DataSheet1_Population pharmacokinetics of everolimus in patients with seizures associated with focal cortical dysplasia.PDF

Background: Everolimus is an inhibitor of mammalian target of rapamycin complex 1. As mutations in TSC1 and TSC2, which cause partial-onset seizures associated with TSC, were found in focal cortical dysplasia type Ⅱ (FCD Ⅱ) patients, a clinical trial has been performed to explore the efficacy and safety of everolimus in FCD patients. However, no dosage regimen was determined to treat FCD II. To recommend an optimal dose regimen for FCD patients, a population pharmacokinetic model of everolimus in FCD patients was developed.Methods: The data of everolimus were collected from September 2017 to May 2020 in a tertiary-level hospital in Korea. The model was developed using NONMEM® software version 7.4.1 (Icon Development Solutions, Ellicott City, MD, United States).Results: The population pharmacokinetics of everolimus was described as the one-compartment model with first-order absorption, with the effect of BSA on clearance. The final model was built as follows: TVCL = 12.5 + 9.71 × (BSA/1.5), TVV = 293, and TVKA = 0.585. As a result of simulation, a dose higher than 7 mg/m2 is needed in patients with BSA 0.5 m2, and a dose higher than 6 mg/m2 is needed in patients with BSA 0.7 m2. A dose of 4.5 mg/m2 is enough in the population with BSA higher than 1.5 m2 to meet the target trough range of 5–15 ng/mL.Conclusion: Based on the developed pharmacokinetics model, the optimal dose of everolimus in practice was recommended by considering the available strengths of Afinitor disperz®, 2 mg, 3 mg, and 5 mg.</p

Table_1_The Efficacy of Ketogenic Diet for Specific Genetic Mutation in Developmental and Epileptic Encephalopathy.DOCX

<p>Objectives: Pathogenic mutations in developmental and epileptic encephalopathy (DEE) are increasingly being discovered. However, little has been known about effective targeted treatments for this rare disorder. Here, we assessed the efficacy of ketogenic diet (KD) according to the genes responsible for DEE.</p><p>Methods: We retrospectively evaluated the data from 333 patients who underwent a targeted next-generation sequencing panel for DEE, 155 of whom had tried KD. Patients showing ≥90% seizure reduction from baseline were considered responders. The KD efficacy was examined at 3, 6, and 12 months after initiation. Patients were divided into those with an identified pathogenic mutation (n = 73) and those without (n = 82). The KD efficacy in patients with each identified pathogenic mutation was compared with that in patients without identified genetic mutations.</p><p>Results: The responder rate to KD in the patients with identified pathogenic mutations (n = 73) was 52.1, 49.3, and 43.8% at 3, 6, and 12 months after initiation, respectively. Patients with mutations in SCN1A (n = 18, responder rate = 77.8%, p = 0.001), KCNQ2 (n = 6, responder rate = 83.3%, p = 0.022), STXBP1 (n = 4, responder rate = 100.0%, p = 0.015), and SCN2A (n = 3, responder rate = 100.0%, p = 0.041) showed significantly better responses to KD than patients without identified genetic mutations. Patients with CDKL5 encephalopathy (n = 10, responder rate = 0.0%, p = 0.031) showed significantly less-favorable responses to KD.</p><p>Conclusions: The responder rate to KD remained consistent after KD in DEE patients with specific pathogenic mutations. KD is effective in patients with DEE with genetic etiology, especially in patients with SCN1A, KCNQ2, STXBP1, and SCN2A mutations, but is less effective in patients with CDKL5 mutations. Therefore, identifying the causative gene can help predict the efficacy of KD in patients with DEE.</p

Additional file 1: Table S1. of Efficient strategy for the molecular diagnosis of intractable early-onset epilepsy using targeted gene sequencing

List of 172 targeted genes included in the epilepsy panel. Table S2. Clinical and demographic information of the patients. Table S3. Quality control matrices of NGS test results for all patients in this study. Table S4. Diagnostic yield of targeted NGS according to types of epilepsy syndrome. Table S5. Clinical factors associated with genetic abnormalities. (XLSX 31 kb

Highly Pure and Expandable PSA-NCAM-Positive Neural Precursors from Human ESC and iPSC-Derived Neural Rosettes

<div><p>Homogeneous culture of neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) would provide a powerful tool for biomedical applications. However, previous efforts to expand mechanically dissected neural rosettes for cultivation of NPCs remain concerns regarding non-neural cell contamination. In addition, several attempts to purify NPCs using cell surface markers have not demonstrated the expansion capability of the sorted cells. In the present study, we show that polysialic acid-neural cell adhesion molecule (PSA-NCAM) is detected in neural rosette cells derived from hPSCs, and employ PSA-NCAM as a marker for purifying expandable primitive NPCs from the neural rosettes. PSA-NCAM-positive NPCs (termed hNPC^PSA-NCAM+) were isolated from the heterogeneous cell population of mechanically harvested neural rosettes using magnetic-based cell sorting. The hNPC^PSA-NCAM+ extensively expressed neural markers such as Sox1, Sox2, Nestin, and Musashi-1 (80∼98% of the total cells) and were propagated for multiple passages while retaining their primitive characteristics in our culture condition. Interestingly, PSA-NCAM-negative cells largely exhibited characteristics of neural crest cells. The hNPC^PSA-NCAM+ showed multipotency and responsiveness to instructive cues towards region-specific neuronal subtypes <em>in vitro</em>. When transplanted into the rat striatum, hNPC^PSA-NCAM+ differentiated into neurons, astrocytes, and oligodendrocytes without particular signs of tumorigenesis. Furthermore, Ki67-positive proliferating cells and non-neural lineage cells were rarely detected in the grafts of hNPC^PSA-NCAM+ compared to those of neural rosette cells. Our results suggest that PSA-NCAM-mediated cell isolation provides a highly expandable population of pure primitive NPCs from hPSCs that will lend themselves as a promising strategy for drug screening and cell therapy for neurodegenerative disorders.</p> </div

hNPC^PSA-NCAM+ exhibited multipotency and differentiated neurons were electrophysiologically functional.

<p>(A–D) hNPC^PSA-NCAM+ were spontaneously differentiated into neurons (A­B), oligodendrocytes (C), and astrocytes (D) in the absence of mitogens. (E) GABA-immunoreactivity was frequently observed among Tuj1-positive cells. (F–G) Voltage-dependent membrane currents: depolarizing voltage steps elicited outward K⁺ currents (F) and fast inward Na⁺ currents (G). (H–I) In current-clamp recordings, short (3 ms) or prolonged (500 ms) depolarizing current injections above supra-threshold elicited the single action potential (H), and the fast action potentials (I). Scale bars: 50 µm.</p

Isolation and differentiation of hNPC^PSA-NCAM+ from iPSCs; spontaneous and directed differentiation from them.

<p>(A–E) Data from WT-iPSC3 and (A’–E’) from PD-iPSC4. (A, A’ and B, B’) Induction of neural rosettes from iPSCs. Neural markers (Sox1, Pax6, and PSA-NCAM) were prominently expressed in rosette structure with distinct Zo-1 expression in lumen. (C–E and C’–E’) hNPC^PSA-NCAM+ were successfully isolated from two hiPSC lines and maintained in adherent culture with the comparative efficiency to that of hESCs. (F–H) hNPC^PSA-NCAM+ derived from PD-iPSC were able to differentiate into neurons, oligodendrocytes and astrocytes. (I–K) PD-specific hNPC^PSA-NCAM+ primed with Shh and FGF8 efficiently gave rise to neurons exhibiting DA neuron phenotypes. Scale bars: 50 µm.</p

Efficient induction of neural rosette cells and isolation of hNPC^PSA-NCAM+.

<p>(A) hESC derived-EBs treated with dorsomorphin (DM) and SB431542 (SB), (B) When EBs treated with DM and SB were attached onto the Matrigel-coated dish, a large number of rosette structures formed in the center of the colonies after 4–5 days. (C–D) Typical neural markers such as Sox1 and Pax6 were strongly expressed in neural rosettes (C) with strong expression of Zo-1 in the central lumens of rosettes (D). (E–F) PSA-NCAM was highly expressed on the surface of neural rosette cells, but not on the cells out-migrating from the rosette clump (indicated with white arrows in E and F). (G–H) PSA-NCAM-positive cells showed typical morphology of NPCs (G), whereas PSA-NCAM-negative cells had flat and large cell bodies, similar to neural crest cells (H). (I) NCAM and PST, a polysialylating enzyme, were more abundantly expressed in PSA-NCAM-positive fraction after cell sorting. (J) A representative image for the culture of hNPC^PSA-NCAM+_. After cell sorting, most of the cells were Sox2/Nestin double-positive, indicating a highly homogeneous culture of NPCs (J, inset) hNPC^PSA-NCAM+ spontaneously formed the neural rosette structure. (K–L) After sorting, the proportion of Sox1-positive cells and PSA-NCAM-positive cells were enriched up to ∼85% and ∼93% of the total cells, respectively. (M-P) Cells in PSA-NCAM negative-fraction were predominantly positive for AP2 (M), HNK1 (N), and P75 (O) which were used to identify neural crest cells, and enhanced the expression of several genes implicated in neural crest development, such as <i>dHand</i>, <i>Snail</i>, and <i>FoxD3</i> (P). Scale bars: 50 µm.</p

Integration and survival of hNPC^PSA-NCAM+ in adult rat striatum.

<p>(A) A series of coronal sections from +1.08 to −0.1 shows survival and integration of hNPC^PSA-NCAM+ grafts cells in the host striatum 4 weeks after transplantation. (B) Dual-label confocal immunofluorescence microscopy with DAPI confirms a large population of HNA- and Tuj1-positive cells within the graft of hNPC^PSA-NCAM+. (C–D) In addition to Tuj1-positive cells, GFAP- and NG2-positive cells within HNA-positive graft sites show the potential of hNPC^PSA-NCAM+ in differentiating into all three neural lineages <i>in vivo</i>. (E–F) Further dual immunohistochemistry for HNA:GABA and HNA:TH indicates the ability of grafted cells to differentiate into various neuronal subtypes. (G–H) A representative area of a neural rosette cell graft indicates a significantly higher number of Ki67-positive cells and clusters whereas only a few Ki67-positive cells with no clusters are observed for hNPC^PSA-NCAM+. (I) Bars indicate the proportions of Ki67-positive cells in the grafts of neural rosette cells and hNPC^PSA-NCAM+ at 4 weeks (blue bars) and 12 weeks (red bars) post-transplantation as mean ± s.e.m. (J–K) Representative images of H/E-stained brain tissues grafted with unsorted neural rosette cells (J) and hNPC^PSA-NCAM+ (K). Melanocyte-like cells stained in dark-brown color were shown only in the grafts of unsorted neural rosette cells (J). Scale bar: 50 µm. Abbreviations: LV, lateral ventricle; A, anterior; P, posterior; D, dorsal; V, ventral. *, p<0.05.</p

hNPC^PSA-NCAM+ were able to differentiate into specific neuronal subtypes using regionalizing cues.

<p>(A) Neurons exhibiting dopaminergic phenotypes were derived from hNPC^PSA-NCAM+. (B–C) The cells treated with Shh and FGF8 for 8 days, followed by treatment with BDNF, GDNF, and ascorbic acid gave rise to a larger number of TH-positive neurons than control cells (non-treated group) without a significant change in total neuronal differentiation. (D–E) TH-positive cells co-expressed with Nurr1 and Pitx3. (F) Quantitative RT-PCR analysis confirmed the enhanced expression of several transcription factors involved in dopaminergic differentiation after treatment with Shh and FGF8 to hNPC^PSA-NCAM+. (G) RA/Shh-exposed hNPC^PSA-NCAM+ showed induction of HoxB4 and HB9, a posterior gene and a motor neuron-specific gene, respectively. Scale bars: 20 µm, n.s., not significant, **, p<0.01.</p