Induced pluripotent stem cells: a new technology to study human diseases

Induced pluripotent stem cells (iPS cells) are somatic cells that have been reprogrammed to a pluripotent state by the introduction of specific factors. They can be generated from cells of different origins such as fibroblasts, keratinocytes, hepatocytes and blood. iPS cells are similar to embryonic stem cells in several aspects such as morphology, expression of pluripotency markers and the capacity to develop teratomas; tumors containing cells of the three germ layers. As pluripotent stem cells they can be differentiated into several lineages including neuronal, cardiac and blood cells. Recently, several groups have successfully generated patient-specific iPS cells from donors suffering different disorders and differentiated them into the cell type affected by the disease. These new human cell-based models cannot only be used to study the dynamics of diseases but also as systems to screen new drugs. Moreover, iPS cells promise to be good candidates for regenerative medicine.     

Overlapping Genes May Control Reprogramming of Mouse Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) and Breast Cancer Stem Cells

Recent findings suggest the possibility that tumors originate from cancer cells with stem cell properties. The cancer stem cell (CSC) hypothesis provides an explanation for why existing cancer therapies often fail in eradicating highly malignant tumors and end with tumor recurrence. Although normal stem cells and CSCs both share the capacity for self-renewal and multi-lineage differentiation, suggesting that CSC may be derived from normal SCs, the cellular origin of transformation of CSCs is debatable. Research suggests that the tightly controlled balance of self-renewal and differentiation that characterizes normal stem cell function is dis-regulated in cancer. Additionally, recent evidence has linked an embryonic stem cell (ESC)-like gene signature with poorly differentiated high-grade tumors, suggesting that regulatory pathways controlling pluripotency may in part contribute to the somatic CSC phenotype. Here, we introduce expression profile bioinformatic analyses of mouse breast cells with CSC properties, mouse embryonic stem (mES) and induced pluripotent stem (iPS) cells with an emphasis on how study of pluripotent stem cells may contribute to the identification of genes and pathways that facilitate events associated with oncogenesis. Global gene expression analysis from CSCs and induced pluripotent stem cell lines represent an ideal model to study cancer initiation and progression and provide insight into the origin cancer stem cells. Additionally, insight into the genetic and epigenomic mechanisms regulating the balance between self-renewal and differentiation of somatic stem cells and cancer may help to determine whether different strategies used to generate iPSCs are potentially safe for therapeutic use.     

Human-Induced Pluripotent Stem Cells: In Quest of Clinical Applications

In the field of regenerative medicine, the development of induced pluripotent stem (iPS) cells may represent a potential strategy to overcome the limitations of human embryonic stem cells (ESCs). iPS cells have the potential to mimic human disease, since they carry the genome of the donor. Hypothetically, with iPS cell technology it is possible to screen patients for a genetic cause of disease (genetic mutation), develop cell lines, reprogram them back to iPS cells, finally differentiate them into one or more cell types that develop the disease. Although the creation of multiple lineages with iPS cells can seem limitless, a number of challenges need to be addressed in order to effectively use these cell lines for disease modeling. These include the low efficiency of iPS cell generation without genetic alterations, the possibility of tumor formation in vivo, the random integration of retroviral-based delivery vectors into the genome, and unregulated growth of the remaining cells that are partially reprogrammed and refractory to differentiation. The establishment of protein or RNA-based reprogramming strategies will help generate human iPS cells without permanent genetic alterations. Finally, direct reprogramming strategies can provide rapid production of models of human ??diseases in a dish??, without first passing the cells through a pluripotent state, so avoiding the challenges of time-consumming and labor-intensive iPS cell line generation. This review will overview methods to develop iPS cells, current strategies for direct reprogramming, and main applications of iPS cells as human disease model, focusing on human cardiovascular diseases, with the aim to be a potential information resource for biomedical scientists and clinicians who exploit or intend to exploit iPS cell technology in a range of applications.     

Genomic analysis of induced pluripotent stem (iPS) cells: routes to reprogramming

The phenomenal proliferation of scientific studies into the nature of induced pluripotent stem (iPS) cells following publication of the findings of Takahashi and Yamanaka little more than 2 years ago, have significantly expanded our understanding of cellular mechanisms relating to cell lineage, differentiation, and proliferation. While the full potential of iPS cell lineages for both scientific tool and therapeutic applications is as yet unclear, findings from several lines of investigation suggests that multipotential and terminally differentiated cells from an array of cell types are competent to undergo epigenetic reprogramming to a pluripotential state. The nature of this pluripotential state appears to be similar to, but not identical with that previously described for embryonic stem (ES) cells. Understanding the nature of this induced reprogrammed state will be critical to determining the full potential of iPS cells. Recently, this issue has been examined through an integrated analysis of the genome in fully and partially reprogrammed iPS cell lineages. These results provide a window onto the temporal components of reprogramming and suggest mechanisms by which the efficacy of reprogramming can be enhanced.     

The promise of human induced pluripotent stem cells for research and therapy

Induced pluripotent stem (iPS) cells are human somatic cells that have been reprogrammed to a pluripotent state. There are several hurdles to be overcome before iPS cells can be considered as a potential patient-specific cell therapy, and it will be crucial to characterize the developmental potential of human iPS cell lines. As a research tool, iPS-cell technology provides opportunities to study normal development and to understand reprogramming. iPS cells can have an immediate impact as models for human diseases, including cancer     

诱导性多潜能干细胞(iPS cells)——现状及前景展望

主要从 iPS细胞发展历程、获得 iPS细胞的几个关键步骤 (如基因导入方式、诱导 iPS细胞所需因子组合与小分子化合物运用和体细胞种类选择等)、病人或疾病特异性 iPS细胞、iPS细胞体内外诱导分化与其衍生物的临床应用和制备无遗传修饰的(genetic modification-free) iPS细胞的可行性与前景等方面对 iPS细胞最新研究进展做评述．日本和美国研究小组先后用4种基因将小鼠(2006年8月)和人(2007年11～12月)的体细胞在体外重编程为诱导性多潜能干细胞(induced pluripotent stem cells,iPS cells),此后在短短两年多时间内,iPS 细胞的研究和关注度呈爆炸式增长．体细胞重编程、去分化和多潜能干细胞来源等一系列热点问题再次成为干细胞和发育生物学等研究的热点和焦点．与胚胎干细胞(embryonic stem cells,ES cells)一样,iPS细胞在体内可分化为3个胚层来源的所有细胞,进而参与形成机体所有组织和器官．迄今,在体外已由 iPS细胞定向诱导分化出功能性的多种成熟细胞．因此,iPS细胞研究不仅具有重要理论意义,而且在再生医学、组织工程和药物发现与评价等方面极具应用价值．     

疾病特异诱导多能干细胞研究进展

通过病毒或非病毒转导体系,在小鼠和人的体细胞中人为表达几个与细胞多能性相关的转录因子,从而使细胞达到类似于胚胎干细胞（embryonic stem cells,ESCs）状态,是近年来新发展起来的体细胞重编程技术。这些被重编程的细胞称为诱导多能干细胞（induced pluripotent stem cells,iPS细胞）。这项技术为获得患者和疾病特异的多能干细胞提供了新的途径。患者和疾病特异的iPS细胞的获得,不仅在避免免疫排斥的宿主特异的细胞移植治疗上有广泛前景,并对了解疾病发生机理、药物筛选和毒性研究有着重要的意义。该文综述从iPS细胞技术的发明入手,着重讨论疾病iPS细胞的研究进展及其在应用于治疗时亟需解决的问题。     

核移植与诱导多能干细胞技术在体细胞重编程研究中的应用

体细胞重编程是指分化的体细胞在特定的条件下被逆转后恢复到多能性或全能性状态,或者形成多能干细胞系,或者形成早期胚胎然后发育成一个新的个体的过程。诱导体细胞重编程的方法有许多,如核移植（nuclear transfer,NT）、细胞融合、细胞培养和通过导入特定因子获得诱导多能干（induced pluripotent stem,iPS）细胞的方法等。其中核移植和iPS技术是到目前为止诱导体细胞为多能干细胞最为完全、最具有运用于临床再生医学潜能的方法。然而,它们的效率都很低,机制也不清楚,如何将两个方法结合在一起,提高重编程的效率,揭示重编程的机制,进而促进其在患者特异性治疗中的运用将是下阶段的努力方向。     

A novel strategy to derive iPS cells from porcine fibroblasts

Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state by over-expressing four reprogramming factors. This technology has created an interest in deriving iPS cells from domesticated animals such as pigs, sheep and cattle. Moloney murine leukemia retrovirus vectors have been widely used to generate and study mouse iPS cells. However, this retrovirus system infects only mouse and rat cells, which limits its use in establishing iPS cells from other mammals. In our study, we demonstrate a novel retrovirus strategy to efficiently generate porcine iPS cells from embryonic fibroblasts. We transfected four human reprogramming factors (Oct4, Sox2, Klf4 and Myc) into fibroblasts in one step by using a VSV-G envelope-coated pantropic retrovirus that was easily packaged by GP2-293 cells. We established six embryonic stem (ES)-like cell lines in human ES cell medium supplemented with bFGF. Colonies showed a similar morphology to human ES cells with a high nuclei-cytoplasm ratio and phase-bright flat colonies. Porcine iPS cells could form embryoid bodies in vitro and differentiate into the three germ layers in vivo by forming teratomas in immunodeficient mice.     

Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors

Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. Here, we demonstrate induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions. Unexpectedly, Nanog was dispensable. These cells, which we designated iPS (induced pluripotent stem) cells, exhibit the morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent stem cells can be directly generated from fibroblast cultures by the addition of only a few defined factors.     

Methods for iPS cell generation for basic research and clinical applications

The induction of pluripotency can be achieved by forced expression of defined factors in somatic cells. The established cells, termed induced pluripotent stem (iPS) cells, have pluripotency and an infinite capacity for self-renewal in common with embryonic stem (ES) cells. Patient-specific iPS cells could be a useful source for drug discovery and cell transplantation therapies; however, the original method for iPS cell generation had several issues that were obstacles to their clinical application. Recent studies have brought about various improvements for iPS cell generation and uncovered several characteristics of iPS cells. Here we summarize the current status of iPS cell studies, with a focus on the improved methods that can be used to generate iPS cells, and also refer to the future challenges.