂交瘤活细胞与死细胞之间的沉降速率差异，活细胞的沉降速率(15．5mm／h)大约是死细胞(9．36mm／h)的1．67倍，约是细胞碎片(1．46mm／h)的10倍；把该方法用于不同种类不同培养时期的脐血造血细胞，发现它们具有不同的沉降速率。这些数据将为细胞连续灌注培养过程的细胞截留研究提供依据。     

Comparative pluripotency analysis of mouse embryonic stem cells derived from wild-type and infertile hermaphrodite somatic cell nuclear transfer blastocysts

Therapeutic cloning, whereby embryonic stem cells （ESCs） are derived from patient-specific cloned blastocysts via somatic cell nuclear transfer （SCNT）, holds great promise for treating many human diseases using regenerative medicine. Teratoma formation and germline transmission have been used to confirm the pluripotency of mouse stem cells, but human embryonic stem cells （hESCs） have not been proven to be fully pluripotent owing to the ethical impossibility of testing for germ line transmis- sion, which would be the strongest evidence for full pluripotency. Therefore, formation of differentiated cells from the three somatic germ layers within a teratoma is taken as the best indicator of pluripotency in hESC lines. The possibility that these lines lack full multi- or pluripotency has not yet been evaluated. In this study, we established 16 mouse ESC lines, including 3 genetically defective nuclear transfer- ESC （ntESC） lines derived from SCNT blastocysts of infertile hermaphrodite F1 mice and 13 ntESC lines derived from SCNT blastocysts of normal F1 mice. We found that the defective ntESCs expressed all in vitro markers of pluripotency and could form teratomas that included derivatives from all three germ layers, but could not be transmitted via the germ line, in contrast with normal ntESCs. Our results in- dicate that teratoma formation assays with hESCs might be an insufficient standard to assess full pluripotency, although they do define multipotency to some degree. More rigorous standards are required to assess the safety of hESCs for therapeutic cloning.