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.     

Biomaterial aided differentiation and maturation of induced pluripotent stem cells

Engineering/reprogramming differentiated adult somatic cells to gain the ability to differentiate into any type of cell lineage are called as induced pluripotent stem cells (iPSCs). Offering unlimited self-renewal and differentiation potential, these iPSC are aspired to meet the growing demands in the field of regenerative medicine, tissue engineering, disease modeling, nanotechnology, and drug discovery. Biomaterial fabrication with the rapid evolution of technology increased their versatility and utility in regenerative medicine and tissue engineering, revolutionizing the stem cell biology research with the property to guide the process of proliferation, differentiation, and morphogenesis. Combining traditional culture platforms of iPSC with biomaterials aids to overcome the limitations associated with derivation, proliferation, and maturation, thereby could improve the clinical translation of iPSC. The present review discusses in brief about the reprogramming techniques for the derivation iPSC and details on several biomaterial guided differentiation of iPSC to different cell types with specific relevance to tissue engineering/regenerative medicine.     

Induced pluripotency and direct reprogramming: a new window for treatment of neurodegenerative diseases

Human embryonic stem cells (hESCs) are pluripotent cells that have the ability of unlimited self-renewal and can be differentiated into different cell lineages, including neural stem (NS) cells. Diverse regulatory signaling pathways of neural stem cells differentiation have been discovered, and this will be of great benefit to uncover the mechanisms of neuronal differentiation in vivo and in vitro. However, the limitations of hESCs resource along with the religious and ethical concerns impede the progress of ESCs application. Therefore, the induced pluripotent stem cells (iPSCs) via somatic cell reprogramming have opened up another new territory for regenerative medicine. iPSCs now can be derived from a number of lineages of cells, and are able to differentiate into certain cell types, including neurons. Patient-specific iPSCs are being used in human neurodegenerative disease modeling and drug screening. Furthermore, with the development of somatic direct reprogramming or lineage reprogramming technique, a more effective approach for regenerative medicine could become a complement for iPSCs.     

Determinants of pluripotency: From avian,rodents, to primates

Since mouse embryonic stem (ES) cells was first derived in 1981, the ability of this unprecedented cell type to self‐renew and differentiate without limit has revolutionized the discovery tools that are used to study gene functions and development. Furthermore, they have inspired others to hunt for similar cells from other species. The derivation of human ES cells in 1998 has accelerated these discoveries and has also widely provoked public interest, due to both the scientific significance of these cells for human tissue regeneration and the ethical disputes over the use of donated early human embryos. However, this is no longer a barrier, with the recent discovery of methods that can convert differentiated somatic cells into ES‐like cells or induced pluripotent stem (iPS) cells, by using defined reprogramming factors. This review attempts to summarize the progresses in the derivation of ES cells (as well as other embryo‐derived pluripotent cells) and iPS cells from various species. We will focus on the molecular and biological features of the cells, as well as the different determinants identified thus far to sustain their pluripotency. J. Cell. Biochem. 109: 16–25, 2010. © 2009 Wiley‐Liss, Inc.     

Mechanism and methods to induce pluripotency

Pluripotent stem cells are able to self-renew indefinitely and differentiate into all types of cells in the body. They can thus be an inexhaustible source for future cell transplantation therapy to treat degenerative diseases which currently have no cure. However, non-autologous cells will cause immune rejection. Induced pluripotent stem cell (iPSC) technology can convert somatic cells to the pluripotent state, and therefore offers a solution to this problem. Since the first generation of iPSCs, there has been an explosion of relevant research, from which we have learned much about the genetic networks and epigenetic landscape of pluripotency, as well as how to manipulate genes, epigenetics, and microRNAs to obtain iPSCs. In this review, we focus on the mechanism of cellular reprogramming and current methods to induce pluripotency. We also highlight new problems emerging from iPSCs. Better understanding of the fundamental mechanisms underlying pluripotenty and refining the methodology of iPSC generation will have a significant impact on future development of regenerative medicine.     

The prospect of pluripotent stem cell-based therapy

Human embryonic stem cells (hESC) are able to maintain pluripotency in culture, to proliferate indefinitely and to differentiate into all somatic cell types. Due to these unique properties, hESC may become an exceptional source of tissues for transplantation and have a great potential for the therapy of incurable diseases. Here, we review new developments in the area of embryonic stem cells and discuss major challenges — standardization of protocols for cell derivation and cultivation, identification of specific molecular markers, development of new approaches for directed differentiation, etc. — which remain to be settled, prior to safe and successful clinical application of stem cells. We appraise several potential approaches in hESC-based therapy including derivation of autologous cells via therapeutic cloning (1), generation of immune tolerance to allogenic donor cells via hematopoetic chimerism (2), and development of the banks of hESC lines compatible with the main antigens and exhibiting equivalent pluripotency (3). In addition, we discuss briefly induced pluripotent cells, which are derived via genetic modification of autologous somatic cells and are analogous to ESC. Our analysis demonstrates that uncontrollable differentiation in vivo and teratogenic potential of hESC are critical limitations of their application in clinical practice. Therefore, the major approach in hESC therapy is derivation of a specific differentiated progeny, which has lower proliferative potential and immune privilege, yet poses fewer risks for organism. The review demonstrates that cell therapy is far more complex and resource-consuming process as compared with drug-based medicine and consequently pluripotent stem cell biology and technology still requires further investigation and development before these cells can be used in clinical practice.     

直接重整细胞核程序的诱导性多能干细胞研究进展

诱导性多能干细胞（induced pluripotent stem cells, iPS）是分化细胞在外源性因子作用下,经直接细胞核程序重整而重新获得分化潜能的干细胞,具有很重要的应用前景。介绍了iPS诱导方法从转录因子、RNA结合蛋白、小分子化合物、到信号传导通路的发展过程,以及在提高生物安全性方面的改进。iPS的生成在细胞学上表现为渐进的、时间依赖的过程,同细胞的分化状态密切相关;然而,iPS同胚胎干细胞表遗传特征并非完全相同。iPS的进展结合基因治疗和细胞治疗的成果已应用到动物疾病模型的治疗。     

Neural stem cells: Mechanisms of fate specification and nuclear reprogramming in regenerative medicine

Recently, intense interest in the potential use of neural stem cells (NSC) in the clinical therapy of brain disease and injury has resulted in rapid progress in research on the properties of NSC, their innate and directed differentiation potential and the induced reprogramming of differentiated somatic cells to revert to a pluripotent NSC-like state. The aim of this review is to give an overview of our current operational definitions of the NSC lineage, the growing understanding of extrinsic and intrinsic mechanisms, including heritable but reversible epigenetic chromatin modifications that regulate the maintenance and differentiation of NSC in vivo, and to emphasize ground-breaking efforts of cellular reprogramming with the view to generating patient-specific stem cells for cell replacement therapy. This is set against a summary of current practical procedures for the isolation, research and application of NSC, and of the state of the art in NSC-based regenerative medicine of the nervous system. Both provide the backdrop for the translation of recent findings into innovative clinical applications, with the hope of increasing the safety, efficiency and ethical acceptability of NSC-based therapies in the near future.     

哺乳动物DNA甲基化及其在体细胞诱导重编程中的作用

诱导多能干细胞(Induced pluripotent stem cell, iPS)技术提供了将终末分化的细胞逆转为多潜能干细胞的可能, 在干细胞基础理论研究和再生医学中具有重要意义。然而, 目前体细胞诱导重编程方法效率极低, 常发生不完全的重编程。研究表明, 在不完全重编程的细胞中存在体细胞的表观遗传记忆, 而DNA甲基化作为相对长期和稳定的表观遗传修饰, 是影响重编程效率和iPS细胞分化能力的重要因素之一。哺乳动物DNA甲基化是指胞嘧啶第五位碳原子上的甲基化修饰, 常发生于CpG位点。DNA甲基化能够调节体细胞特异基因和多能性基因的表达, 因此其在哺乳动物基因调控、胚胎发育和细胞重编程过程中发挥着重要作用。此外, 异常DNA甲基化可能导致iPS细胞基因印记的异常和X染色体的失活。文章重点围绕DNA甲基化的机制、分布特点、及其在体细胞诱导重编程中的作用进行了综述。     

Specific lectin biomarkers for isolation of human pluripotent stem cells identified through array-based glycomic analysis

Rapid and dependable methods for isolating human pluripotent stem cell (hPSC) populations are urgently needed for quality control in basic research and in cell-based therapy applications. Using lectin arrays, we analyzed glycoproteins extracted from 26 hPSC samples and 22 differentiated cell samples, and identified a small group of lectins with distinctive binding signatures that were sufficient to distinguish hPSCs from a variety of non-pluripotent cell types. These specific biomarkers were shared by all the 12 human embryonic stem cell and the 14 human induced pluripotent stem cell samples examined, regardless of the laboratory of origin, the culture conditions, the somatic cell type reprogrammed, or the reprogramming method used. We demonstrated a practical application of specific lectin binding by detecting hPSCs within a differentiated cell population with lectin-mediated staining followed by fluorescence microscopy and flow cytometry, and by enriching and purging viable hPSCs from mixed cell populations using lectin-mediated cell separation. Global gene expression analysis showed pluripotency-associated differential expression of specific fucosyltransferases and sialyltransferases, which may underlie these differences in protein glycosylation and lectin binding. Taken together, our results show that protein glycosylation differs considerably between pluripotent and non-pluripotent cells, and demonstrate that lectins may be used as biomarkers to monitor pluripotency in stem cell populations and for removal of viable hPSCs from mixed cell populations.     

细胞重编程与表观遗传学调控

细胞重编程是生命科学研究的热点之一,目前体细胞核移植、细胞融合和特定转录因子诱导等方法都可以实现体外细胞重编程,而在细胞重编程过程中表观遗传学发挥关键的调控作用,因此对重编程过程中表观遗传学调控机制开展深入研究具有重要的意义。本文简要综述细胞重编程的研究现状和表观遗传学调控细胞重编程机制的研究进展,并对小分子化合物和microRNA提高细胞重编程效率的最新进展进行了介绍。     

The emerging functions of the p53-miRNA network in stem cell biology

The p53 pathway plays an essential role in tumor suppression, regulating multiple cellular processes coordinately to maintain genome integrity in both somatic cells and stem cells. Despite decades of research dedicated to p53 function in differentiated somatic cells, we are just starting to understand the complexity of the p53 pathway in the biology of pluripotent stem cells and tissue stem cells. Recent studies have demonstrated that p53 suppresses proliferation, promotes differentiation of embryonic stem (ES) cells and constitutes an important barrier to somatic reprogramming. In addition, emerging evidence reveals the role of the p53 network in the self-renewal, proliferation and genomic integrity of adult stem cells. Interestingly, non-coding RNAs, and microRNAs in particular, are integral components of the p53 network, regulating multiple p53-controlled biological processes to modulate the self-renewal and differentiation potential of a variety of stem cells. Thus, elucidation of the p53-miRNA axis in stem cell biology may generate profound insights into the mechanistic overlap between malignant transformation and stem cell biology.     

The emerging functions of the p53-miRNA network in stem cell biology

The p53 pathway plays an essential role in tumor suppression, regulating multiple cellular processes coordinately to maintain genome integrity in both somatic cells and stem cells. Despite decades of research dedicated to p53 function in differentiated somatic cells, we are just starting to understand the complexity of the p53 pathway in the biology of pluripotent stem cells and tissue stem cells. Recent studies have demonstrated that p53 suppresses proliferation, promotes differentiation of embryonic stem (ES) cells and constitutes an important barrier to somatic reprogramming. In addition, emerging evidence reveals the role of the p53 network in the self-renewal, proliferation and genomic integrity of adult stem cells. Interestingly, non-coding RNAs, and microRNAs in particular, are integral components of the p53 network, regulating multiple p53-controlled biological processes to modulate the self-renewal and differentiation potential of a variety of stem cells. Thus, elucidation of the p53-miRNA axis in stem cell biology may generate profound insights into the mechanistic overlap between malignant transformation and stem cell biology.     

Genomic instability in induced stem cells

The ability to reprogram adult cells into stem cells has raised hopes for novel therapies for many human diseases. Typical stem cell reprogramming protocols involve expression of a small number of genes in differentiated somatic cells with the c-Myc and Klf4 proto-oncogenes typically included in this mix. We have previously shown that expression of oncogenes leads to DNA replication stress and genomic instability, explaining the high frequency of p53 mutations in human cancers. Consequently, we wondered whether stem cell reprogramming also leads to genomic instability. To test this hypothesis, we examined stem cells induced by a variety of protocols. The first protocol, developed specifically for this study, reprogrammed primary mouse mammary cells into mammary stem cells by expressing c-Myc. Two other previously established protocols reprogrammed mouse embryo fibroblasts into induced pluripotent stem cells by expressing either three genes, Oct4, Sox2 and Klf4, or four genes, OSK plus c-Myc. Comparative genomic hybridization analysis of stem cells derived by these protocols revealed the presence of genomic deletions and amplifications, whose signature was suggestive of oncogene-induced DNA replication stress. The genomic aberrations were to a significant degree dependent on c-Myc expression and their presence could explain why p53 inactivation facilitates stem cell reprogramming.