体细胞重编程为多能干细胞的研究进展

体细胞通过重编程转变成其他类型的细胞,在再生医学方面具有重要的应用前景。细胞重编程的方法主要有体细胞核移植、细胞融合、细胞提取物诱导、限定因子诱导等,这些方法可以不同程度地改变细胞命运。最近,限定因子诱导的多能干细胞（induced pluripotent stem cell。iPS）为重编程提供了一种崭新的方法,不仅可以避免伦理争议,还提供了一种更为便利的技术,为再生医学开辟了新的天地;同时,iPS技术为研究基因表达调控、蛋白质互作、机体生长发育等提供了一个非常重要的研究手段。本文主要论述了体细胞重编程的方法及iPS细胞的进展、面临的问题和应用前景。     

中国细胞重编程和多能干细胞研究进展

近几十年是干细胞领域飞速发展的重要时期。随着中国经济实力的发展壮大,科研实力也在稳步增强,干细胞研究领域达到了国际并跑甚至领跑的水平。本文从体细胞核移植、诱导多能干细胞、单倍体多能干细胞和胚胎早期发育研究4个方面,对中国细胞重编程和干细胞领域的研究进展进行了历史性回顾,总结了中国科学家在相关领域所取得的重要科研成果。随着单细胞测序技术的发展,各种发育过程将实现更为深入的解读,干细胞的临床应用在中国也会大放异彩。     

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.     

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.     

Post-irradiation promotes susceptibility to reprogramming to pluripotent state in human fibroblasts

Ionizing radiation causes not only targeted effects in cells that have been directly irradiated but also non-targeted effects in several cell generations after initial exposure. Recent studies suggest that radiation can enrich for a population of stem cells, derived from differentiated cells, through cellular reprogramming. Here, we elucidate the effect of irradiation on reprogramming, subjected to two different responses, using an induced pluripotent stem cell (iPSC) model. iPSCs were generated from non-irradiated cells, directly-irradiated cells, or cells subsequently generated after initial radiation exposure. We found that direct irradiation negatively affected iPSC induction in a dose-dependent manner. However, in the post-irradiated group, after five subsequent generations, cells became increasingly sensitive to the induction of reprogramming compared to that in non-irradiated cells as observed by an increased number of Tra1-81-stained colonies as well as enhanced alkaline phosphatase and Oct4 promoter activity. Comparative analysis, based on reducing the number of defined factors utilized for reprogramming, also revealed enhanced efficiency of iPSC generation in post-irradiated cells. Furthermore, the phenotypic acquisition of characteristics of pluripotent stem cells was observed in all resulting iPSC lines, as shown by morphology, the expression of pluripotent markers, DNA methylation patterns of pluripotency genes, a normal diploid karyotype, and teratoma formation. Overall, these results suggested that reprogramming capability might be differentially modulated by altered radiation-induced responses. Our findings provide that susceptibility to reprogramming in somatic cells might be improved by the delayed effects of non-targeted response, and contribute to a better understanding of the biological effects of radiation exposure.     

Novel imprinted single CpG sites found by global DNA methylation analysis in human parthenogenetic induced pluripotent stem cells

Genomic imprinting is the process of epigenetic modification whereby genes are expressed in a parent-of-origin dependent manner; it plays an important role in normal growth and development. Parthenogenetic embryos contain only the maternal genome. Parthenogenetic embryonic stem cells could be useful for studying imprinted genes. In humans, mature cystic ovarian teratomas originate from parthenogenetic activation of oocytes; they are composed of highly differentiated mature tissues containing all three germ layers. To establish human parthenogenetic induced pluripotent stem cell lines (PgHiPSCs), we generated parthenogenetic fibroblasts from ovarian teratoma tissues. We compared global DNA methylation status of PgHiPSCs with that of biparental human induced pluripotent stem cells by using Illumina Infinium HumanMethylation450 BeadChip array. This analysis identified novel single imprinted CpG sites. We further tested DNA methylation patterns of two of these sites using bisulfite sequencing and described novel candidate imprinted CpG sites. These results confirm that PgHiPSCs are a powerful tool for identifying imprinted genes and investigating their roles in human development and diseases.     

Diversity of dermal fibroblasts as major determinant of variability in cell reprogramming

Induced pluripotent stem cells (iPSCs) are adult somatic cells genetically reprogrammed to an embryonic stem cell‐like state. Notwithstanding their autologous origin and their potential to differentiate towards cells of all three germ layers, iPSC reprogramming is still affected by low efficiency. As dermal fibroblast is the most used human cell for reprogramming, we hypothesize that the variability in reprogramming is, at least partially, because of the skin fibroblasts used. Human dermal fibroblasts harvested from five different anatomical sites (neck, breast, arm, abdomen and thigh) were cultured and their morphology, proliferation, apoptotic rate, ability to migrate, expression of mesenchymal or epithelial markers, differentiation potential and production of growth factors were evaluated in vitro. Additionally, gene expression analysis was performed by real‐time PCR including genes typically expressed by mesenchymal cells. Finally, fibroblasts isolated from different anatomic sites were reprogrammed to iPSCs by integration‐free method. Intriguingly, while the morphology of fibroblasts derived from different anatomic sites differed only slightly, other features, known to affect cell reprogramming, varied greatly and in accordance with anatomic site of origin. Accordingly, difference also emerged in fibroblasts readiness to respond to reprogramming and ability to form colonies. Therefore, as fibroblasts derived from different anatomic sites preserve positional memory, it is of great importance to accurately evaluate and select dermal fibroblast population prior to induce reprogramming.     

Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions

Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.     

诱导多能干细胞及其在神经退行性疾病研究中的应用

用干细胞转录因子OCT4、SOX2、c-MYC和KLF4进行体细胞重编程产生具有胚胎干细胞特性的诱导多能干细胞(iPS细胞)是干细胞研究领域的突破性进展。近年来,iPS细胞的研究从产生方法、重编程机理及实际应用方面不断取得进展。由于iPS细胞的产生可取自体细胞,因而克服了胚胎干细胞应用的伦理学和免疫排斥等缺陷,为iPS细胞的临床应用开辟了广阔的前景。该文将对iPS细胞的产生方法、重编程机理及其在神经性退行性疾病的研究与应用进行文献综述,反映近几年iPS细胞最新研究成果,并阐述了用病人iPS细胞模型探讨帕金森氏病、老年性痴呆症、脊髓侧索硬化症、脊髓肌肉萎缩症及舞蹈症等5种常见神经性退行性疾病发病机理的研究现状。     

Transgenic animals as models for human diseasereport of an EC Study Group

ContributorsThis report results from the discussion of an Expert Group convened in Edinburgh on 29–30 October 1992 for a workshop on that subject sponsored and organized by the Commission of the European Communities, Directorate General XII (CEC-DG XII). The experts taking part in the workshop were: R. Lathe and J.J. Mullins, Coordinators (AFRC Centre for Genome Research, University of Edinburgh); G.N. Fracchia, Secretary (Medical Research-Pharmaceuticals, CEC-DG XII, Brussels); and the participants; C. Babinet (Dept d'Immunologie, Institut Pasteur, Paris); P. Eliard (EFPIA, Brussels); C. Benoist (LGME du CNRS/INSERM, Strasbourg); G. Bianchi (Ospedale San Raffaele, Universita di Milano, Milan); E. Boncinelli (DIBIT, Ospedale San Raffaele, Milan); G. Brem (Universitat München); G. Cossu (Institute of Histology, School of Medicine, University of Rome); N. Dillon (MRC National Institute for Medical Research, London); V. Episkopou (Dept of Biochemistry & Molecular Genetics, St Mary's Hospital Medical School, London); M. Evans (Wellcome/CRC Institute, Cambridge); R. Forster (Italfarmaco Research Centre, Cinisello Balsamo, Milan); D. Ganten (Max-Delbrück-Zentrum für Molekulare Medizin, Berlin); A. Gossler (Max-Delbrück-Laboratorium in der Max-Planck-Gesellschaft, Köln); J. Gray (Dept Psychology, Institute of Psychiatry, London); R. Hammer (Howard Hughes Medical Institute, University of Texas, Dallas, USA); A. Hobden (Genetics Unit, Glaxo Group Research Ltd, Middlesex); G. Kollias (Laboratory of Molecular Genetics, Hellenic Pasteur Institute, Athens); D. Lamy (Transgène SA, Strasbourg); D. Lincoln (MRC Reproductive Biology Unit, Edinburgh); J. Mallet (CNRS/LNCM, Gif-sur-Yvette); D. Melton (ICMB, University of Edinburgh, Edinburgh); J.M. Moalic (U127 INSERM, Hôpital Laraboisire, Paris); S. Mockrin (Dept Health & Human Services, National Institutes of Health, Bethesda, MA, USA); J. Ottesen (Biopharmaceuticals Division, Dept of Gene Technology and Virology, Novo Industrie, Denmark); D. Porteous (MRC Human Genetics Unit, Western General Hospital, Edinburgh); P. Rae (Pharmaceutical Division, Miles, West Haven, USA); F. Theuring (Schering AG, Pharmaceutical Research, Berlin); G. Tremp (Rhone-Poulenc Rorer SA, Centre de Recherche de Vitry-Alfortville, Vitry-sur-Seine); H. Van der Putten (Dept Biotechnology, Ciba-Geigy AG, Basel); R. Wolf (ICRF Molecular Pharmacology Group, Biochemistry Dept, Edinburgh). Further supporting documentation and commentary were received from P. Dement (Amsterdam), U. Habenicht (Berlin), P. Grüss (Gottingen), M. Lyon (Oxford), C.C.J. Miller (London), W.-D. Schleuning (Berlin) and R. Williamson (London) and their contributions are gratefully acknowledged.