Maturation of Spinal Motor Neurons Derived from Human Embryonic Stem Cells

Our understanding of motor neuron biology in humans is derived mainly from investigation of human postmortem tissue and more indirectly from live animal models such as rodents. Thus generation of motor neurons from human embryonic stem cells and human induced pluripotent stem cells is an important new approach to model motor neuron function. To be useful models of human motor neuron function, cells generated in vitro should develop mature properties that are the hallmarks of motor neurons in vivo such as elaborated neuronal processes and mature electrophysiological characteristics. Here we have investigated changes in morphological and electrophysiological properties associated with maturation of neurons differentiated from human embryonic stem cells expressing GFP driven by a motor neuron specific reporter (Hb9::GFP) in culture. We observed maturation in cellular morphology seen as more complex neurite outgrowth and increased soma area over time. Electrophysiological changes included decreasing input resistance and increasing action potential firing frequency over 13 days in vitro. Furthermore, these human embryonic stem cell derived motor neurons acquired two physiological characteristics that are thought to underpin motor neuron integrated function in motor circuits; spike frequency adaptation and rebound action potential firing. These findings show that human embryonic stem cell derived motor neurons develop functional characteristics typical of spinal motor neurons in vivo and suggest that they are a relevant and useful platform for studying motor neuron development and function and for modeling motor neuron diseases

Improvement of Severe COVID-19 in an Elderly Man by Sequential Use of Antiviral Drugs

Although a variety of existing drugs are being tested for patients with coronavirus disease 2019 (COVID-19), no efficacious treatment has been found so far, particularly for severe cases. We report successful recovery in an elderly patient with severe pneumonia requiring mechanical ventilation and extracorporeal membrane oxygenation (ECMO). Despite administration of multiple antiviral drugs, including lopinavir/ritonavir, chloroquine, and favipiravir, the patient’s condition did not improve. However, after administration of another antiviral drug, remdesivir, we were able to terminate invasive interventions, including ECMO, and subsequently obtained negative polymerase chain reaction results. Although further validation is needed, remdesivir might be effective in treating COVID-19

Representative morphology and membrane potential responses to current step injection in hESMNs at 3 different times <i>in vitro</i>.

<p>Imaging of cells fixed after patch-clamp recordings indicate that recorded cells express the <i>Hb9</i>::GFP reporter transgene (A-C,E,G,and I). Voltage responses and imaging in the same rows are taken from same neurons. The neurons for A-C are same as that shown in F and G. D,F,H show examples of voltage responses to current steps recorded from 3 neurons current-clamped at −58 mV, −60 mV, and −55 mV, respectively. Bottom traces in D,F, and H show injected currents. Scale bars in images are 50 μm.</p

Spike frequency adaptation (SFA) and rebound action potentials (RAPs) in hESMNs.

<p>(A) An example of the change in instantaneous frequency during a train of action potentials evoked with positive current injection for 1 sec. Inset shows APs (upper) and injected currents (bottom). APs from which ‘a’ and ‘b’ ISI values were measure for SFA calculation are indicated. (B) SFA ratio, calculated as the maximum value of normalized ISIs after any amplitude of positive current injection, increased with DIV (n  = 8, R  = 0.73, P<0.05, Pearson’s linear regression). (C) RAPs were observed in a large subset of hESMNs. Upper trace shows voltage change after negative current injection. Bottom trace shows injected negative current steps. RAP follows the return of current to baseline after the hyperpolarizing step. (D) Incidence of RAPs in hESMNs at 4 different ages as indicated in Fig. 3 legend (n  = 29). Negative current steps with 5 pA increments (to at least 20 pA) were injected while checking for RAPs.</p

Developmental changes in intrinsic membrane properties of hESMNs.

<p>(A) Input resistance decreased over days <i>in vitro</i> (n  = 28, P<0.01, one-way ANOVA). ∗∗ P<0.01, Tukey’s <i>post hoc</i> test. (B) Resting membrane potential and (C) rheobase did not change (n  = 27 and 29, respectively). Positive current steps were injected in 5 pA increments to distinguish small differences in rheobase among individual neurons. (D) Half-width of action potentials (APs),, changed over time <i>in vitro</i> (n  = 26, P<0.001, one-way ANOVA). ∗∗ P<0.01, ∗∗∗ P<0.001, Tukey’s <i>post hoc</i> test. (E) Maximum frequency of APs after current injection increased over time <i>in vitro</i> (n  = 28, P<0.05, one-way ANOVA). ∗P<0.05, Tukey’s <i>post hoc</i> test. Dots shows frequency values for individual neurons. The numbers in parentheses indicate the number of neurons used for analysis taken from 22 dishes in total. In all panels, the first bar represents data from 31+2 DIV, 2^nd bar is 31+4 DIV, 3^rd bar is 31+8/31+9 DIV and 4^th bar is 31+12/31+13 DIV.</p

Human ES-derived motor neurons show increasing morphological complexity as they mature <i>in vitro</i>.

<p>(A) Top, schematic of ES cell directed differentiation to motor neurons shows timing of addition of the inductive cues retinoic acid (RA), and sonic hedgehog (SHH). Bottom, timing of morphometric and electrophysiological analyses. (B) Representative image of day 31+5 hESMN showing mature neuronal morphology and co-expression of GFP with motor neuron marker HB9. GFP intensity distinguished hESMN cell bodies (arrow, ∼65,000 gray levels (g.l.)), neurites (arrowhead, ∼18,000 g.l.), and cytoplasmic GFP background in non-MNs (star, ∼800 g.l.). Scale bar 50 µm. (C) Representative camera lucida (Metamorph) neurite traces from 5 randomly chosen (every 8^th) image fields at day 31+2, 31+5, and 31+9 show increasing neurite size and complexity. Scale bar 40 µm. (D-F) Soma area, branches, total neurite outgrowth and processes (not shown) were quantified (number of cells analyzed at each timepoint shown in brackets in D), median (grey line), mean (red line), 25–75 percentile (grey box), 10–90 percentiles (whisker bars), all outliers (+) are shown for each day from which measurements were made. The values for each morphometric parameter on each day were distributed non-normally (Shapiro-Wilk test, P<0.05) and Kruskal-Wallis One Way Analysis of Variance on Ranks showed significant changes in (D) cell soma area (H = 43.885, 2 d.f., P<0.001), (E) complexity or branches/cell H = 309.245, 2 d.f., P<0.001), (F) total neurite outgrowth (H = 161.287, 2 d.f., P<0.001), and (not shown) number of primary neurites (median, 25^th–75^th percentile: day 33: 3, 2–5; day 36: 6, 5–9; day 40: 8, 6–12, H = 442.555, 2 d.f., P<0.001). All significant <i>post hoc</i> pairwise comparisons, Dunn’s Method, are shown by black bars on graphs. and all pairwise comparisons for primary neurite number were significant, P<0.05.</p

Comparative epidemiology of suspected perioperative hypersensitivity reactions

Suspected perioperative hypersensitivity reactions are rare but contribute significantly to the morbidity and mortality of surgical procedures. Recent publications have highlighted the differences between countries concerning the respective risk of different drugs, and changes in patterns of causal agents and the emergence of new allergens. This review summarises recent information on the epidemiology of perioperative hypersensitivity reactions, with specific consideration of differences between geographic areas for the most frequently involved offending agents