Identification of cell surface markers and establishment of monolayer differentiation to retinal pigment epithelial cells.

In vitro differentiation of human pluripotent stem cells into functional retinal pigment epithelial (RPE) cells provides a potentially unlimited source for cell based reparative therapy of age-related macular degeneration. Although the inherent pigmentation of the RPE cells have been useful to grossly evaluate differentiation efficiency and allowed manual isolation of pigmented structures, accurate quantification and automated isolation has been challenging. To address this issue, here we perform a comprehensive antibody screening and identify cell surface markers for RPE cells. We show that these markers can be used to isolate RPE cells during in vitro differentiation and to track, quantify and improve differentiation efficiency. Finally, these surface markers aided to develop a robust, direct and scalable monolayer differentiation protocol on human recombinant laminin-111 and -521 without the need for manual isolation.

Preclinical safety studies of human embryonic stem cell-derived retinal pigment epithelial cells for the treatment of age-related macular degeneration.

As pluripotent stem cell (PSC)-based reparative cell therapies are reaching the bedside, there is a growing need for the standardization of studies concerning safety of the derived products. Clinical trials using these promising strategies are in development, and treatment for age-related macular degeneration is one of the first that has reached patients. We have previously established a xeno-free and defined differentiation protocol to generate functional human embryonic stem cells (hESCs)-derived retinal pigment epithelial (RPE) cells. In this study, we perform preclinical safety studies including karyotype and whole-genome sequencing (WGS) to assess genome stability, single-cell RNA sequencing to ensure cell purity, and biodistribution and tumorigenicity analysis to rule out potential migratory or tumorigenic properties of these cells. WGS analysis illustrates that existing germline variants load is higher than the introduced variants acquired through in vitro culture or differentiation, and enforces the importance to examine the genome integrity at a deeper level than just karyotype. Altogether, we provide a strategy for preclinical evaluation of PSC-based therapies and the data support safety of the hESC-RPE cells generated through our in vitro differentiation methodology.

Human induced pluripotent stem cells from two azoospermic patients with Klinefelter syndrome show similar X chromosome inactivation behavior to female pluripotent stem cells.

STUDY QUESTION: Does the X chromosome inactivation (XCI) of Klinefelter syndrome (KS)-derived human induced pluripotent stem cells (hiPSCs) correspond to female human pluripotent stem cells (hPSCs) and reflect the KS genotype? SUMMARY ANSWER: Our results demonstrate for the first time that KS-derived hiPSCs show similar XCI behavior to female hPSCs in culture and show biological relevance to KS genotype-related clinical features. WHAT IS KNOWN ALREADY: So far, assessment of XCI of KS-derived hiPSCs was based on H3K27me3 staining and X-inactive specific transcript gene expression disregarding the at least three XCI states (XaXi with XIST coating, XaXi lacking XIST coating, and XaXe (partially eroded XCI)) that female hPSCs display in culture. STUDY DESIGN, SIZE, DURATION: The study used hiPSC lines generated from two azoospermic patients with KS and included two healthy male (HM) and one healthy female donor. PARTICIPANTS/MATERIALS, SETTING, METHODS: In this study, we derived hiPSCs by reprograming fibroblasts with episomal plasmids and applying laminin 521 as culture substrate. hiPSCs were characterized by karyotyping, immunocytochemistry, immunohistochemistry, quantitative PCR, teratoma formation, and embryoid body differentiation. XCI and KS hiPSC relevance were assessed by whole genome transcriptomics analysis and immunocytochemistry plus FISH of KS, HM and female fibroblast, and their hiPSC derivatives. MAIN RESULTS AND THE ROLE OF CHANCE: Applying whole genome transcriptomics analysis, we could identify differentially expressed genes (DEGs) between KS and HM donors with enrichment in gene ontology terms associated with fertility, cardiovascular development, ossification, and brain development, all associated with KS genotype-related clinical features. Furthermore, XCI analysis based on transcriptomics data, RNA FISH, and H3K27me3 staining revealed variable XCI states of KS hiPSCs similar to female hiPSCs, showing either normal (XaXi) or eroded (XaXe) XCI. KS hiPSCs with normal XCI showed nevertheless upregulated X-linked genes involved in nervous system development as well as synaptic transmission, supporting the potential use of KS-derived hiPSCs as an in vitro model for KS. LIMITATIONS, REASONS FOR CAUTION: Detailed clinical information for patients included in this study was not available. Although a correlation between DEGs and the KS genotype could be observed, the biological relevance of these cells has to be confirmed with further experiments. In addition, karyotype analysis for two hiPSC lines was performed at passage 12 but not repeated at a later passage. Nevertheless, since all XCI experiments for those lines were performed between passage 11 and 15 the authors expect no karyotypic changes for those experiments. WIDER IMPLICATIONS OF THE FINDINGS: As KS patients have variable clinical phenotypes that are influenced by the grade of aneuploidy, mosaicism, origin of the X chromosome, and XCI 'escapee' genes, which vary not only among individuals but also among different tissues within the same individual, differentiated KS hiPSCs could be used for a better understanding of KS pathogenesis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Knut and Alice Wallenberg Foundation (2016.0121 and 2015.0096), Ming Wai Lau Centre for Reparative Medicine (2-343/2016), Ragnar Söderberg Foundation (M67/13), Swedish Research Council (2013-32485-100360-69), the Centre for Innovative Medicine (2-388/2016-40), Kronprinsessan Lovisas Förening För Barnasjukvård/Stiftelsen Axel Tielmans Minnesfond, Samariten Foundation, Jonasson Center at the Royal Institute of Technology, Sweden, and Initial Training Network Marie Curie Program 'Growsperm' (EU-FP7-PEOPLE-2013-ITN 603568). The authors declare no conflicts of interest.

Cytotoxicity Study of Ionophore-Based Membranes: Toward On-Body and in Vivo Ion Sensing.

We present the most complete study to date comprising in vitro cytotoxicity tests of ion-selective membranes (ISMs) in terms of cell viability, proliferation, and adhesion assays with human dermal fibroblasts. ISMs were prepared with different types of plasticizers and ionophores to be tested in combination with assays that focus on the medium-term and long-term leaching of compounds. Furthermore, the ISMs were prepared in different configurations considering (i) inner-filling solution-type electrodes, (ii) all-solid-state electrodes based on a conventional drop-cast of the membrane, (iii) peeling after the preparation of a wearable sensor, and (iv) detachment from a microneedle-based sensor, thus covering a wide range of membrane shapes. One of the aims of this study, other than the demonstration of the biocompatibility of various ISMs and materials tested herein, is to create an awareness in the scientific community surrounding the need to perform biocompatibility assays during the very first steps of any sensor development with an intended biomedical application. This will foster meeting the requirements for subsequent on-body application of the sensor and avoiding further problems during massive validations toward the final in vivo use and commercialization of such devices.

Wearable All-Solid-State Potentiometric Microneedle Patch for Intradermal Potassium Detection.

A new analytical all-solid-state platform for intradermal potentiometric detection of potassium in interstitial fluid is presented here. Solid microneedles are modified with different coatings and polymeric membranes to prepare both the potassium-selective electrode and reference electrode needed for the potentiometric readout. These microneedle-based electrodes are fixed in an epidermal patch suitable for insertion into the skin. The analytical performances observed for the potentiometric cell (Nernstian slope, limit of detection of 10-4.9 potassium activity, linear range of 10-4.2 to 10-1.1, drift of 0.35 ± 0.28 mV h-1), together with a fast response time, adequate selectivity, and excellent reproducibility and repeatability, are appropriate for potassium analysis in interstitial fluid within both clinical and harmful levels. The potentiometric response is maintained after several insertions into animal skin, confirming the resiliency of the microneedle-based sensor. Ex vivo tests based on the intradermal detection of potassium in chicken and porcine skin demonstrate that the microneedle patch is suitable for monitoring potassium changes inside the skin. In addition, the dimensions of the microneedles modified with the corresponding layers necessary to enhance robustness and provide sensing capabilities (1000 µm length, 45° tip angle, 15 µm thickness in the tip, and 435 µm in the base) agree with the required ranges for a painless insertion into the skin. In vitro cytotoxicity experiments showed that the patch can be used for at least 24 h without any side effect for the skin cells. Overall, the developed concept constitutes important progress in the intradermal analysis of ions related to an electrolyte imbalance in humans, which is relevant for the control of certain types of diseases.