Establishment and characterization of a novel human induced pluripotent stem cell line stably expressing the iRFP720 reporter.

Stem cell therapy has great potential for replacing beta-cell loss in diabetic patients. However, a key obstacle to cell therapy's success is to preserve viability and function of the engrafted cells. While several strategies have been developed to improve engrafted beta-cell survival, tools to evaluate the efficacy within the body by imaging are limited. Traditional labeling tools, such as GFP-like fluorescent proteins, have limited penetration depths in vivo due to tissue scattering and absorption. To circumvent this limitation, a near-infrared fluorescent mutant version of the DrBphP bacteriophytochrome, iRFP720, has been developed for in vivo imaging and stem/progenitor cell tracking. Here, we present the generation and characterization of an iRFP720 expressing human induced pluripotent stem cell (iPSC) line, which can be used for real-time imaging in various biological applications. To generate the transgenic cells, the CRISPR/Cas9 technology was applied. A puromycin resistance gene was inserted into the AAVS1 locus, driven by the endogenous PPP1R12C promoter, along with the CAG-iRFP720 reporter cassette, which was flanked by insulator elements. Proper integration of the transgene into the targeted genomic region was assessed by comprehensive genetic analysis, verifying precise genome editing. Stable expression of iRFP720 in the cells was confirmed and imaged by their near-infrared fluorescence. We demonstrated that the reporter iPSCs exhibit normal stem cell characteristics and can be efficiently differentiated towards the pancreatic lineage. As the genetically modified reporter cells show retained pluripotency and multilineage differentiation potential, they hold great potential as a cellular model in a variety of biological and pharmacological applications.

Environmental Alterations during Embryonic Development: Studying the Impact of Stressors on Pluripotent Stem Cell-Derived Cardiomyocytes.

Non-communicable diseases (NCDs) sauch as diabetes, obesity and cardiovascular diseases are rising rapidly in all countries world-wide. Environmental maternal factors (e.g., diet, oxidative stress, drugs and many others), maternal illnesses and other stressors can predispose the newborn to develop diseases during different stages of life. The connection between environmental factors and NCDs was formulated by David Barker and colleagues as the Developmental Origins of Health and Disease (DOHaD) hypothesis. In this review, we describe the DOHaD concept and the effects of several environmental stressors on the health of the progeny, providing both animal and human evidence. We focus on cardiovascular diseases which represent the leading cause of death worldwide. The purpose of this review is to discuss how in vitro studies with pluripotent stem cells (PSCs), such as embryonic and induced pluripotent stem cells (ESC, iPSC), can underpin the research on non-genetic heart conditions. The PSCs could provide a tool to recapitulate aspects of embryonic development "in a dish", studying the effects of environmental exposure during cardiomyocyte (CM) differentiation and maturation, establishing a link to molecular mechanism and epigenetics.

Integration of nano- and biotechnology for beta-cell and islet transplantation in type-1 diabetes treatment.

Regenerative medicine using human or porcine ß-cells or islets has an excellent potential to become a clinically relevant method for the treatment of type-1 diabetes. High-resolution imaging of the function and faith of transplanted porcine pancreatic islets and human stem cell-derived beta cells in large animals and patients for testing advanced therapy medicinal products (ATMPs) is a currently unmet need for pre-clinical/clinical trials. The iNanoBIT EU H2020 project is developing novel highly sensitive nanotechnology-based imaging approaches allowing for monitoring of survival, engraftment, proliferation, function and whole-body distribution of the cellular transplants in a porcine diabetes model with excellent translational potential to humans. We develop and validate the application of single-photon emission computed tomography (SPECT) and optoacoustic imaging technologies in a transgenic insulin-deficient pig model to observe transplanted porcine xeno-islets and in vitro differentiated human beta cells. We are progressing in generating new transgenic reporter pigs and human-induced pluripotent cell (iPSC) lines for optoacoustic imaging and testing them in transplantable bioartificial islet devices. Novel multifunctional nanoparticles have been generated and are being tested for nuclear imaging of islets and beta cells using a new, high-resolution SPECT imaging device. Overall, the combined multidisciplinary expertise of the project partners allows progress towards creating much needed technological toolboxes for the xenotransplantation and ATMP field, and thus reinforces the European healthcare supply chain for regenerative medicinal products.

Systematic in vitro and in vivo characterization of Leukemia-inhibiting factor- and Fibroblast growth factor-derived porcine induced pluripotent stem cells.

Derivation and stable maintenance of porcine induced pluripotent stem cells (piPSCs) is challenging. We herein systematically analyzed two piPSC lines, derived by lentiviral transduction and cultured under either leukemia inhibitory factor (LIF) or fibroblast growth factor (FGF) conditions, to shed more light on the underlying biological mechanisms of porcine pluripotency. LIF-derived piPSCs were more successful than their FGF-derived counterparts in the generation of in vitro chimeras and in teratoma formation. When LIF piPSCs chimeras were transferred into surrogate sows and allowed to develop, only their prescence within the embryonic membranes could be detected. Whole-transcriptome analysis of the piPSCs and porcine neonatal fibroblasts showed that they clustered together, but apart from the two pluripotent cell populations of early porcine embryos, indicating incomplete reprogramming. Indeed, bioinformatic analysis of the pluripotency-related gene network of the LIF- versus FGF-derived piPSCs revealed that ZFP42 (REX1) expression was absent in both piPSC-like cells, whereas it was expressed in the porcine inner cell mass at Day 7/8. A second striking difference was the expression of ATOH1 in piPSC-like cells, which was absent in the inner cell mass. Moreover, our gene expression analyses plus correlation analyses of known pluripotency genes identified unique relationships between pluripotency genes in the inner cell mass, which are to some extent, in the piPSC-like cells. This deficiency in downstream gene activation and divergent gene expression may be underlie the inability to derive germ line-transmitting piPSCs, and provides unique insight into which genes are necessary to achieve fully reprogrammed piPSCs. 84: 229-245, 2017. © 2016 Wiley Periodicals, Inc.

Functionality of endothelial cells and pericytes from human pluripotent stem cells demonstrated in cultured vascular plexus and zebrafish xenografts.

OBJECTIVE: Endothelial cells (ECs), pericytes, and vascular smooth muscle cells (vSMCs) are essential for vascular development, and their dysfunction causes multiple cardiovascular diseases. Primary vascular cells for research are, however, difficult to obtain. Human-induced pluripotent stem cells (hiPSCs) derived from somatic tissue are a renewable source of ECs and vSMCs; however, their use as disease models has been limited by low and inconsistent efficiencies of differentiation and the lack of phenotypic bioassays. APPROACH AND RESULTS: Here, we developed defined conditions for simultaneous derivation of ECs and pericytes with high efficiency from hiPSCs of different tissue origin. The protocol was equally efficient for all lines and human embryonic stem cells (hESCs). The ECs could undergo sequential passage and were phenotypically indistinguishable, exhibiting features of arterial-like embryonic ECs. Moreover, hiPSC-derived ECs formed an authentic vascular plexus when cocultured with hiPSC-derived pericytes. The coculture system recapitulated (1) major steps of vascular development including EC proliferation and primary plexus remodeling, and (2) EC-mediated maturation and acquisition of contractile vSMC phenotype by pericytes. In addition, hiPSC-derived ECs integrated into developing vasculature as xenografts in zebrafish. This contrasts with more widely used ECs from human umbilical vein, which form only unstable vasculature and were completely unable to integrate into zebrafish blood vessels. CONCLUSIONS: We demonstrate that vascular derivatives of hiPSC, such as ECs and pericytes, are fully functional and can be used to study defective endothelia-pericyte interactions in vitro for disease modeling and studies on tumor angiogenesis.

Generation of mouse induced pluripotent stem cells from different genetic backgrounds using Sleeping beauty transposon mediated gene transfer.

Induced pluripotent stem (iPS) cell technology involves reprogramming somatic cells to a pluripotent state. The original technology used to produce these cells requires viral gene transduction and results in the permanent integration of exogenous genes into the genome. This can lead to the development of abnormalities in the derived iPS cells. Here, we report that non-viral transfection of a Sleeping Beauty (SB) transposon containing the coding sequences Oct3/4 (Pouf1), Sox-2, Klf-4 and c-Myc (OSKM) linked with 2A peptides, can reprogram mouse fibroblasts. We have established reprogrammed mouse cell lines from three different genetic backgrounds: (1) ICR-outbred, (2) C57BL/6-inbred and (3) F1-hybrid (C57BL/6 x DBA/2J), with parallel robust expression of all exogenous (Oct3/4, Sox-2, Klf-4, and c-Myc) and endogenous (e.g. Oct3/4 and Nanog) pluripotency genes. The iPS cell lines exhibited characteristics typical for undifferentiated embryonic stem (ES) cell lines: ES cell-like morphology, alkaline phosphatase (ALP) positivity and gene expression pattern (shown by reverse transcription PCR, and immunofluorescence of ES cell markers-e.g. Oct3/4, SSEA1, Nanog). Furthermore, cells were able to form embryoid bodies (EBs), to beat rhythmically, and express cardiac (assayed by immunofluorescence, e.g. cardiac Troponin T, desmin) and neuronal (assayed by immunofluorescence e.g. nestin, Tuj1) markers. The in vitro differentiation potential was found to be the highest in the ICR-derived iPS lines (ICR-iPS). Interestingly, the ICR-iPS lines had even higher differentiation potential than the ICR-ES cell lines: the rate of EBs forming rhythmically beating cardiomyocytes was 4% in ICR-ES and 79% in ICR-iPS cells, respectively. In vivo, the ICR and F1 hybrid iPS cells formed chimeras and one of the iPS cells from the F1 hybrid background transmitted to the germline. Our results suggest that iPS technology may be useful for generating pluripotent stem cells from genetic backgrounds of which good quality ES cell generation is difficult. These studies provide insights into viral-free iPS technology and may contribute towards defining future cell-based therapies, drug-screening methods and production of transgenic animals using genetically modified iPS cells.

Generation of neuronal progenitor cells and neurons from mouse sleeping beauty transposon-generated induced pluripotent stem cells.

Mouse embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells can be used as models of neuronal differentiation for the investigation of mammalian neurogenesis, pharmacological testing, and development of cell-based therapies. Recently, mouse iPS cell lines have been generated by Sleeping Beauty (SB) transposon-mediated transgenesis (SB-iPS). In this study, we determined for the first time the differentiation potential of mouse SB-iPS cells to form neuronal progenitor cells (NPCs) and neurons. Undifferentiated SB-iPS and ES cells were aggregated into embryoid bodies (EBs) and cultured in neuronal differentiation medium supplemented with 5 µM all-trans retinoic acid. Thereafter, EBs were dissociated and plated to observe further neuronal differentiation. Samples were fixed on days 10 and 14 for immunocytochemistry staining using the NPC markers Pax6 and Nestin and the neuron marker ßIII-tubulin/Tuj1. Nestin-labeled cells were analyzed further by flow cytometry. Our results demonstrated that SB-iPS cells can generate NPCs and differentiate further into neurons in culture, although SB-iPS cells produced less nestin-positive cells than ESCs (6.12 ± 1.61 vs. 74.36 ± 1.65, respectively). In conclusion, the efficiency of generating SB-iPS cells-derived NPCs needs to be improved. However, given the considerable potential of SB-iPS cells for drug testing and as therapeutic models in neurological disorders, continuing investigation of their neuronal differentiation ability is required.