Due to the risk of insertional mutagenesis, viral transduction has been increasingly replaced by nonviral methods to generate induced pluripotent stem (iPS) cells. One technique that has not yet been explored is the use of "minicircle" DNA, a novel compact vector that is free of bacterial DNA and capable of persistent high level expression in cells. Here, we report the use of a single minicircle vector to generate transgene-free iPS cells from adult human cells. Keywords minicircle DNA; reprogramming; iPS cells; viral-free; human adipose stem cellsNon-viral methods for generating iPS cells using adenovirus 1 , plasmids 2 , or excision of reprogramming factors using Cre/LoxP 3,4 or piggyBAC transposition 5 have been reported, but in general are restricted to mouse, suffer from low reprogramming efficiencies (<0.003%), and may leave behind residual vector sequences. Recently, successful reprogramming of human neonatal foreskin fibroblasts was reported using episomal vectors derived from the EpsteinBarr virus6. However, this technique required three individual plasmids carrying a total of seven factors, including the oncogene SV40, and has not been shown to successfully reprogram cells from adult donors, a more clinically-relevant target population. Further, expression of the EBNA1 protein, as was required for this technique, may increase immune cell recognition of transfected cells7, thus potentially limiting clinical application if the transgene is not completely removed. Protein-based iPS cell generation in mouse8 and human9 fetal and neonatal cells has also been published, but required either chemical treatment (valproic acid) 8 NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript methods require only minimal molecular biology background, and so remain a more attractive option for a wider population of researchers interested in cellular reprogramming.Compared to plasmids, minicircle DNA benefits from higher transfection efficiencies and longer ectopic expression due to its lower activation of exogenous silencing mechanisms10 , 11 , and thus may represent an ideal mechanism for generating iPS cells. We constructed a plasmid (P2PhiC31-LGNSO) that contained a single cassette of four reprogramming factors (Oct4, Sox2, Lin28, Nanog) plus a green fluorescent protein (GFP) reporter gene, each separated by selfcleavage peptide 2A sequences 12, 13 ( Supplementary Fig. 1a,b). We next took advantage of the PhiC31-based intramolecular recombination system that allows the plasmid backbone to be excluded and degraded in bacteria, and the minicircle to be purified and isolated as described 10,11 ( Supplementary Fig. 1c). Expression of individual protein factors was validated in 293FT cells ( Supplementary Fig. 2). To determine the reprogramming ability of the minicircle vector, we chose to induce pluripotency in human adipose stem cells (hASCs). hASCs have a number of advantages over other somatic cell types such as fibroblasts since they can be isolated in large quantities (100 ml of human adipo...

Background-MicroRNAs are involved in various critical functions, including the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis. We hypothesize that microRNA-210 can rescue cardiac function after myocardial infarction by upregulation of angiogenesis and inhibition of cellular apoptosis in the heart. Methods and Results-Using microRNA microarrays, we first showed that microRNA-210 was highly expressed in live mouse HL-1 cardiomyocytes compared with apoptotic cells after 48 hours of hypoxia exposure. We confirmed by polymerase chain reaction that microRNA-210 was robustly induced in these cells. Gain-of-function and loss-of-function approaches were used to investigate microRNA-210 therapeutic potential in vitro. After transduction, microRNA-210 can upregulate several angiogenic factors, inhibit caspase activity, and prevent cell apoptosis compared with control. Afterward, adult FVB mice underwent intramyocardial injections with minicircle vector carrying microRNA-210 precursor, minicircle carrying microRNA-scramble, or sham surgery. At 8 weeks, echocardiography showed a significant improvement of left ventricular fractional shortening in the minicircle vector carrying microRNA-210 precursor group compared with the minicircle carrying microRNA-scramble control. Histological analysis confirmed decreased cellular apoptosis and increased neovascularization. Finally, 2 potential targets of microRNA-210, Efna3 and Ptp1b, involved in angiogenesis and apoptosis were confirmed through additional experimental validation. Conclusion-MicroRNA-210 can improve angiogenesis, inhibit apoptosis, and improve cardiac function in a murine model of myocardial infarction. It represents a potential novel therapeutic approach for treatment of ischemic heart disease. (Circulation. 2010;122[suppl 1]:S124 -S131.)Key Words: gene therapy Ⅲ ischemic heart disease Ⅲ microRNA Ⅲ minicircle vector I schemic heart disease is the number 1 cause of morbidity and mortality in the United States owing to aging, obesity, diabetes, and other comorbid diseases. One potent therapeutic approach for ischemic heart disease is to reduce oxygen consumption, inhibit cardiomyocyte apoptosis, increase coronary flow, and induce revascularization. MicroRNAs (miRNAs), representing approximately 1% of the eukaryotic transcriptome, is an evolutionarily conserved family of noncoding RNAs of 20 to 22 nucleotides that negatively regulate the expression of protein-coding genes through translational inhibition and RNA decay. miRNAs are involved in diverse biological progresses, including cellular differentiation, proliferation, angiogenesis, and apoptosis. 1 To date, 721 miRNAs have been discovered in human and 597 miRNAs in the mouse according to the miRBase Sequence Database Release 14 (www.mirbase.org/). miRNAs can regulate approximately 30% human protein-coding genes. 2 Importantly, the successful suppression of murine liver cancer by systemic delivery of miR-26a suggests the potential of using miRNAs as a novel therapeutic tool. 3 In th...

Ectopic expression of transcription factors can reprogram somatic cells to a pluripotent state. However, most of the studies used skin fibroblasts as the starting population for reprogramming, which usually take weeks for expansion from a single biopsy. We show here that induced pluripotent stem (iPS) cells can be generated from adult human adipose stem cells (hASCs) freshly isolated from patients. Furthermore, iPS cells can be readily derived from adult hASCs in a feeder-free condition, thereby eliminating potential variability caused by using feeder cells. hASCs can be safely and readily isolated from adult humans in large quantities without extended time for expansion, are easy to maintain in culture, and therefore represent an ideal autologous source of cells for generating individual-specific iPS cells.differentiation ͉ pluripotency ͉ reprogramming

MicroRNAs (miRNAs) are a newly discovered endogenous class of small noncoding RNAs that play important posttranscriptional regulatory roles by targeting mRNAs for cleavage or translational repression. Accumulating evidence now supports the importance of miRNAs for human embryonic stem cell (hESC) self-renewal, pluripotency, and differentiation. However, with respect to induced pluripotent stem cells (iPSC), in which embryonic-like cells are reprogrammed from adult cells using defined factors, the role of miRNAs during reprogramming has not been well-characterized. Determining the miRNAs that are associated with reprogramming should yield significant insight into the specific miRNA expression patterns that are required for pluripotency. To address this lack of knowledge, we use miRNA microarrays to compare the "microRNA-omes" of human iPSCs, hESCs, and fetal fibroblasts. We confirm the presence of a signature group of miRNAs that is up-regulated in both iPSCs and hESCs, such as the miR-302 and 17-92 clusters. We also highlight differences between the two pluripotent cell types, as in expression of the miR-371/372/373 cluster. In addition to histone modifications, promoter methylation, transcription factors, and other regulatory control elements, we believe these miRNA signatures of pluripotent cells likely represent another layer of regulatory control over cell fate decisions, and should prove important for the cellular reprogramming field.

Human induced pluripotent stem cells (hiPSCs) derived from patient samples have tremendous potential for innovative approaches to disease pathology investigation and regenerative medicine therapies. However, most hiPSC derivation techniques utilize integrating viruses, which may leave residual transgene sequences as part of the host genome, thereby unpredictably altering cell phenotype in downstream applications. Here we describe a protocol for hiPSC derivation by transfection of a simple, nonviral minicircle DNA construct into human adipose stromal cells (hASCs). Minicircle DNA vectors are free of bacterial DNA and thereby capable of high expression in mammalian cells. Their repeated transfection into hASCs, an abundant somatic cell source that is amenable to efficient reprogramming, results in transgene-free hiPSCs. This protocol requires only readily available molecular biology reagents and expertise, and produces hiPSC colonies from an adipose tissue sample in ~4 weeks.

Background-MicroRNAs (miRNAs) are a newly discovered endogenous class of small, noncoding RNAs that play important posttranscriptional regulatory roles by targeting messenger RNAs for cleavage or translational repression. Human embryonic stem cells are known to express miRNAs that are often undetectable in adult organs, and a growing body of evidence has implicated miRNAs as important arbiters of heart development and disease. Methods and Results-To better understand the transition between the human embryonic and cardiac "miRNA-omes," we report here the first miRNA profiling study of cardiomyocytes derived from human embryonic stem cells. Analyzing 711 unique miRNAs, we have identified several interesting miRNAs, including miR-1, -133, and -208, that have been previously reported to be involved in cardiac development and disease and that show surprising patterns of expression across our samples. We also identified novel miRNAs, such as miR-499, that are strongly associated with cardiac differentiation and that share many predicted targets with miR-208. Overexpression of miR-499 and -1 resulted in upregulation of important cardiac myosin heavy-chain genes in embryoid bodies; miR-499 overexpression also caused upregulation of the cardiac transcription factor MEF2C. Conclusions-Taken together, our data give significant insight into the regulatory networks that govern human embryonic stem cell differentiation and highlight the ability of miRNAs to perturb, and even control, the genes that are involved in cardiac specification of human embryonic stem cells. (Circ Cardiovasc Genet. 2010;3:426-435.)Key Words: microRNA Ⅲ microarrays Ⅲ human embryonic stem cells Ⅲ differentiation Ⅲ cardiomyocyte Ⅲ heart H uman embryonic stem cell-derived cardiomyocytes (hESC-CMs) hold great promise for myocardial regeneration after infarction. A number of groups have reported successful transplantation of hESC-CMs into rodent models of myocardial infarction. [1][2][3] However, a significant obstacle still exists with consistent derivation of purified hESC-CM populations. To precisely drive differentiation of hESCs into cardiac cells, a more comprehensive understanding of the regulatory networks that are involved in this process is needed. hESC-CM transcriptional profiling has already been reported 2 ; however, no group has yet focused on the rapidly evolving field of micro-RNAs (miRNAs) and their expression profiles in hESC-CMs. Clinical Perspective on p 435miRNAs play important posttranscriptional regulatory roles by targeting messenger RNAs for cleavage or translational repression. 4 Hundreds of human miRNAs have been discovered, and each is predicted to target tens or hundreds of different messenger RNAs. hESCs are known to express miRNAs that are often undetectable in adult organs, [5][6][7] and some of these miRNAs are believed to regulate ESC selfrenewal and differentiation. 8 A growing body of evidence has also implicated miRNAs in heart disease and development. 9 As examples, have demonstrated roles in heart failure, hypertro...

BackgroundDifferentiation of human embryonic stem cells into endothelial cells (hESC-ECs) has the potential to provide an unlimited source of cells for novel transplantation therapies of ischemic diseases by supporting angiogenesis and vasculogenesis. However, the endothelial differentiation efficiency of the conventional embryoid body (EB) method is low while the 2-dimensional method of co-culturing with mouse embryonic fibroblasts (MEFs) require animal product, both of which can limit the future clinical application of hESC-ECs. Moreover, to fully understand the beneficial effects of stem cell therapy, investigators must be able to track the functional biology and physiology of transplanted cells in living subjects over time.MethodologyIn this study, we developed an extracellular matrix (ECM) culture system for increasing endothelial differentiation and free from contaminating animal cells. We investigated the transcriptional changes that occur during endothelial differentiation of hESCs using whole genome microarray, and compared to human umbilical vein endothelial cells (HUVECs). We also showed functional vascular formation by hESC-ECs in a mouse dorsal window model. Moreover, our study is the first so far to transplant hESC-ECs in a myocardial infarction model and monitor cell fate using molecular imaging methods.ConclusionTaken together, we report a more efficient method for derivation of hESC-ECs that express appropriate patterns of endothelial genes, form functional vessels in vivo, and improve cardiac function. These studies suggest that hESC-ECs may provide a novel therapy for ischemic heart disease in the future.

Background Although stem cell therapy has provided a promising treatment for myocardial infarction, the low survival of the transplanted cells in the infarcted myocardium is possibly a primary reason for failure of long-term improvement. Therefore, the development of novel pro-survival strategies to boost stem cell survival will be of significant benefit to this field. Method and Results Cardiac progenitor cells (CPCs) were isolated from transgenic mice which constitutively express firefly luciferase and green fluorescent protein. The CPCs were transduced with individual lentivirus carrying the precursor of miR-21, miR-24, and miR-221, a cocktail of these 3 miRNA precursors, or GFP as control. After challenge in serum free medium, CPCs treated with the 3 miRNA cocktail showed significantly higher viability compared to untreated CPCs. Following intramuscular and intramyocardial injections, in vivo bioluminescence imaging (BLI) showed that miRNA cocktail-treated CPCs survived significantly longer after transplantation. Following left anterior descending artery ligation, miRNA cocktail-treated CPCs boosts the therapeutic efficacy in terms of functional recovery. Histological analysis confirmed increased myocardial wall thickness and CPC engraftment in the myocardium with miRNA cocktail. Finally, we used bioinformatics analysis and experimental validation assays to show that Bim, a critical apoptotic activator, is an important target gene of the miRNA cocktail, which collectively can bind to the 3’UTR region of Bim and suppress its expression. Conclusion We have demonstrated that a miRNA pro-survival cocktail (miR-21, miR-24, and miR-221) can improve the engraftment of transplanted cardiac progenitor cells and therapeutic efficacy for treatment of ischemic heart disease.