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.

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.

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.

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.

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.

Autologous platelet-rich plasma: a biological supplement to enhance adipose-derived mesenchymal stem cell expansion.

Currently the use of non-autologous cell culture media (e.g., animal-derived or allogeneic serum) for clinical applications of mesenchymal stem cells (MSCs) is criticized by regulatory agencies. Autologous platelet-rich plasma (PRP) is proposed as a safer alternative medium supplement for adipose-derived mesenchymal stem cells (AT-MSC) culture. To study its efficiency on cell proliferation, AT-MSCs were cultured for 10 days in media supplemented with different concentrations of autologous non-activated PRP (nPRP) or thrombin-activated PRP (tPRP) (1-60%). AT-MSC proliferation, cell phenotype, multipotency capacity, and chromosome stability were assessed and compared to AT-MSCs expanded in a classical medium supplemented with 10% of fetal bovine serum (FBS). Culture media supplemented with nPRP showed dose-dependent higher AT-MSC proliferation than did FBS or tPRP. Twenty percent nPRP was the most effective concentration to promote cell proliferation. This condition increased 13.9 times greater AT-MSC number in comparison to culture with FBS, without changing the AT-MSC phenotype, differentiation capacity, and chromosome status. We concluded that 20% autologous nPRP is a safe, efficient, and cost-effective supplement for AT-MSC expansion. It should be considered as an alternative to FBS or other nonautologous blood derivatives. It could serve as a potent substitute for the validation of future clinical protocols as it respects good manufacturing practices and regulatory agencies' standards.

Human stem cell-based three-dimensional microtissues for advanced cardiac cell therapies.

Cardiac stem cell therapy has been proposed as a therapy option to treat the diseased myocardium. However, the low retention rate of transplanted single-cell suspensions remains a major issue of current therapy strategies. Therefore, the concept of scaffold-free cellular self-assembly into three-dimensional microtissues (3D-MTs) prior to transplantation may be beneficial to enhance retention and survival. We compared clinically relevant, human stem cell sources for their ability to generate 3D-MTs with particular regards to formation characteristics, proliferation-activity, viability and extracellular-matrix production. Single-cell suspensions of human bone marrow- and adipose tissue-derived mesenchymal stem cells (hBMMSCs and hATMSCs), Isl1(+) cardiac progenitors derived from human embryonic stem cells (hESC-Isl1(+) cells), and undifferentiated human induced pluripotent cells (hiPSCs) were characterized before to generate 3D-MTs using a hanging-drop culture. Besides the principal feasibility of cell-specific 3D-MT formation, a detailed head-to-head comparison between cell sources was performed using histology, immunocyto- and histo-chemistry as well as flow cytometry. Round-oval shaped and uniform 3D-MTs could be successfully generated from all cell types starting with a loose formation within the first 24 h that fully stabilized after 3 days and resulting in a mean 3D-MT diameter of 194.56 ± 18.01 µm (hBMMSCs), 194.56 ± 16.30 µm (hATMSCs), 159.73 ± 19.20 µm (hESC-Isl1(+) cells) and 120.95 ± 7.97 µm (hiPSCs). While all 3D-MTs showed a homogenous cell distribution, hiPSC-derived 3D-MTs displayed a compact cell formation primarily located at the outer margin. hESC-Isl1(+) and hiPSC-derived 3D-MTs maintained their proliferation-activity which was rather limited in the MSC-based 3D-MTs. All four 3D-MT types revealed a comparable viability in excess of 70% and showed a cell-specific expression profile being comparable to their single-cell counterparts. Extracellular matrix (ECM) production during 3D-MT formation was observed for all cell-specific 3D-MTs, with hiPSC-derived 3D-MTs being the fastest one. Interestingly, ECM distribution was homogenous for hATMSC- and hiPSC-based 3D-MTs, while it appeared to be primarily concentrated within in the center of hESC-Isl1(+) and hBMMSC-based 3D-MTs. The results of this head-to-head comparative study indicated that 3D-MTs can be successfully generated from hESC-derived Isl1(+) cells, hiPSCs and MSC lines upon hanging drop culture. Cell-specific 3D-MTs displayed sufficient viability and instant ECM formation. The concept of 3D-MT in vitro generation prior to cell transplantation may represent a promising delivery format for future strategies to enhance cellular engraftment and survival.

Embryonic stem cell-based cardiopatches improve cardiac function in infarcted rats.

Pluripotent stem cell-seeded cardiopatches hold promise for in situ regeneration of infarcted hearts. Here, we describe a novel cardiopatch based on bone morphogenetic protein 2-primed cardiac-committed mouse embryonic stem cells, embedded into biodegradable fibrin matrices and engrafted onto infarcted rat hearts. For in vivo tracking of the engrafted cardiac-committed cells, superparamagnetic iron oxide nanoparticles were magnetofected into the cells, thus enabling detection and functional evaluation by high-resolution magnetic resonance imaging. Six weeks after transplantation into infarcted rat hearts, both local (p < .04) and global (p < .015) heart function, as well as the left ventricular dilation (p < .0011), were significantly improved (p < .001) as compared with hearts receiving cardiopatches loaded with iron nanoparticles alone. Histological analysis revealed that the fibrin scaffolds had degraded over time and clusters of myocyte enhancer factor 2-positive cardiac-committed cells had colonized most of the infarcted myocardium, including the fibrotic area. De novo CD31-positive blood vessels were formed in the vicinity of the transplanted cardiopatch. Altogether, our data provide evidence that stem cell-based cardiopatches represent a promising therapeutic strategy to achieve efficient cell implantation and improved global and regional cardiac function after myocardial infarction.

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.

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.

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.

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.

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.

Fetal bovine serum enables cardiac differentiation of human embryonic stem cells.

During development, cardiac commitment within the mesoderm requires endoderm-secreted factors. Differentiation of embryonic stem cells into the three germ layers in vitro recapitulates developmental processes and can be influenced by supplements added to culture medium. Hence, we investigated the effect of fetal bovine serum (FBS) and KnockOut serum replacement (SR) on germ layers specification and cardiac differentiation of H1 human embryonic stem cells (hESC) within embryoid bodies (EB). At the time of EB formation, FBS triggered an increased apoptosis. As assessed by quantitative PCR on 4-, 10-, and 20-day-old EB, FBS promoted a faster down-regulation of pluripotency marker Oct4 and an increased expression of endodermal (Sox17, alpha-fetoprotein, AFP) and mesodermal genes (Brachyury, CSX). While neuronal and hematopoietic differentiation occurred in both supplements, spontaneously beating cardiomyocytes were only observed in FBS. Action potential (AP) morphology of hESC-derived cardiomyocytes indicated that ventricular cells were present only after 2 months of culture. However, quantification of myosin light chain 2 ventricular (mlc2v)-positive areas revealed that mlc2v-expressing cardiomyocytes could be detected already after 2 weeks of differentiation, but not in all beating clusters. In conclusion, FBS enabled cardiac differentiation of hESC, likely in an endodermal-dependent pathway. Among cardiac cells, ventricular cardiomyocytes differentiated over time, but not as the predominant cardiac cell subtype.

Developmental changes in cardiomyocytes differentiated from human embryonic stem cells: a molecular and electrophysiological approach.

Cardiomyocytes derived from human embryonic stem cells constitute a promising cell source for the regeneration of damaged hearts. The assessment of their in vitro functional properties is mandatory to envisage appropriate cardiac cell-based therapies. In this study, we characterized human embryonic stem cell-derived cardiomyocytes over a 3-month period, using patch-clamp or intracellular recordings to assess their functional maturation and reverse transcriptase-polymerase chain reaction to evaluate the expression of ion channel-encoding subunits. I(to1) and I(K1), the transient outward and inward rectifier potassium currents, were present in cardiomyocytes only, whereas the rapid delayed rectifier potassium current (I(Kr)), pacemaker current (I(f)), and L-type calcium current (I(Ca,L)) could be recorded both in undifferentiated human embryonic stem cells and in cardiomyocytes. Most of the currents underwent developmental maturation in cardiomyocytes, as assessed by modifications in current density (I(to1), I(K1), and I(Ca,L)) and properties (I(f)). Ion-channel mRNAs were always present when the current was recorded. Intracellular recordings in spontaneously beating clusters of cardiomyocytes revealed changes in action potential parameters and in response to pharmacological tools according to time of differentiation. In summary, human embryonic stem cell-derived cardiomyocytes mature over time during in vitro differentiation, approaching an adult phenotype. Disclosure of potential conflicts of interest is found at the end of this article.

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.

Rapid generation of stable transgenic embryonic stem cell lines using modular lentivectors.

Generation of stable transgenic embryonic stem (ES) cell lines by classic transfection is still a difficult task, requiring time-consuming clonal selection, and hampered by clonal artifacts and gene silencing. Here we describe a novel system that allows construction of lentivectors and generation of stable ES cell lines with > 99% transgene expression within a very short time frame. Rapid insertion of promoters and genes of interest is obtained through a modular recombinational cloning system. Vectors contain central polypurine tract from HIV-1 element and woodchuck hepatitis virus post-transcriptional regulatory element as well as antibiotic resistance to achieve optimal and homogenous transgene expression. We show that the system 1) is functional in mouse and human ES cells, 2) allows the generation of ES cells expressing genes of interest under the control of ubiquitous or tissue-specific promoters, and 3) allows ES cells expressing two constructs through selection with different antibiotics to be obtained. The technology described herein should become a useful tool in stem cell research.

Embryonic stem cells: new possible therapy for degenerative diseases that affect elderly people.

The capacity of embryonic stem (ES) cells for virtually unlimited self renewal and differentiation has opened up the prospect of widespread applications in biomedical research and regenerative medicine. The use of these cells would overcome the problems of donor tissue shortage and implant rejection, if the cells are made immunocompatible with the recipient. Since the derivation in 1998 of human ES cell lines from preimplantation embryos, considerable research is centered on their biology, on how differentiation can be encouraged toward particular cell lineages, and also on the means to enrich and purify derivative cell types. In addition, ES cells may be used as an in vitro system not only to study cell differentiation but also to evaluate the effects of new drugs and the identification of genes as potential therapeutic targets. This review will summarize what is known about animal and human ES cells with particular emphasis on their application in four animal models of human diseases. Present studies of mouse ES cell transplantation reveal encouraging results but also technical barriers that have to be overcome before clinical trials can be considered.