hiPSC-Derived Epidermal Keratinocytes from Ichthyosis Patients Show Altered Expression of Cornification Markers

Inherited ichthyoses represent a large heterogeneous group of skin disorders characterised by impaired epidermal barrier function and disturbed cornification. Current knowledge about disease mechanisms has been uncovered mainly through the use of mouse models or human skin organotypic models. However, most mouse lines suffer from severe epidermal barrier defects causing neonatal death and human keratinocytes have very limited proliferation ability in vitro. Therefore, the development of disease models based on patient derived human induced pluripotent stem cells (hiPSCs) is highly relevant. For this purpose, we have generated hiPSCs from patients with congenital ichthyosis, either non-syndromic autosomal recessive congenital ichthyosis (ARCI) or the ichthyosis syndrome trichothiodystrophy (TTD). hiPSCs were successfully differentiated into basal keratinocyte-like cells (hiPSC-bKs), with high expression of epidermal keratins. In the presence of higher calcium concentrations, terminal differentiation of hiPSC-bKs was induced and markers KRT1 and IVL expressed. TTD1 hiPSC-bKs showed reduced expression of FLG, SPRR2B and lipoxygenase genes. ARCI hiPSC-bKs showed more severe defects, with downregulation of several cornification genes. The application of hiPSC technology to TTD1 and ARCI demonstrates the successful generation of in vitro models mimicking the disease phenotypes, proving a valuable system both for further molecular investigations and drug development for ichthyosis patients

Dynamic Support Culture of Murine Skeletal Muscle-derived Stem Cells Improves Their Cardiogenic Potential In Vitro

Ischemic heart disease is the main cause of death in western countries and its burden is increasing worldwide. It typically involves irreversible degeneration and loss of myocardial tissue leading to poor prognosis and fatal outcome. Autologous cells with the potential to regenerate damaged heart tissue would be an ideal source for cell therapeutic approaches. Here, we compared different methods of conditional culture for increasing the yield and cardiogenic potential of murine skeletal muscle-derived stem cells. A subpopulation of nonadherent cells was isolated from skeletal muscle by preplating and applying cell culture conditions differing in support of cluster formation. In contrast to static culture conditions, dynamic culture with or without previous hanging drop preculture led to significantly increased cluster diameters and the expression of cardiac specific markers on the protein and mRNA level. Whole-cell patch-clamp studies revealed similarities to pacemaker action potentials and responsiveness to cardiac specific pharmacological stimuli. This data indicates that skeletal muscle-derived stem cells are capable of adopting enhanced cardiac muscle cell-like properties by applying specific culture conditions. Choosing this route for the establishment of a sustainable, autologous source of cells for cardiac therapies holds the potential of being clinically more acceptable than transgenic manipulation of cells

Rapid establishment of the European Bank for induced Pluripotent Stem Cells (EBiSC):The Hot Start experience

A fast track “Hot Start” process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community. eTOC: The report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field

Induced pluripotent stem cells as cardiac arrhythmic in vitro models and the impact for drug discovery

Introduction: The development of new antiarrhythmic agents is challenging and is hampered by high attrition rate of novel drug candidates. One of the reasons for this is limited predictability of existing preclinical models for drug assessment. Cardiomyocytes (CMs) derived from disease-specific induced pluripotent stem cells (iPSC) represent a novel in vitro cellular model of cardiac arrhythmias with an unprecedented potential for generating new mechanistic insight into disease pathophysiology and improving the process of drug development. Areas covered: This review outlines recent studies demonstrating the suitability and limitations of iPSC-derived CMs (iPS-CMs) for in vitro modeling inherited arrhythmias and drug testing. The authors focus on channelopathies and outline the properties of iPS-CMs, highlighting their utility and limitations for investigating the mechanism of cardiac arrhythmias and drug discovery. Expert opinion: The iPS-CMs represent a valuable addition to the already existing armamentarium of cardiac arrhythmic models. However, the superiority of iPS-CMs over other arrhythmia models has not yet been rigorously established and the limitations of the model must be overcome before its full potential for antiarrhythmic drug discovery can be realized. Nevertheless, iPS cell-based platforms hold a great potential for increasing our knowledge about cellular arrhythmia mechanisms and improving the drug discovery process

Acid-Sensitive Ion Channels Are Expressed in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are potential sources for cardiac regeneration and drug development. hiPSC-CMs express all the cardiac ion channels and the unique cardiac Ca2+-signaling phenotype. In this study, we tested for expression of acid sensing ion channels (ASICs) in spontaneously beating cardiomyocytes derived from three different hiPSC lines (IMR-90, iPSC-K3, and Ukki011-A). Rapid application of solutions buffered at pH 6.7, 6.0, or 5.0 triggered rapidly activating and slowly inactivating voltage-independent inward current that reversed at voltages positive to E-Na, was suppressed by 5 mu M amiloride and withdrawal of [Na+](o), like neuronal ASIC currents. ASIC currents were expressed at much lower percentages and densities in undifferentiated hiPSC and in dermal fibroblasts. ASIC1 mRNA and protein were measured in first 60 days but decreased in 100 days postdifferentiation hiPSC cultures. Hyperacidification (pH 5 and 6) also triggered large Ca2+ transients in intact hiPSC-CMs that were neither ruthenium red nor amiloride-sensitive, but were absent in whole cell-clamped hiPSC-CMs. Neither ASIC1 current nor its protein was detected in rat adult cardiomyocytes, but hyperacidification did activate smaller and slowly activating currents with drug sensitivity similar to TRPV channels. Considering ASIC expression in developing but not adult myocardium, a role in heart development is likely

From Early Embryonic to Adult Stage: Comparative Study of Action Potentials of Native and Pluripotent Stem Cell-Derived Cardiomyocytes

Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPS-CMs) are promising candidates for cell therapy, drug screening, and developmental studies. It is known that iPS-CMs possess immature electrophysiological properties, but an exact characterization of their developmental stage and subtype differentiation is hampered by a lack of knowledge of electrophysiological properties of native CMs from different developmental stages and origins within the heart. Thus, we sought to systematically investigate action potential (AP) properties of native murine CMs and to establish a database that allows classification of stem cell-derived CMs. Hearts from 129S2PasCrl mice were harvested at days 9-10, 12-14, and 16-18 postcoitum, as well as 1 day, 3-4 days, 1-2 weeks, 3-4 weeks, and 6 weeks postpartum. AP recordings in left and right atria and at apical, medial, and basal left and right ventricles were performed with sharp glass microelectrodes. Measurements revealed significant changes in AP morphology during pre-and postnatal murine development and significant differences between atria and ventricles, enabling a classification of developmental stage and subtype differentiation of stem cell-derived CMs based on their AP properties. For iPS-CMs derived from cell line TiB7.4, a typical ventricular phenotype was demonstrated at later developmental stages, while there were electrophysiological differences from atrial as well as ventricular native CMs at earlier stages. This finding supports that iPS-CMs can develop AP properties similar to native CMs, but points to differences in the maturation process between iPS-CMs and native CMs, which may be explained by dissimilar conditions during in vitro differentiation and in vivo development

Murine transgenic iPS cell line for monitoring and selection of cardiomyocytes

We report here a transgenic murine induced pluripotent stem cell (iPSC) line expressing puromycin N-acetyl-transferase (PAC) and enhanced green fluorescent protein (EGFP) under the control of a-myosin heavy chain promoter. This transgenic cell line reproducibly differentiates into EGFP-expressing cardiomyocytes (CMs) which can be generated at high purity with puromycin treatment and exhibit molecular and functional properties of immature heart muscle cells. This genetically modified iPSC line can be used for assessment of the utility of CMs for myocardial repair, pharmacological and toxicological applications and development of improved cardiac differentiation protocols. (C) 2016 The Authors. Published by Elsevier B.V