A Time Course Analysis of the Electrophysiological Properties of Neurons Differentiated from Human Induced Pluripotent Stem Cells (iPSCs)

Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function, little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet, understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore, we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs, from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties, including resting membrane potential, action potential, sodium and potassium channel currents, somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons, the resting membrane potential became more negative, the expression of voltage-gated sodium channels increased, the membrane became capable of generating action potentials following adequate depolarization and, at day 48–55, 50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step, of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca2+ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology, as electrophysiological properties of iPSC-derived neurons mature over time

Sweeping away barriers to interdisciplinary research:recommendations based on X-Net project outcomes - March 2024

X-Net is an interdisciplinary research network whose main aim is to understand barriers to interdisciplinary research, before offering solutions to overcome them. X-Net recommends a 13-step programme of targeted multi-level interventions drawn from evidence gathered by the network in 2022- 2023. The 13 interventions would deeply weave interdisciplinarity into UK scientific research culture and free the flow of ideas and expertise across traditional disciplinary boundaries and sectors

Overcoming barriers to cross-disciplinary research

Interdisciplinary research can create many scientific opportunities but may also face challenges and barriers. X-Net’s main objective is helping interdisciplinary scientists to overcome those barriers providing guidance and resources, particularly to early career researchers. We organised an online workshop “Overcoming barriers to cross-disciplinary research” (6th July, 2022) with the purpose of identifying the main obstacles of interdisciplinary research (IDR) in the UK. The workshop incorporated a pre-workshop anonymous survey that allowed participants to identify and share some of their personal experiences of cross-disciplinary research. The workshop then used these experiences to find themes or challenges in common. It also allowed participants to consider, through action learning, what specific cross-disciplinary barrier(s) they sought advice on. The survey questionnaire was designed to focus on the opinions of individual scientists regarding the barriers or incentives for interdisciplinary research and to receive diverse perspectives. Researchers with early or ongoing experience in interdisciplinarity entering biomedical sciences from STEM were approached for their opinions

Genotype effects contribute to variation in longitudinal methylome patterns in older people

Background: DNA methylation levels change along with age, but few studies have examined the variation in the rate of such changes between individuals. Methods: We performed a longitudinal analysis to quantify the variation in the rate of change of DNA methylation between individuals using whole blood DNA methylation array profiles collected at 2-4 time points (N = 2894) in 954 individuals (67-90 years). Results: After stringent quality control, we identified 1507 DNA methylation CpG sites (rsCpGs) with statistically significant variation in the rate of change (random slope) of DNA methylation among individuals in a mixed linear model analysis. Genes in the vicinity of these rsCpGs were found to be enriched in Homeobox transcription factors and the Wnt signalling pathway, both of which are related to ageing processes. Furthermore, we investigated the SNP effect on the random slope. We found that 4 out of 1507 rsCpGs had one significant (P < 5 × 10/1507) SNP effect and 343 rsCpGs had at least one SNP effect (436 SNP-probe pairs) reaching genome-wide significance (P < 5 × 10). Ninety-five percent of the significant (P < 5 × 10) SNPs are on different chromosomes from their corresponding probes. Conclusions: We identified CpG sites that have variability in the rate of change of DNA methylation between individuals, and our results suggest a genetic basis of this variation. Genes around these CpG sites have been reported to be involved in the ageing process

Adult-Derived Stem Cells from the Liver Become Myocytes in the Heart in Vivo

Recent evidence suggests that adult-derived stem cells, like their embryonic counterparts, are pluripotent. These simple, undifferentiated and uncommitted cells are able to respond to signals from their host tissue microenvironment and differentiate, producing progeny that display a phenotype characteristic of the mature cells of that tissue. We used a clonal stem cell line (termed WB-F344) that was derived from an adult male rat liver to investigate the possibility that uncommitted stem cells from a nonmyogenic tissue source would respond to the tissue microenvironment of the heart in vivo and differentiate into cardiac myocytes. Male WB-F344 cells that carry the Escherichia coli β-galactosidase gene were identified in the left ventricular myocardium of adult female nude mice 6 weeks after transplantation. We confirmed the presence of a rat Y-chromosome-specific repetitive DNA sequence exclusively in the β-galactosidase-positive myocytes by polymerase chain reaction and fluorescence in situ hybridization. Immunohistochemistry, using a cardiac troponin T-specific monoclonal antibody, and ultrastructural analysis confirmed a cardiac myocyte phenotype of the stem cell-derived myocytes. The β-galactosidase-positive myocytes ranged from <20 μm to 110 μm in length. The longer of these cells contained well-organized sarcomeres and myofibrils, and formed intercalated disks and gap junctions with endogenous (host-derived) myocytes, suggesting that WB-F344-derived myocytes participate in the function of the cardiac syncytium. These results demonstrate that adult liver-derived stem cells respond to the tissue microenvironment of the adult heart in vivo and differentiate into mature cardiac myocytes

CRISPR/Cas9-Correctable mutation-related molecular and physiological phenotypes in iPSC-derived Alzheimer’s PSEN2 N141I neurons

Abstract Basal forebrain cholinergic neurons (BFCNs) are believed to be one of the first cell types to be affected in all forms of AD, and their dysfunction is clinically correlated with impaired short-term memory formation and retrieval. We present an optimized in vitro protocol to generate human BFCNs from iPSCs, using cell lines from presenilin 2 (PSEN2) mutation carriers and controls. As expected, cell lines harboring the PSEN2 N141I mutation displayed an increase in the Aβ42/40 in iPSC-derived BFCNs. Neurons derived from PSEN2 N141I lines generated fewer maximum number of spikes in response to a square depolarizing current injection. The height of the first action potential at rheobase current injection was also significantly decreased in PSEN2 N141I BFCNs. CRISPR/Cas9 correction of the PSEN2 point mutation abolished the electrophysiological deficit, restoring both the maximal number of spikes and spike height to the levels recorded in controls. Increased Aβ42/40 was also normalized following CRISPR/Cas-mediated correction of the PSEN2 N141I mutation. The genome editing data confirms the robust consistency of mutation-related changes in Aβ42/40 ratio while also showing a PSEN2-mutation-related alteration in electrophysiology

Spontaneous calcium transients increase in iPSC-derived neurons during development.

<p>A) After imaging of spontaneous Ca²⁺- transients iPSC-derived neurons were fixed and stained for Tuj1 at day 32 and day 55, revealing more complex morphology at day 55 (scale bar = 40 µm). B) Representative Fluo-4 spontaneous Ca²⁺- transients from 3 iPSC-derived neurons at day 32 and at day 55 (ΔFluo = ΔFluorescence; a.u. = arbitrary unit). Neurons exhibit slow broad transients, consistent with early developing neurons at day 32. By day 55, calcium transients take on a more spike-like morphology reflecting what has been reported for maturing neurons. Group data demonstrates that iPSC-derived neurons exhibit a significant increase in calcium transients at day 55 as compared to day 32 (n = 113 neurons/10 coverslips for both groups, p<0.001 comparing day 32 with day 55). C) The average number of Ca²⁺ transients at day 55 was dramatically reduced following perfusion with 1 µM TTX with recovery following washout of the toxin (n = 20/3 coverslips, p<0.001 compared with average number of Ca²⁺ transients prior to TTX perfusion). A sample trace derived from a representative experiment is shown on the left).</p

Timeline of the experiments.

<p>The different steps of the experiments are represented, as well as the time-points for the electrophysiological, calcium imaging, immunofluorescence and FACS analyses. The 3 different timing groups for statistical purposes are the following: t1  =  from day 31 to day 38; t2  =  from day 41 to day 45; t3  =  from day 48 to day 55.</p

Evolution of basal membrane properties over time in iPSC-derived neurons plated on POL.

<p>A) The average resting membrane potential (RMP in mV) decreased over time. The number of cells examined for these experiments are shown inside the bar in these and the following figures. The vertical lines correspond to the SEM in this and the following figures. **: p<0.001; *: 0.0010.05 for these and the following figures. B) The membrane input resistance decreased over time (GΩ). C) The membrane time constant decreased over time (ms).</p

Characterization of the APs in iPSC-derived neurons plated on POL.

<p>A) Representative APs in response to step current injections of 20 pA (upper panel) in current clamp mode. The cell shown was recorded at day 45. APs were observed in 4 out of 10 cells. Following initial recording, APs were blocked by 1 µM TTX. B) Multiple firing of APs in response to step current injections in 20 pA increments was observed in cells from day 45. The trace shown in this figure was obtained from a cell recorded at 55 days. The inset shows a high magnification view of the APs. C) At day 55, 13 out of 22 cells had APs, and 3 of them had rebound APs like the one shown on the inset of this panel. Multiple APs in response to depolarizing current injections and “rebound” APs at the end of hyperpolarizing current injections were also visible. After perfusion with 1 µM TTX, the APs, due to Na⁺ channel dependence, was blocked, but rebound APs persisted in 4 out of 17 cells with rebound APs.</p