Autologous Cell Therapy Approach for Duchenne Muscular Dystrophy using PiggyBac Transposons and Mesoangioblasts.

Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting disease currently without cure. We investigated the use of the PiggyBac transposon for full-length dystrophin expression in murine mesoangioblast (MABs) progenitor cells. DMD murine MABs were transfected with transposable expression vectors for full-length dystrophin and transplanted intramuscularly or intra-arterially into mdx/SCID mice. Intra-arterial delivery indicated that the MABs could migrate to regenerating muscles to mediate dystrophin expression. Intramuscular transplantation yielded dystrophin expression in 11%-44% of myofibers in murine muscles, which remained stable for the assessed period of 5 months. The satellite cells isolated from transplanted muscles comprised a fraction of MAB-derived cells, indicating that the transfected MABs may colonize the satellite stem cell niche. Transposon integration site mapping by whole-genome sequencing indicated that 70% of the integrations were intergenic, while none was observed in an exon. Muscle resistance assessment by atomic force microscopy indicated that 80% of fibers showed elasticity properties restored to those of wild-type muscles. As measured in vivo, transplanted muscles became more resistant to fatigue. This study thus provides a proof-of-principle that PiggyBac transposon vectors may mediate full-length dystrophin expression as well as functional amelioration of the dystrophic muscles within a potential autologous cell-based therapeutic approach of DMD.

Functional Characterization and Comparison of Intercellular Communication in Stem Cell-Derived Cardiomyocytes.

One novel treatment strategy for the diseased heart focuses on the use of pluripotent stem cell-derived cardiomyocytes (SC-CMs) to overcome the heart's innate deficiency for self-repair. However, targeted application of SC-CMs requires in-depth characterization of their true cardiogenic potential in terms of excitability and intercellular coupling at cellular level and in multicellular preparations. In this study, we elucidated the electrical characteristics of single SC-CMs and intercellular coupling quality of cell pairs, and concomitantly compared them with well-characterized murine native neonatal and immortalized HL-1 cardiomyocytes. Firstly, we investigated the electrical properties and Ca(2+) signaling mechanisms specific to cardiac contraction in single SC-CMs. Despite heterogeneity of the new cardiac cell population, their electrophysiological activity and Ca(2+) handling were similar to native cells. Secondly, we investigated the capability of paired SC-CMs to form an adequate subunit of a functional syncytium and analyzed gap junctions and signal transmission by dye transfer in cell pairs. We discovered significantly diminished coupling in SC-CMs compared with native cells, which could not be enhanced by a coculture approach combining SC-CMs and primary CMs. Moreover, quantitative and structural analysis of gap junctions presented significantly reduced connexin expression levels compared with native CMs. Strong dependence of intercellular coupling on gap junction density was further confirmed by computational simulations. These novel findings demonstrate that despite the cardiogenic electrophysiological profile, SC-CMs present significant limitations in intercellular communication. Inadequate coupling may severely impair functional integration and signal transmission, which needs to be carefully considered for the prospective use of SC-CMs in cardiac repair. Stem Cells 2015;33:2208-2218.

Perturbations of heart development and function in cardiomyocytes from human embryonic stem cells with trisomy 21.

Congenital heart defects (CHD) occur in approximately 50% of patients with Down syndrome (DS); the mechanisms for this occurrence however remain unknown. In order to understand how these defects evolve in early development in DS, we focused on the earliest stages of cardiogenesis to ascertain perturbations in development leading to CHD. Using a trisomy 21 (T21) sibling human embryonic stem cell (hESC) model of DS, we show that T21-hESC display many significant differences in expression of genes and cell populations associated with mesodermal, and more notably, secondary heart field (SHF) development, in particular a reduced number of ISL1(+) progenitor cells. Furthermore, we provide evidence for two candidate genes located on chromosome 21, ETS2 and ERG, whose overexpression during cardiac commitment likely account for the disruption of SHF development, as revealed by downregulation or overexpression experiments. Additionally, we uncover an abnormal electrophysiological phenotype in functional T21 cardiomyocytes, a result further supported by mRNA expression data acquired using RNA-Seq. These data, in combination, revealed a cardiomyocyte-specific phenotype in T21 cardiomyocytes, likely due to the overexpression of genes such as RYR2, NCX, and L-type Ca(2+) channel. These results contribute to the understanding of the mechanisms involved in the development of CHD. Stem Cells 2015;33:1434-1446.

FastSkin® Concept: A Novel Treatment for Complex Acute and Chronic Wound Management.

Successful treatments for acute and chronic skin wounds remain challenging. The goal of this proof-of-concept study was to assess the technical feasibility and safety of a novel wound treatment solution, FastSkin®, in a pig model. FastSkin® was prepared from skin micrografts patterned in blood using acoustic waves. Upon coagulation, the graft was transferred on a silicone sheet and placed on wounds. Six full-thickness wounds were created at the back of two pigs and treated with either FastSkin®, split-thickness skin graft (positive control), a gauze coverage (negative control, NC1), or blood patterned without micrografts (negative control, NC2). Silicone sheets were removed after 7, 14, and 21 days. Wound healing was monitored for six weeks and evaluated macroscopically for re-epithelialization and morphometrically for residual wound area and wound contraction. Tissue regeneration was assessed with histology after six weeks. Re-epithelialization was faster in wounds covered with FastSkin® treatments compared to NC2 and in NC2 compared to NC1. Importantly, an enhanced collagen organization was observed in FastSkin® in contrast to NC treatments. In summary, two clinically approved skin wound treatments, namely micrografting and blood clot graft, were successfully merged with sound-induced patterning of micrografts to produce an autologous, simple, and biologically active wound treatment concept.

The first reported generation of several induced pluripotent stem cell lines from homozygous and heterozygous Huntington's disease patients demonstrates mutation related enhanced lysosomal activity.

Neuronal disorders, like Huntington's disease (HD), are difficult to study, due to limited cell accessibility, late onset manifestations, and low availability of material. The establishment of an in vitro model that recapitulates features of the disease may help understanding the cellular and molecular events that trigger disease manifestations. Here, we describe the generation and characterization of a series of induced pluripotent stem (iPS) cells derived from patients with HD, including two rare homozygous genotypes and one heterozygous genotype. We used lentiviral technology to transfer key genes for inducing reprogramming. To confirm pluripotency and differentiation of iPS cells, we used PCR amplification and immunocytochemistry to measure the expression of marker genes in embryoid bodies and neurons. We also analyzed teratomas that formed in iPS cell-injected mice. We found that the length of the pathological CAG repeat did not increase during reprogramming, after long term growth in vitro, and after differentiation into neurons. In addition, we observed no differences between normal and mutant genotypes in reprogramming, growth rate, caspase activation or neuronal differentiation. However, we observed a significant increase in lysosomal activity in HD-iPS cells compared to control iPS cells, both during self-renewal and in iPS-derived neurons. In conclusion, we have established stable HD-iPS cell lines that can be used for investigating disease mechanisms that underlie HD. The CAG stability and lysosomal activity represent novel observations in HD-iPS cells. In the future, these cells may provide the basis for a powerful platform for drug screening and target identification in HD.

High-speed tracking of murine cardiac stem cells by harmonic nanodoublers.

Potassium niobate nonlinear nanoparticles are used for the first time to monitor the evolution of embryonic stem cells (ESC) by second harmonic microscopy. These particles feature the complete absence of photo-bleaching and unlimited excitation wavelength flexibility. The potential of this approach is made evident for tissue-regeneration studies and applications, by capturing a high-speed movie of ESC-derived cardiomyocytes autonomously beating within a cluster. Time-resolved data are analyzed to retrieve 3D information of the contraction pattern at the cellular level.

Mathematical modelling of the action potential of human embryonic stem cell derived cardiomyocytes.

BACKGROUND: Human embryonic stem cell derived cardiomyocytes (hESC-CMs) hold high potential for basic and applied cardiovascular research. The development of a reliable simulation platform able to mimic the functional properties of hESC-CMs would be of considerable value to perform preliminary test complementing in vitro experimentations. METHODS: We developed the first computational model of hESC-CM action potential by integrating our original electrophysiological recordings of transient-outward, funny, and sodium-calcium exchanger currents and data derived from literature on sodium, calcium and potassium currents in hESC-CMs. RESULTS: The model is able to reproduce basal electrophysiological properties of hESC-CMs at 15 40 days of differentiation (Early stage). Moreover, the model reproduces the modifications occurring through the transition from Early to Late developmental stage (50-110, days of differentiation). After simulated blockade of ionic channels and pumps of the sarcoplasmic reticulum, Ca2+ transient amplitude was decreased by 12% and 33% in Early and Late stage, respectively, suggesting a growing contribution of a functional reticulum during maturation. Finally, as a proof of concept, we tested the effects induced by prototypical channel blockers, namely E4031 and nickel, and their qualitative reproduction by the model. CONCLUSIONS: This study provides a novel modelling tool that may serve useful to investigate physiological properties of hESC-CMs.

Cardiac Organoids and Gastruloids to Study Physio-Pathological Heart Development.

Ethical issues restrict research on human embryos, therefore calling for in vitro models to study human embryonic development including the formation of the first functional organ, the heart. For the last five years, two major models have been under development, namely the human gastruloids and the cardiac organoids. While the first one mainly recapitulates the gastrulation and is still limited to investigate cardiac development, the second one is becoming more and more helpful to mimic a functional beating heart. The review reports and discusses seminal works in the fields of human gastruloids and cardiac organoids. It further describes technologies which improve the formation of cardiac organoids. Finally, we propose some lines of research towards the building of beating mini-hearts in vitro for more relevant functional studies.

Transplantation of mouse embryonic stem cells induces hematopoietic and tissue chimerism in rats.

Embryonic stem cells (ESC) can differentiate into all cell lineages, and ESC-like cells were shown to induce hematopoietic chimerism and tolerance in allogeneic models. The aim of our study was to test the capacity of mouse ESC (mESC) to engraft in rats in a xenotransplantation setting. Forty-six rats were transplanted intravenously with 1 million mESC, without immunosuppression (group 1, n = 23) or with cyclosporine (group 2, n = 23). Three months after mESC transplantation, skin grafts were performed from allogeneic, xenogeneic identical to mESC, or xenogeneic third party donors. At day 27 post-transplant, we detected circulating mouse cells in the blood of 4/23 and 5/23 animals of group 1 and group 2, respectively. Chimerism was confirmed by PCR. We also identified long-term surviving murine cells within livers of chimeric animals. Skin grafts showed no difference in survival between allogeneic and xenogeneic donors. Transplantation of xenogeneic mouse ESC induced short-term chimerism in the blood and persistent tissue chimerism in the liver of recipient rats, but did not induce tolerance to skin grafts. Improved immunosuppressive protocols should be tested to prolong chimerism and allow tolerance.

Human fetal mesoangioblasts reveal tissue-dependent transcriptional signatures.

Mesoangioblasts (MABs) derived from adult skeletal muscles are well-studied adult stem/progenitor cells that already entered clinical trials for muscle regeneration in genetic diseases; however, the transcriptional identity of human fetal MABs (fMABs) remains largely unknown. Herein we analyzed the transcriptome of MABs isolated according to canonical markers from fetal atrium, ventricle, aorta, and skeletal muscles (from 9.5 to 13 weeks of age) to uncover specific gene signatures correlating with their peculiar myogenic differentiation properties inherent to their tissue of origin. RNA-seq analysis revealed for the first time that human MABs from fetal aorta, cardiac (atrial and ventricular), and skeletal muscles display subsets of differentially expressed genes likely representing distinct expression signatures indicative of their original tissue. Identified GO biological processes and KEGG pathways likely account for their distinct differentiation outcomes and provide a set of critical genes possibly predicting future specific differentiation outcomes. This study reveals novel information regarding the potential of human fMABs that may help to improve specific differentiation outcomes relevant for therapeutic muscle regeneration.

Fate of undifferentiated mouse embryonic stem cells within the rat heart: role of myocardial infarction and immune suppression.

Abstract It has recently been suggested that the infarcted rat heart microenvironment could direct pluripotent mouse embryonic stem cells to differentiate into cardiomyocytes through an in situ paracrine action. To investigate whether the heart can function as a cardiogenic niche and confer an immune privilege to embryonic stem cells, we assessed the cardiac differentiation potential of undifferentiated mouse embryonic stem cells (mESC) injected into normal, acutely or chronically infarcted rat hearts. We found that mESC survival depended on immunosuppression both in normal and infarcted hearts. However, upon Cyclosporin A treatment, both normal and infarcted rat hearts failed to induce selective cardiac differentiation of implanted mESC. Instead, teratomas developed in normal and infarcted rat hearts 1 week and 4 weeks (50% and 100%, respectively) after cell injection. Tight control of ESC commitment into a specific cardiac lineage is mandatory to avoid the risk of uncontrolled growth and tumourigenesis following transplantation of highly plastic cells into a diseased myocardium.

Immortalized human skin fibroblast feeder cells support growth and maintenance of both human embryonic and induced pluripotent stem cells.

BACKGROUND: Feeder cells are frequently used for the early-stage of derivation and culture of human embryonic stem cell (hESC) lines. METHODS: We established a conditionally immortalized human foreskin fibroblast line that secreted basic fibroblast growth factor (bFGF). These cells were used as feeder cells for hESC culture and induced pluripotent stem (iPS) cell derivation and expansion. This conditional immortalization was performed using lentiviral vector (LV) mediated transduction of Bmi-1 and human telomerase reverse transcriptase genes and the resulting cell line was further modified by LV-mediated transduction of a secreted form of bFGF gene product. Three different laboratories have tested whether this feeder cell line could support the maintenance of four different hESC lines. RESULTS: Immortalized fibroblasts secreting stable amounts of bFGF supported the growth of all hESC lines, which remained pluripotent and had a normal karyotype for at least 10 passages. Even at high passage (p56), these modified cells, when used as feeders, could support iPS derivation and propagation. Derived iPS cells expressed pluripotency markers, had hESC morphology and produced tissue components of the three germ layers when differentiated in vitro. CONCLUSION: These modified fibroblasts are useful as a genetically-defined feeder cell line for reproducible and cost-effective culture of both hESC and iPS cells.

Calreticulin reveals a critical Ca(2+) checkpoint in cardiac myofibrillogenesis.

Calreticulin (crt) is an ubiquitously expressed and multifunctional Ca(2+)-binding protein that regulates diverse vital cell functions, including Ca(2+) storage in the ER and protein folding. Calreticulin deficiency in mice is lethal in utero due to defects in heart development and function. Herein, we used crt(-/-) embryonic stem (ES) cells differentiated in vitro into cardiac cells to investigate the molecular mechanisms underlying heart failure of knockout embryos. After 8 d of differentiation, beating areas were prominent in ES-derived wild-type (wt) embryoid bodies (EBs), but not in ES-derived crt(-/-) EBs, despite normal expression levels of cardiac transcription factors. Crt(-/-) EBs exhibited a severe decrease in expression and a lack of phosphorylation of ventricular myosin light chain 2 (MLC2v), resulting in an impaired organization of myofibrils. Crt(-/-) phenotype could be recreated in wt cells by chelating extracellular or cytoplasmic Ca(2+) with EGTA or BAPTA, or by inhibiting Ca(2+)/calmodulin-dependent kinases (CaMKs). An imposed ionomycin-triggered cystolic-free Ca(2+) concentration ([Ca(2+)](c)) elevation restored the expression, phosphorylation, and insertion of MLC2v into sarcomeric structures and in turn the myofibrillogenesis. The transcription factor myocyte enhancer factor C2 failed to accumulate into nuclei of crt(-/-) cardiac cells in the absence of ionomycin-triggered [Ca(2+)](c) increase. We conclude that the absence of calreticulin interferes with myofibril formation. Most importantly, calreticulin deficiency revealed the importance of a Ca(2+)-dependent checkpoint critical for early events during cardiac myofibrillogenesis.

The NADPH oxidase NOX4 drives cardiac differentiation: Role in regulating cardiac transcription factors and MAP kinase activation.

Reactive oxygen species (ROS) generated by the NOX family of NADPH oxidases have been described to act as second messengers regulating cell growth and differentiation. However, such a function has hitherto not been convincingly demonstrated. We investigated the role of NOX-derived ROS in cardiac differentiation using mouse embryonic stem cells. ROS scavengers prevented the appearance of spontaneously beating cardiac cells within embryoid bodies. Down-regulation of NOX4, the major NOX isoform present during early stages of differentiation, suppressed cardiogenesis. This was rescued by a pulse of low concentrations of hydrogen peroxide 4 d before spontaneous beating appears. Mechanisms of ROS-dependent signaling included p38 mitogen-activated protein kinase (MAPK) activation and nuclear translocation of the cardiac transcription factor myocyte enhancer factor 2C (MEF2C). Our results provide first molecular evidence that the NOX family of NADPH oxidases regulate vertebrate developmental processes.

Three-dimensional extracellular matrix-directed cardioprogenitor differentiation: systematic modulation of a synthetic cell-responsive PEG-hydrogel.

We show that synthetic three-dimensional (3D) matrix metalloproteinase (MMP)-sensitive poly(ethylene glycol) (PEG)-based hydrogels can direct differentiation of pluripotent cardioprogenitors, using P19 embryonal carcinoma (EC) cells as a model, along a cardiac lineage in vitro. In order to systematically probe 3D matrix effects on P19 EC differentiation, matrix elasticity, MMP-sensitivity and the concentration of a matrix-bound RGDSP peptide were modulated. Soft matrices (E=322+/-64.2 Pa, stoichiometric ratio: 0.8), mimicking the elasticity of embryonic cardiac tissue, increased the fraction of cells expressing the early cardiac transcription factor Nkx2.5 around 2-fold compared to embryoid bodies (EB) in suspension. In contrast, stiffer matrices (E=4,036+/-419.6 Pa, stoichiometric ratio: 1.2) decreased the number of Nkx2.5-positive cells significantly. Further indicators of cardiac maturation were promoted by ligation of integrins relevant in early cardiac development (alpha(5)beta(1,) alpha(v)beta(3)) by the RGDSP ligand in combination with the MMP-sensitivity of the matrix, with a 6-fold increased amount of myosin heavy chain (MHC)-positive cells as compared to EB in suspension. This precisely controlled 3D culture system thus may serve as a potential alternative to natural matrices for engineering cardiac tissue structures for cell culture and potentially therapeutic applications.

Derivation of the first Swiss human embryonic stem cell line from a single blastomere of an arrested four-cell stage embryo.

PRINCIPLES: Human embryonic stem cells (hESC) hold enormous potential for regenerative medicine. So far, the majority of hESC lines have been derived from the isolated inner cell mass (ICM) of blastocysts of variable quality, and several of them from low-grade embryos. Moreover, most of the lines have been obtained in media containing animal components such as foetal bovine serum. We aimed to derive hESC lines in xeno-free conditions using spare embryos frozen in Switzerland before 2001. METHODS: In cooperation with Swiss IVF centres we collected up to 199 donated embryos frozen between 1988 and 2000 at different stages of development. RESULTS: Embryo quality at thawing showed wide variability, reduced quality and low survival upon culture. Using early arrested embryos (n=46), we report here the first Swiss hESC line, called CH-ES1, derived from a single blastomere of an arrested four-cell-stage embryo. Despite its polyploidy, already present at the third passage, CH-ES1 expressed ESC markers of pluripotency and differentiated into all three germ layers in embryoid bodies in vitro and in teratomas in vivo. CONCLUSIONS: As the destruction of viable developing embryos, even spare ones, raises serious ethical concerns, deriving hESC lines from arrested embryos may be an alternative approach to avoid embryo destruction. However, given the reduced derivation efficiency they should not be considered a unique and/or selective source of hESC lines.

[MR imaging of embryonic stem cells labeled by superparamagnetic iron oxide].

OBJECTIVE: To evaluate the labeling efficiency of superparamagnetic iron oxide (SPIO) nanoparticles and its toxicity to mouse embryonic stem cells (ESCs) and (embryoid body (ES)-derived cardiomyocytes. METHODS: Mouse ESCs of the line CGR8 were cultured and induced to differentiate into ES-derived cardiomyocytes. The EB-derived cardiomyocytes were coincubated with SPIO contrast agent at different concentrations (1, 8, 9.3, 14, 28, and 56 mg/L) and transfection agent for 24 and 48 hours for cell labeling. Cells not labeled by SPIO and cells labeled by SPIO without transfection agent were used as controls. Spectrophotometer was used to detect the iron concentration in the cells. Confocal microscopy was used to test the intracellular calcium levels ([Ca(2+)] i). The ultrastructure of the cells was observed by electron microscopy. Ex vivo MRI was used to observe the signals of the cells. RESULTS: Iron-containing intracytoplasmic vesicles could be observed clearly with electron microscopy. The intracellular iron concentration was higher in the cells treated with transfection agent than in the cells not treated with transfection agent. The iron concentration of the cells treated with the SPIO at the concentration of 9.3 microg/ml for 24 hours was the highest. There were no differences in the morphology, contractile areas (chi(2) = 1.32; P = 0.25), and the beating frequency (t = 1.73; P = 0.10) between the EBs from iron-labeled ESCs and from the control ESCs. The rhythmic intracellular free Ca(2+) fluctuation in the labeled cardiomyocytes was similar to that of the controls. The MR images with T(2)WI and T(2)WI sequences, especially those with T(2)WI sequence, of the ESCs showed that the signals of the SPIO labeled cells were lower than those of the SPIO-labeled cells. CONCLUSION: SPIO labeling of ESCs and ES-derived cardiomyocytes does not influence the cell viability and proliferation. The standard 1.5T MR equipment can image the labeled cells, thus offering the possibility of cell tracking and migration monitoring in MRI.

Human Hepatocyte-Derived Induced Pluripotent Stem Cells: MYC Expression, Similarities to Human Germ Cell Tumors, and Safety Issues.

Induced pluripotent stem cells (iPSC) are a most promising approach to the development of a hepatocyte transplantable mass sufficient to induce long-term correction of inherited liver metabolic diseases, thus avoiding liver transplantation. Their intrinsic self-renewal ability and potential to differentiate into any of the three germ layers identify iPSC as the most promising cell-based therapeutics, but also as drivers of tumor development. Teratoma development currently represents the gold standard to assess iPSC pluripotency. We analyzed the tumorigenic potential of iPSC generated from human hepatocytes (HEP-iPSC) and compared their immunohistochemical profiles to that of tumors developed from fibroblast and hematopoietic stem cell-derived iPSC. HEP-iPSC generated tumors significantly presented more malignant morphological features than reprogrammed fibroblasts or CD34+ iPSC. Moreover, the protooncogene myc showed the strongest expression in HEP-iPSC, compared to only faint expression in the other cell subsets. Random integration of transgenes and the use of potent protooncogenes such as myc might be a risk factor for malignant tumor development if hepatocytes are used for reprogramming. Nonviral vector delivery systems or reprogramming of cells obtained from less invasive harvesting methods would represent interesting options for future developments in stem cell-based approaches for liver metabolic diseases.

Ca2+ signalling in cardiogenesis.

A variety of InsP3-dependent Ca2+ signals coded in time, amplitude and space occur during the process of cardiac cell differentiation and heart development. Studies performed in both embryos and embryonic stem cell differentiating in cardiomyocytes have uncovered that Ca2+ regulates multiple steps of these biological processes in vertebrates. These include secretion of cardiogenic factors, cardiac transcriptional cascades and in turn gene expression, myofibrillogenesis and initiation of embryonic pacemaker activity. Evidence is accumulating to foresee Ca2+ as a major second messenger in directing the fate of stem cells and patterning the heart in the embryo.

Cardiac tissue engineering: regeneration of the wounded heart.

New solutions are needed to regenerate hearts damaged by myocardial infarction, to overcome bad prognosis of patients with heart failure, and to address the shortage of heart donors. In the past few years, cardiac tissue engineering has emerged as a new and ambitious approach that combines knowledge from material chemistry with cell biology and medicine. In this short review, we present an overview on the most promising materials and cell-therapy strategies used in the past few years for the regeneration of the wounded heart.