Induced Pluripotent Stem Cells Are Sensitive to DNA Damage

Induced pluripotent stem cells(iPSCs)resemble embryonic stem cells(ESCs)in morphology,gene expression and in vitro differentiation,raising new hope for personalized clinical therapy.While many efforts have been made to improve reprogramming effciency,signifcant problems such as genomic instability of iPSCs need to be addressed before clinical therapy.In this study,we try to fgure out the real genomic state of iPSCs and their DNA damage response to ionizing radiation(IR).We found that iPSC line 3FB4-1 had lower DNA damage repair ability than mouse embryonic fbroblast(MEF)cells,from which 3FB4-1line was derived.After the introduction of DNA damage by IR,the number of c-H2AX foci in 3FB4-1 increased modestly compared to a large increase seen in MEF,albeit both signifcantly(P<0.01).In addition,whole-genome sequencing analysis showed that after IR,3FB4-1 possessed more point mutations than MEF and the point mutations spread all over chromosomes.These observations provide evidence that iPSCs are more sensitive to ionizing radiation and their relatively low DNA damage repair capacity may account for their high radiosensitivity.The compromised DNA damage repair capacity of iPSCs should be considered when used in clinical therapy.     

Pluripotency of Induced Pluripotent Stem Cells

Induced pluripotent stem （iPS） cells can be generated by forced expression of four pluripotency factors in somatic cells. This has received much attention in recent years since it may offer us a promising donor cell source for cell transplantation therapy. There has been great progress in iPS cell research in the past few years. However, several issues need to be further addressed in the near future before the clinical application of iPS cells, like the immunogenieity of iPS cells, the variability of differentiation potential and most importantly tumor formation of the iPS derivative cells. Here, we review recent progress in research into the pluripotency of iPS cells.     

诱导多能干细胞的研究进展

体细胞诱导成为多能性干细胞(induced pluripotent stem cell,iPS cell)的研究成果被国际生命科学界誉为具有里程碑意义的创新之举.在短短3年多的时间里,这项研究已经在细胞重编程的机理研究、探索疾病的发生发展机制以及临床医学的应用等领域引发了很多突破性的进展,而且,这一非克隆干细胞技术的诞生,成功地避开了长期以来争论不休的伦理问题,极大地推动该领域和相关科学领域的发展.从iPS细胞的研究历程、iPS细胞的构建机理、iPS细胞研究的最新应用成果以及iPS细胞的发展前景和研究方向等方面进行了评.     

NEUROD1 Intrinsically Initiates Differentiation of Induced Pluripotent Stem Cells into Neural Progenitor Cells

Cell type specification is a delicate biological event in which every step is under tight regulation. From a molecular point of view, cell fate commitment begins with chromatin alteration, which kickstarts lineage-determining factors to initiate a series of genes required for cell specification. Several important neuronal differentiation factors have been identified from ectopic over-expression studies. However, there is scarce information on which DNA regions are modified during induced pluripotent stem cell (iPSC) to neuronal progenitor cell (NPC) differentiation, the cis regulatory factors that attach to these accessible regions, or the genes that are initially expressed. In this study, we identified the DNA accessible regions of iPSCs and NPCs via the Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq). We identified which chromatin regions were modified after neuronal differentiation and found that the enhancer regions had more active histone modification changes than the promoters. Through motif enrichment analysis, we found that NEUROD1 controls iPSC differentiation to NPC by binding to the accessible regions of enhancers in cooperation with other factors such as the Hox proteins. Finally, by using Hi-C data, we categorized the genes that directly interacted with the enhancers under the control of NEUROD1 during iPSC to NPC differentiation.     

Repression of SIRT1 Promotes the Differentiation of Mouse Induced Pluripotent Stem Cells into Neural Stem Cells

The use of transplanting functional neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) has increased for the treatment of brain diseases. As such, it is important to understand the molecular mechanisms that promote NSCs differentiation of iPSCs for future NSC-based therapies. Sirtuin 1 (SIRT1), a NAD⁺-dependent protein deacetylase, has attracted significant attention over the past decade due to its prominent role in processes including organ development, longevity, and cancer. However, it remains unclear whether SIRT1 plays a role in the differentiation of mouse iPSCs toward NSCs. In this study, we produced NSCs from mouse iPSCs using serum-free medium supplemented with retinoic acid. We then assessed changes in the expression of SIRT1 and microRNA-34a, which regulates SIRT1 expression. Moreover, we used a SIRT1 inhibitor to investigate the role of SIRT1 in NSCs differentiation of iPSCs. Data revealed that the expression of SIRT1 decreased, whereas miRNAs-34a increased, during this process. In addition, the inhibition of SIRT1 enhanced the generation of NSCs and mature neurocytes. This suggests that SIRT1 negatively regulated the differentiation of mouse iPSCs into NSCs, and that this process may be regulated by miRNA-34a.     

Generation,Purification and Transplantation of Photoreceptors Derived from Human Induced Pluripotent Stem Cells

Background

Inherited and acquired retinal degenerations are frequent causes of visual impairment and photoreceptor cell replacement therapy may restore visual function to these individuals. To provide a source of new retinal neurons for cell based therapies, we developed methods to derive retinal progenitors from human ES cells.

Methodology/Physical Findings

In this report we have used a similar method to direct induced pluripotent stem cells (iPS) from human fibroblasts to a retinal progenitor fate, competent to generate photoreceptors. We also found we could purify the photoreceptors derived from the iPS cells using fluorescence activated cell sorting (FACS) after labeling photoreceptors with a lentivirus driving GFP from the IRBP cis-regulatory sequences. Moreover, we found that when we transplanted the FACS purified iPSC derived photoreceptors, they were able to integrate into a normal mouse retina and express photoreceptor markers.

Conclusions

This report provides evidence that enriched populations of human photoreceptors can be derived from iPS cells.     

Transplantation of Induced Pluripotent Stem Cells Improves Functional Recovery in Huntington's Disease Rat Model

The purpose of this study was to determine the functional recovery of the transplanted induced pluripotent stem cells in a rat model of Huntington''s disease with use of ¹⁸F-FDG microPET/CT imaging.

Methods

In a quinolinic acid-induced rat model of striatal degeneration, induced pluripotent stem cells were transplanted into the ipsilateral lateral ventricle ten days after the quinolinic acid injection. The response to the treatment was evaluated by serial ¹⁸F-FDG PET/CT scans and Morris water maze test. Histological analyses and Western blotting were performed six weeks after stem cell transplantation.

Results

After induced pluripotent stem cells transplantation, higher ¹⁸F-FDG accumulation in the injured striatum was observed during the 4 to 6-weeks period compared with the quinolinic acid-injected group, suggesting the metabolic recovery of injured striatum. The induced pluripotent stem cells transplantation improved learning and memory function (and striatal atrophy) of the rat in six week in the comparison with the quinolinic acid-treated controls. In addition, immunohistochemical analysis demonstrated that transplanted stem cells survived and migrated into the lesioned area in striatum, and most of the stem cells expressed protein markers of neurons and glial cells.

Conclusion

Our findings show that induced pluripotent stem cells can survive, differentiate to functional neurons and improve partial striatal function and metabolism after implantation in a rat Huntington''s disease model.     

Induced Pluripotent Stem Cell-Derived Neural Cells Survive and Mature in the Nonhuman Primate Brain

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Highlights? Autologous iPSC-derived neural progenitors survive in the monkey brain for 6 months ? Grafted cells differentiate into neurons, astrocytes, and oligodendrocytes ? Autologous transplantation elicits minimal immune and glial response     

Special Issue on Induced Pluripotent Stem Cells

The journal Genomics Proteomics & Bioinformatics (GPB) is now inviting submissions for a special issue (to be published around summer in 2013) on the topic of Induced Pluripotent Stem Cells. The induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cells artificially derived from non-pluripotent cells, typically adult somatic cells, by inducing a forced expression of specific genes. iPSCs are useful tools for drug     

诱导多功能干细胞的影响因素

将体细胞诱导为多功能干细胞为人类的再生医学提供了一个全新的研究手段,从而可以不用损坏胚胎就能获得可用于治疗各种特殊疾病的细胞。本文比较了近年来关于生成诱导性多能干细胞(induced pluripotent stem cells,iPS细胞)的诱导方法及重编程效率,总结了这些方法的共同点;另外通过对每个不同试验过程的影响因素进行比较,归纳了影响iPS细胞重编程过程的几个因素。     

诱导性多能干细胞与肝样细胞分化

肝脏疾病正逐渐成为全球棘手的医疗问题。肝细胞是肝脏生理活动的主要承担者,在肝脏疾病的研究以及药物的研发和测试方面有着举足轻重的作用。然而,体外分离培养的原代肝细胞面临在体外不能无限增殖和稳定表达肝脏特异基因等问题。有强大的自我更新能力和三胚层分化潜能的诱导性多能肝细胞(iPSCs)能被诱导因子、外源基因和小分子化合物等定向诱导分化为功能性肝细胞。同时,还避免了伦理、宗教以及免疫排斥等诸多问题。本文简要综述了从不同策略诱导iPSCs成为功能性肝细胞的研究方法和成果,并对该领域进行小结和展望。     

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)¹. While improvements in several gene delivery methods have been described^2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor¹¹, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.     

Human Induced Pluripotent Stem Cell-Derived Models to Investigate Human Cytomegalovirus Infection in Neural Cells

Human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS) cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs), neural progenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate.     

不同组织来源的细胞建立诱导多能干细胞系的研究

目的:比较不同组织来源的细胞生成iPS细胞的效率,获得高效制备iPS细胞的组织类型.方法:通过四种逆转录病毒(OCT4/SOX2/KLF4/c-MYC)转染羊水细胞、绒毛细胞和皮肤成纤维细胞,建立不同组织来源的iPS细胞系.结果:我们建立了羊水、绒毛细胞和皮肤细胞三个不同组织来源的iPS细胞系,并对其多能性基因Oct4、Nanog以及分子表面标记Tra-1.60以及体外分化为三个胚层能力进行鉴定,发现利用羊水细胞建立iPS细胞的效率显著高于绒毛细胞和皮肤细胞.结论:羊水细胞可能是制备iPS细胞的理想细胞类型.     

Human Induced Pluripotent Stem Cells on Autologous Feeders

Background

For therapeutic usage of induced Pluripotent Stem (iPS) cells, to accomplish xeno-free culture is critical. Previous reports have shown that human embryonic stem (ES) cells can be maintained in feeder-free condition. However, absence of feeder cells can be a hostile environment for pluripotent cells and often results in karyotype abnormalities. Instead of animal feeders, human fibroblasts can be used as feeder cells of human ES cells. However, one still has to be concerned about the existence of unidentified pathogens, such as viruses and prions in these non-autologous feeders.

Methodology/Principal Findings

This report demonstrates that human induced Pluripotent Stem (iPS) cells can be established and maintained on isogenic parental feeder cells. We tested four independent human skin fibroblasts for the potential to maintain self-renewal of iPS cells. All the fibroblasts tested, as well as their conditioned medium, were capable of maintaining the undifferentiated state and normal karyotypes of iPS cells. Furthermore, human iPS cells can be generated on isogenic parental fibroblasts as feeders. These iPS cells carried on proliferation over 19 passages with undifferentiated morphologies. They expressed undifferentiated pluripotent cell markers, and could differentiate into all three germ layers via embryoid body and teratoma formation.

Conclusions/Significance

These results suggest that autologous fibroblasts can be not only a source for iPS cells but also be feeder layers. Our results provide a possibility to solve the dilemma by using isogenic fibroblasts as feeder layers of iPS cells. This is an important step toward the establishment of clinical grade iPS cells.     

体细胞重编程为诱导多能干细胞

诱导多功能性干细胞（induced pluripotent stem cells,iPS细胞）是通过导入特定的转录因子（如Oct3/4、Sox2、c-Myc和Klf4等）将体细胞诱导重编程为多能性干细胞,其功能与胚胎干细胞相似.iPS细胞的建立,在生命科学领域引起了新的轰动.目前,iPS细胞的研究领域在转录因子的优化、iPS细胞的筛选、载体的运用、体细胞种类的选择和iPS细胞的应用等方面取得突破进展,但仍然存在致癌性、效率低等一系列急需解决的问题.     

Integration-free Methods for Generating Induced Pluripotent Stem Cells

Induced pluripotent stem （iPS） cells by exogenous expression of four factors, Oct4, can be generated from mouse or human fibroblasts Sox2, Klf4 and c-Myc, and hold great potential for transplantation therapies and regenerative medicine. However, use of retroviral vectors during iPS cell generation has limited the techniques clinical application due to the potential risks resulting from genome integration of transgenes, including insertional mutations and altered differentiation potentials of the target cells, which may lead to pathologies such as tumorigenesis. Here we review recent progress in generating safer transgene-free or integration-free iPS cells, including the use of non-integrating vectors, excision of vectors after integration, DNA-free delivery of factors and chemical induction of pluripotency.     

Transdifferentiation-Induced Neural Stem Cells Promote Recovery of Middle Cerebral Artery Stroke Rats

Induced neural stem cells (iNSCs) can be directly transdifferentiated from somatic cells. One potential clinical application of the iNSCs is for nerve regeneration. However, it is unknown whether iNSCs function in disease models. We produced transdifferentiated iNSCs by conditional overexpressing Oct4, Sox2, Klf4, c-Mycin mouse embryonic fibroblasts. They expanded readily in vitro and expressed NSC mRNA profile and protein markers. These iNSCs differentiated into mature astrocytes, neurons and oligodendrocytes in vitro. Importantly, they reduced lesion size, promoted the recovery of motor and sensory function as well as metabolism status in middle cerebral artery stroke rats. These iNSCs secreted nerve growth factors, which was associated with observed protection of neurons from apoptosis. Furthermore, iNSCs migrated to and passed through the lesion in the cerebral cortex, where Tuj1+ neurons were detected. These findings have revealed the function of transdifferentiated iNSCs in vivo, and thus provide experimental evidence to support the development of personalized regenerative therapy for CNS diseases by using genetically engineered autologous somatic cells.     

Low Immunogenicity of Neural Progenitor Cells Differentiated from Induced Pluripotent Stem Cells Derived from Less Immunogenic Somatic Cells

The groundbreaking discovery of induced pluripotent stem cells (iPS cells) provides a new source for cell therapy. However, whether the iPS derived functional lineages from different cell origins have different immunogenicity remains unknown. It had been known that the cells isolated from extra-embryonic tissues, such as umbilical cord mesenchymal cells (UMCs), are less immunogenic than other adult lineages such as skin fibroblasts (SFs). In this report, we differentiated iPS cells from human UMCs and SFs into neural progenitor cells (NPCs) and analyzed their immunogenicity. Through co-culture with allologous peripheral blood mononuclear cells (PBMCs), we showed that UMCs were indeed less immunogenic than skin cells to simulate proliferation of PBMCs. Surprisingly, we found that the NPCs differentiated from UMC-iPS cells retained low immunogenicity as the parental UMCs based on the PBMC proliferation assay. In cytotoxic expression assay, reactions in most kinds of immune effector cells showed more perforin and granzyme B expression with SF-NPCs stimulation than that with UMC-NPCs stimulation in PBMC co-culture system, in T cell co-culture system as well. Furthermore, through whole genome expression microarray analysis, we showed that over 70 immune genes, including all members of HLA-I, were expressed at lower levels in NPCs derived from UMC-iPS cells than that from SF-iPS cells. Our results demonstrated a phenomenon that the low immunogenicity of the less immunogenic cells could be retained after cell reprogramming and further differentiation, thus provide a new concept to generate functional lineages with lower immunogenicity for regenerative medicine.