Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells

During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellular response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.     

Integration-free iPS cells engineered using human artificial chromosome vectors

Human artificial chromosomes (HACs) have unique characteristics as gene-delivery vectors, including episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. Two HAC vectors (iHAC1 and iHAC2) were constructed. Both carried four reprogramming factors, and iHAC2 also encoded a p53-knockdown cassette. iHAC1 partially reprogrammed MEFs, and iHAC2 efficiently reprogrammed MEFs. Global gene expression patterns showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. Under non-selecting conditions, we established iHAC-free iPS cells by isolating cells that spontaneously lost iHAC2. Analyses of pluripotent markers, teratomas and chimeras confirmed that these iHAC-free iPS cells were pluripotent. Moreover, iHAC-free iPS cells with a re-introduced HAC encoding Herpes Simplex virus thymidine kinase were eliminated by ganciclovir treatment, indicating that the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a built-in safeguard system.     

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.     

Regulation of somatic cell reprogramming through inducible mir-302 expression

Global demethylation is required for early zygote development to establish stem cell pluripotency, yet our findings reiterate this epigenetic reprogramming event in somatic cells through ectopic introduction of mir-302 function. Here, we report that induced mir-302 expression beyond 1.3-fold of the concentration in human embryonic stem (hES) H1 and H9 cells led to reprogramming of human hair follicle cells (hHFCs) to induced pluripotent stem (iPS) cells. This reprogramming mechanism functioned through mir-302-targeted co-suppression of four epigenetic regulators, AOF2 (also known as KDM1 or LSD1), AOF1, MECP1-p66 and MECP2. Silencing AOF2 also caused DNMT1 deficiency and further enhanced global demethylation during somatic cell reprogramming (SCR) of hHFCs. Re-supplementing AOF2 in iPS cells disrupted such global demethylation and induced cell differentiation. Given that both hES and iPS cells highly express mir-302, our findings suggest a novel link between zygotic reprogramming and SCR, providing a regulatory mechanism responsible for global demethylation in both events. As the mechanism of conventional iPS cell induction methods remains largely unknown, understanding this microRNA (miRNA)-mediated SCR mechanism may shed light on the improvements of iPS cell generation.     

Increased dosage of tumor suppressors limits the tumorigenicity of iPS cells without affecting their pluripotency

Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a promising therapeutic tool for many diseases, including aged tissues and organs at high risk of failure. However, the intrinsic self-renewal and pluripotency of ES and iPS cells make them tumorigenic, and hence, the risk of tumor development hinders their clinical application. Here, we present a novel approach to limit their tumorigenicity and increase their safety through increased copy number of tumor suppressors. iPS containing an extra copy of the p53 or Ink4a/ARF locus show normal pluripotency, as determined by in vitro and in vivo differentiation assays. Yet, while retaining full pluripotency, they also possess an improved engagement of the p53 pathway during teratocarcinoma formation, which leads to a reduced tumorigenic potential in various in vitro and in vivo assays. Furthermore, they show an improved response to anticancer drugs, which could aid in their elimination in case tumors arise with no adverse effects on cell function or aging. Our system provides a model for studying tumor suppressor pathways during reprogramming, differentiation, and cell therapy applications. This offers an improved understanding of the pathways involved in tumor growth from engrafted pluripotent stem cells, which could facilitate the use of ES and iPS cells in regenerative medicine.     

Role of leukaemia inhibitory factor in the induction of pluripotent stem cells in mice

iPS (induced pluripotent stem) cells can be induced from somatic cells in mice by genetic manipulation. Most previously established mouse iPS cell lines have been derived using feeder layers supplemented with exogenous LIF (leukaemia inhibitory factor). Although a feeder‐free induction system has been developed in recent studies, LIF is still required for reprogramming, but its role in the generation of mouse iPS cells has remained elusive. In this study, we investigated its contribution to the induction of pluripotency. Our results showed that LIF activates AP (alkaline phosphatase) through a c‐Myc‐dependent mechanism. Moreover, it acts as a protective factor during the transition from AP‐positive colonies to Oct3/4‐positive cells. These findings illustrate a mechanism by which LIF may integrate signalling into reprogramming.     

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.     

microRNA在诱导体细胞重编程中的作用

microRNA是调控基因转录后水平的一类长度约为22个核苷酸的非编码小分子RNA。大量研究证实, microRNAs广泛分布于真核生物, 其在细胞的分化发育、生长代谢等各种活动中都起着重要的调节作用。诱导多能性干细胞(Induced pluripotent stem cell, iPS)是将体细胞诱导成为具有胚胎干细胞性质的多潜能干细胞。iPS过程的核心为体细胞表观遗传状态发生重编程, 因此, 探明体细胞重编程机制对建立完善的iPS技术具有重要理论和实际意义。利用病毒载体将Oct4、Sox2、Klf4和c-Myc等因子导入体细胞的方法已不断发展, 但“基因组整合”及原癌基因的参与增加了诱导细胞的致癌率。随着使用腺病毒、质粒或蛋白诱导等“非整合型”方法及L-myc的替换均可获得具有多潜能性的干细胞, 癌变的风险大大降低。但其发生的理论机制仍不十分清楚。最近的研究证实, microRNAs影响体细胞的重编程过程, 特别是miR302/367、miR200、miR-34和miR290/295等家族的microRNAs在体细胞诱导为iPS过程中发挥重要作用。文章就近年microRNA在诱导多能干细胞中的作用进行综述。     

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。     

Proliferation Rate of Somatic Cells Affects Reprogramming Efficiency

The discovery of induced pluripotent stem (iPS) cells provides not only new approaches for cell replacement therapy, but also new ways for drug screening. However, the undefined mechanism and relatively low efficiency of reprogramming have limited the application of iPS cells. In an attempt to further optimize the reprogramming condition, we unexpectedly observed that removing c-Myc from the Oct-4, Sox-2, Klf-4, and c-Myc (OSKM) combination greatly enhanced the generation of iPS cells. The iPS cells generated without c-Myc attained salient pluripotent characteristics and were capable of producing full-term mice through tetraploid complementation. We observed that forced expression of c-Myc induced the expression of many genes involved in cell cycle control and a hyperproliferation state of the mouse embryonic fibroblasts during the early stage of reprogramming. This enhanced proliferation of mouse embryonic fibroblasts correlated negatively to the overall reprogramming efficiency. By applying small molecule inhibitors of cell proliferation at the early stage of reprogramming, we were able to improve the efficiency of iPS cell generation mediated by OSKM. Our data demonstrated that the proliferation rate of the somatic cell plays critical roles in reprogramming. Slowing down the proliferation of the original cells might be beneficial to the induction of iPS cells.