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-Embryonic

Real-time imaging of transplanted stem cells is essential for understanding their interactions in vivo with host environments, for tracking cell fate and function and for successful delivery and safety monitoring in the clinical setting. In this study, we used bioluminescence (BLI) and magnetic resonance imaging (MRI) to visualize the fate of grafted human embryonic stem cell (hESC)-derived human neural stem cells (hNSCs) in stroke-damaged rat brain. The hNSCs were genetically engineered with a lentiviral vector carrying a double fusion (DF) reporter gene that stably expressed enhanced green fluorescence protein (eGFP) and firefly luciferase (fLuc) reporter genes. The hNSCs were self-renewable, multipotent, and expressed markers for neural stem cells. Cell survival was tracked noninvasively by MRI and BLI for 2 months after transplantation and confirmed histologically. Electrophysiological recording from grafted GFP(+) cells and immuno-electronmicroscopy demonstrated connectivity. Grafted hNSCs differentiated into neurons, into oligodendrocytes in stroke regions undergoing remyelination and into astrocytes extending processes toward stroke-damaged vasculatures. Our data suggest that the combination of BLI and MRI modalities provides reliable real-time monitoring of cell fate.

Mesenchymal stem cells (MSCs) have shown their therapeutic potency for treatment of cardiovascular diseases owing to their low immunogenicity, ease of isolation and expansion, and multipotency. As multipotent progenitors, MSCs have revealed their ability to differentiate into various cell types and could promote endogenous angiogenesis via microenvironmental modulation. Studies on cardiovascular diseases have demonstrated that transplanted MSCs could engraft at the injured sites and differentiate into cardiomyocytes and endothelial cells as well. Accordingly, several clinical trials using MSCs have been performed and revealed that MSCs may improve relevant clinical parameters in patients with vascular diseases. To fully comprehend the characteristics of MSCs, understanding their intrinsic property and associated modulations in tuning their behaviors as well as functions is indispensable for future clinical translation of MSC therapy. This review will focus on recent progresses on endothelial differentiation and potential clinical application of MSCs, with emphasis on therapeutic angiogenesis for treatment of cardiovascular diseases.

Low cell retention and engraftment after transplantation limit the successful application of stem cell therapy for AKI. Engineered microenvironments consisting of a hydrogel matrix and growth factors have been increasingly successful in controlling stem cell fate by mimicking native stem cell niche components. Here, we synthesized a bioactive hydrogel by immobilizing the C domain peptide of IGF-1 (IGF-1C) on chitosan, and we hypothesized that this hydrogel could provide a favorable niche for adipose-derived mesenchymal stem cells (ADSCs) and thereby enhance cell survival in an AKI model. In vitro studies demonstrated that compared with no hydrogel or chitosan hydrogel only, the chitosan-IGF-1C hydrogel increased cell viability through paracrine effects. In vivo, cotransplantation of the chitosan-IGF-1C hydrogel and ADSCs in ischemic kidneys ameliorated renal function, likely by the observed promotion of stem cell survival and angiogenesis, as visualized by bioluminescence imaging and attenuation of fibrosis. In conclusion, IGF-1C immobilized on a chitosan hydrogel provides an artificial microenvironment for ADSCs and may be a promising therapeutic approach for AKI.