Generation of iPS cells using defined factors linked via the self-cleaving 2A sequences in a single open reading frame

Generation of induced pluripotent stem （iPS） cells from somatic cells has been achieved successfully by simultaneous viral transduction of defined reprogramming transcription factors （TFs）. However, the process requires multiple viral vectors for gene delivery. As a result, generated iPS cells harbor numerous viral integration sites in their genomes. This can increase the probability of gene mutagenesis and genomic instability, and present significant barriers to both research and clinical application studies of iPS cells. In this paper, we present a simple lentivirus reprogramming system in which defined factors are fused in-frame into a single open reading frame （ORF） via self-cleaving 2A sequences. A GFP marker is placed downstream of the transgene to enable tracking of transgene expression. We demonstrate that this polycistronic expression system efficiently generates iPS cells. The generated iPS cells have normal karyotypes and are similar to mouse embryonic stem cells in morphology and gene expression. Moreover, they can differentiate into cell types of the three embryonic germ layers in both in vitro and in vivo assays. Remarkably, most of these iPS cells only harbor a single copy of viral vector. This system provides a valuable tool for generation of iPS cells, and our data suggest that the balance of expression of transduced reprogramming TFs in each cell is essential for the reprogramming process. More importantly, when delivered by non-integrating gene-delivery systems, this re-engineered single ORF will facilitate efficient generation of human iPS cells free of genetic modifications.

iPS cells—Alternative pluripotent cells to embryo stem cells

Since the first murine and human embryonic stem cell lines were established by Drs. Evans and Kaufman [1] and Thomson et al. [2], respectively, great progress has been make in the field

Effects of docosahexaenoic acid or arachidonic acid supplementation on the behavior of cardiomyocytes derived from human pluripotent stem cells

Background:Human heart changes its energetic substrates from lactate and glucose to fatty acids during the neonatal period.Noticing the lack of fatty acids in media for the culture of cardiomyocytes derived from human pluripotent stem cells(hiPS-CM),researchers have supplemented mixtures of fatty acids to hiPS-CM and reported the enhancement in the maturation of hiPS-CM.In our previous studies,we separately supplemented two polyunsaturated fatty acids(PUFAs),docosahexaenoic acid(DHA)or arachidonic acid(AA),to rat fetal cardiomyocytes and found that the supplementations upregulated the expressions of mRNAs for cardiomyocyte differentiation,fatty acid metabolism,and cellular adhesion.The enhancement in cellular contractility was attributed to the improvement in intercellular connection rather than a direct enhancement of the contractile force.Methods:This study reports the successive results of the effects of DHA or AA supplementation on hiPS-CM.In addition to the contractile force and mRNA measurements used in the previous study,we further investigated the effect of different cellular aggregations on the contractile force output by means of finite element analysis,measured glucose and fatty acids metabolites,and assessed cTNT and MLC2v expressions through immunofluorecsence evaluation.Results:It showed that the sole supplementation of albumin-conjugated DHA or AA can be taken up by hiPS-CM without other uptake-enhancing factors,and the supplementations may activate the CD36_ERRγmetabolic pathway.DHA or AA supplementation increased the cellular contractile ratio on collagen gels and AA supplementation stimulated hiPS-CM aggregation to form cellular clusters.The enhancement effect on the hiPS-CM contractile force was modest since the increase in contractile force was not significant.AA supplementation was more effective than DHA supplementation because it significantly upregulated mRNA expressions of P300 and CD36.However,finite element analysis showed that the formation of clusters on a collagen gel attenuated the contractile force exerted by the gel on its surroundings.Conclusion:DHA and AA,as having been supplemented in infant formulas,have no direct and significant enhancement effect on the performance of the hiPS-CM when they were supplemented individually,although they were able to enter the cellular metabolic system.The AA supplementation showed some auxiliary effect on the maturation of hiPS-CM,which is worthy of further investigation under the consideration of membrane composition alteration and remodeling of membrane molecules.

Human induced pluripotent stem cells labeled with fluorescent magnetic nanoparticles for targeted imaging and hyperthermia therapy for gastric cancer

Objective: Human induced pluripotent stem(i PS) cells exhibit great potential for generating functional human cells for medical therapies. In this paper, we report for use of human i PS cells labeled with fluorescent magnetic nanoparticles(FMNPs) for targeted imaging and synergistic therapy of gastric cancer cells in vivo. Methods: Human i PS cells were prepared and cultured for 72 h. The culture medium was collected, and then was coincubated with MGC803 cells. Cell viability was analyzed by the MTT method. FMNP-labeled human i PS cells were prepared and injected into gastric cancer-bearing nude mice. The mouse model was observed using a small-animal imaging system. The nude mice were irradiated under an external alternating magnetic field and evaluated using an infrared thermal mapping instrument. Tumor sizes were measured weekly. Results: iP S cells and the collected culture medium inhibited the growth of MGC803 cells. FMNP-labeled human iP S cells targeted and imaged gastric cancer cells in vivo, as well as inhibited cancer growth in vivo through the external magnetic field. Conclusion: FMNP-labeled human i PS cells exhibit considerable potential in applications such as targeted dual-mode imaging and synergistic therapy for early gastric cancer.

RNA Wave for the HIV Therapy: Foods, Stem Cells and the RNA Information Gene

The microRNA (miRNA) gene is small RNA molecule, approximate 20 nucleotides (nts) in length, and also the miRNA is information in a cell as well as the mobile genetic information;therefore, when only one kind of tumor suppressor RNA information gene (Rig) was intravenously administrated, tumorigenic cells can be retuned to the normal cells in vivo. Although the processes of oncogenic have multiple ways, Rig can control its complex system, such as cell cycle with tuning to translation and transcription processing systems. In quite recent experiments, human breast milk and bovine milk have contained Rigs into their microvesicular components. Both also contain the infant nutrient elements. Further, the siRNA genes in artificial nanoparticles were delivered via oral and could restore mouse intestinal inflamemation. In general, Rigs in the diet were found stable to orally affect the digested animals, therefore, the xenotropic Rigs in Rig transgenic plants could also protect from HIV-1 infection by the edible vaccine via intestinal cells. Because orally delivered miRNA as information could be incorporated into intestinal cells and transmitted into intra- and inter-cells and between individuals to wave the system of translation and transcription. Given these mobile characters of Rigs, even though there is the xenotropic miRNA issue, edible Rig agents in plants as a vaccine would be applicable for the Rig diseases (RigDs) by the information technology-based therapy (iTBT) cooperated with system-based therapies such as stem cell therapy and chemotherapy.

A Metric Approach to Hot Topics in Biomedicine via Keyword Co-occurrence

Purpose:To reveal the research hotpots and relationship among three research hot topics in b iomedicine,namely CRISPR,iPS(induced Pluripotent Stem)cell and Synthetic biology.Design/methodology/approach:We set up their keyword co-occurrence networks with using three indicators and information visualization for metric analysis.Findings:The results reveal the main research hotspots in the three topics are different,but the overlapping keywords in the three topics indicate that they are mutually integrated and interacted each other.Research limitations:All analyses use keywords,without any other forms.Practical implications:We try to find the information distribution and structure of these three hot topics for revealing their research status and interactions,and for promoting biomedical developments.Originality/value:We chose the core keywords in three research hot topics in biomedicine by using h-index.

The applications of induced pluripotent stem(iPS)cells in drug development

The introduction of induced pluripotent stem(iPS)cells has been a milestone in the field of regenerative medicine and drug discovery.iPS cells can provide a continuous and individualized source of stem cells and are considered to hold great potential for economically feasible personalized stem cell therapy.Various diseases might potentially be cured by iPS cell-based therapy including Parkinson’s disease,Alzheimer’s disease,Huntington disease,ischemic heart disease,diabetes and so on.Moreover,iPS cells derived from patients suffering from unique incurable diseases can be developed into patient-and disease-specific cell lines.These cells can be used as an effective approach to study the mechanisms of diseases,providing useful tools for drug discovery,development and evaluation.The development of suitable methods for the culture and expansion of iPS cells and their differentiated progenies make feasible modern drug discovery techniques such as high-throughput screening.Furthermore,iPS cells can be applied in the field of toxicological and pharmacokinetics tests.This review focuses on the applications of iPS cells in the field of pharmaceutical industry.

Shared Gene Regulation during Human Somatic Cell Reprogramming

Human induced pluripotent stem （iPS） cells have the ability to differentiate into all somatic cells and to maintain unlimited self- renewal. Therefore, they have great potential in both basic research and clinical therapy for many diseases. To identify potentially universal mechanisms of human somatic cell reprogramming, we studied gene expression changes in three types of cells undergoing reprogramming. The set of 570 genes commonly regulated during induction of iPS cells includes known embryonic stem （ES） cell markers and pluripotency related genes. We also identified novel genes and biological categories which may be related to somatic cell reprogramming. For example, some of the down-regulated genes are predicted targets of the pluripotency microRNA cluster miR302/367, and the proteins from these putative target genes interact with the stem cell pluripotency factor POU5F1 according to our network analysis. Our results identified candidate gene sets to guide research on the mechanisms operating during somatic cell reprogramming.

Reprogramming towards endothelial cells for vascular regeneration

Endothelial damage and dysfunction are implicated in cardiovascular pathological changes and the development of vascular diseases.In view of the fact that the spontaneous endothelial cell(EC)regeneration is a slow and insufficient process,it is of great significance to explore alternative cell sources capable of generating functional ECs to repair damaged endothelium.Indeed,recent achievements of cell reprogramming to convert somatic cells to other cell types provide new powerful approaches to study endothelial regeneration.Based on progress in the research field,the present review aims to summarize the strategies and mechanisms of generating endothelial cells through reprogramming from somatic cells,and to examine what this means for the potential application of cell therapy in the clinic.

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.

CRISPR-mediated genome editing and human diseases

CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)technology has emerged as a powerful technology for genome editing and is now widely used in basic biomedical research to explore gene function.More recently,this technology has been increasingly applied to the study or treatment of human diseases,including Barth syndrome effects on the heart,Duchenne muscular dystrophy,hemophilia,b-Thalassemia,and cystic fibrosis.CRISPR/Cas9(CRISPR-associated protein 9)genome editing has been used to correct diseasecausing DNA mutations ranging from a single base pair to large deletions in model systems ranging from cells in vitro to animals in vivo.In addition to genetic diseases,CRISPR/Cas9 gene editing has also been applied in immunology-focused applications such as the targeting of C-C chemokine receptor type 5,the programmed death 1 gene,or the creation of chimeric antigen receptors in T cells for purposes such as the treatment of the acquired immune deficiency syndrome(AIDS)or promoting anti-tumor immunotherapy.Furthermore,this technology has been applied to the genetic manipulation of domesticated animals with the goal of producing biologic medical materials,including molecules,cells or organs,on a large scale.Finally,CRISPR/Cas9 has been teamed with induced pluripotent stem(iPS)cells to perform multiple tissue engineering tasks including the creation of disease models or the preparation of donor-specific tissues for transplantation.This review will explore the ways in which the use of CRISPR/Cas9 is opening new doors to the treatment of human diseases.

Telomerase as a “stemness” enzyme

Pluripotent or multipotent stem cells are involved in development and tissue homeostasis;they have the ability to self-renew and differentiate into various types of functional cells.To maintain these properties,stem cells must undergo sustained or unlimited proliferation that requires the stabilization of telomeres,which are essential for chromosome end protection.Telomerase,an RNA-dependent DNA polymerase,synthesizes telomeric DNA.Through the lengthening of telomeres the lifespans of cells are extended,or indefinite proliferation is conferred;this is intimately associated with stem cell phenotype.This review highlights our current understanding of telomerase as a"stemness"enzyme and discusses the underlying implications.

曼氏血吸虫虫卵白细胞介素-4诱导因子的原核表达及多克隆抗体的制备

目的原核表达曼氏血吸虫虫卵白细胞介素-4诱导因子(IL-4-inducing principle of schistosoma eggs,IPSE),并制备小鼠多克隆抗体。方法人工合成IPSE基因,采用PCR技术克隆其成熟体开放阅读框,插入原核表达载体pET-28a中,构建重组表达质粒pET-28a-IPSE。转化E.coli BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE和Western blot分析后,应用组氨酸标签蛋白纯化柱纯化包涵体蛋白,免疫BALB/c鼠,制备多克隆抗体。结果克隆的IPSE基因与GenBank中登录的基因序列同源性为100%;重组表达质粒pET-28a-IPSE经PCR及双酶切鉴定证明构建正确;表达的重组蛋白相对分子质量约为16 000,以包涵体形式表达,表达量约占菌体总蛋白的30%,可与鼠抗His单抗特异性结合;用纯化的包涵体蛋白免疫BALB/c小鼠后,获得的特异性抗血清效价为2×10-4,该血清可与IPSE蛋白发生特异性反应。结论已原核表达、纯化了曼氏血吸虫IPSE蛋白,并制备了高滴度的特异鼠抗血清,为进一步从日本血吸虫中筛选相似蛋白及研究其功能奠定了基础。

Differential effects of recombinant fusion proteins TAT-OCT4 and TAT-NANOG on adult human fibroblasts

OCT4 and NANOG are two important tran-scription factors for maintaining the pluripotency and self-renewal abilities of embryonic stem(ES)cells.Meanwhile they play key roles in the induced pluripotent stem(iPS)cells.In this study,recombinant transcript factors TAT-NANOG and TAT-OCT4,which contained a fused powerful protein transduction domain(PTD)TAT from human immunodeficiency virus(HIV),were produced.Each fusion protein could be transported into human adultfibroblasts(HAF)successfully and activated the endogen-ous transcription of both nanog and oct4.Our study revealed the inter-regulation and autoregulation abilities of solo oct4 or nanog in the process of iPS cell reprogram-ming.Meanwhile the transduction of TAT-NANOG could accelerate the growth rate of HAF cells,and the key cell cycle regulator cdc25a was up-regulated.Thus cdc25a may be involved in the regulation of cell growth by NANOG.In addition,the TAT fusion protein technology provided a novel way to improve cell growth that is more controllable and safer.