GPCR標的治療薬に対する評価系の構築と薬効の発現機構に関する研究

学位の種別: 論文博士審査委員会委員 : （主査）東京大学教授 堅田 利明, 東京大学教授 一條 秀憲, 東京大学教授 堀 昌平, 東京大学准教授 名黒 功, 東京大学講師 福山 征光University of Tokyo(東京大学

CDK8/19 inhibition plays an important role in pancreatic β-cell induction from human iPSCs

サイクリン依存性キナーゼCDK8/19阻害はヒトiPS細胞からの膵島様細胞への分化誘導において重要な役割を果たす. 京都大学プレスリリース. 2023-02-27.A safer method of generating pancreatic islet-like cells from human iPS cells by inhibiting cyclin-dependent kinase CDK8/19. 京都大学プレスリリース. 2023-03-08.[Background] Transplantation of differentiated cells from human-induced pluripotent stem cells (hiPSCs) holds great promise for clinical treatments. Eliminating the risk factor of malignant cell transformation is essential for ensuring the safety of such cells. This study was aimed at assessing and mitigating mutagenicity that may arise during the cell culture process in the protocol of pancreatic islet cell (iPIC) differentiation from hiPSCs. [Methods] We evaluated the mutagenicity of differentiation factors used for hiPSC-derived pancreatic islet-like cells (iPICs). We employed Ames mutagenicity assay, flow cytometry analysis, immunostaining, time-resolved fluorescence resonance energy transfer-based (TR-FRET) cell-free dose–response assays, single-cell RNA-sequencing and in vivo efficacy study. [Results] We observed a mutagenic effect of activin receptor-like kinase 5 inhibitor II (ALK5iII). ALK5iII is a widely used β-cell inducer but no other tested ALK5 inhibitors induced β-cells. We obtained kinase inhibition profiles and found that only ALK5iII inhibited cyclin-dependent kinases 8 and 19 (CDK8/19) among all ALK5 inhibitors tested. Consistently, CDK8/19 inhibitors efficiently induced β-cells in the absence of ALK5iII. A combination treatment with non-mutagenic ALK5 inhibitor SB431542 and CDK8/19 inhibitor senexin B afforded generation of iPICs with in vitro cellular composition and in vivo efficacy comparable to those observed with ALK5iII. [Conclusion] Our findings suggest a new risk mitigation approach for cell therapy and advance our understanding of the β-cell differentiation mechanism

Chronic expanding hematoma in the chest: A case report

Chronic expanding hematoma (CEH) is a rare disease that is usually present as a large solitary pulmonary nodule. CEHs are slow growing, but processes underlying their development remain unknown. The present study herein reports the case of a 76‑year‑old male patient with CEH and discusses a number of CEH cases published in the literature. The majority of these previously described patients were Asians. The CEH in the present case was not a successfully resected one, but the patient\u27s clinical course provided information concerning the natural history of the disease. During the clinical course, the patient underwent several chest computed tomography scans. For the present case report, the doubling time and volume change of the mass was calculated, which revealed that the lesion had an inconstant growth rate and that its onset was between 8.2‑11.0 years before the patient succumbed to this disease. Accumulation of knowledge about this rare disease will help to elucidate it further

Characterization and reduction of non-endocrine cells accompanying islet-like endocrine cells differentiated from human iPSC

ヒトiPS細胞から作製した膵島様細胞に混入する目的外細胞の特性評価にもとづく除去. 京都大学プレスリリース. 2022-04-07.Elimination of non-target cells mixed into islet-like cells generated from human iPS cells based on characterization. 京都大学プレスリリース. 2022-04-15.The differentiation of pancreatic endocrine cells from human pluripotent stem cells has been thoroughly investigated for their application in cell therapy against diabetes. Although non-endocrine cells are inevitable contaminating by-products of the differentiation process, a comprehensive profile of such cells is lacking. Therefore, we characterized non-endocrine cells in iPSC-derived pancreatic islet cells (iPIC) using single-cell transcriptomic analysis. We found that non-endocrine cells consist of (1) heterogeneous proliferating cells, and (2) cells with not only pancreatic traits but also liver or intestinal traits marked by FGB or AGR2. Non-endocrine cells specifically expressed FGFR2, PLK1, and LDHB. We demonstrated that inhibition of pathways involving these genes selectively reduced the number of non-endocrine cells in the differentiation process. These findings provide useful insights into cell purification approaches and contribute to the improvement of the mass production of endocrine cells for stem cell-derived cell therapy for diabetes

Temporal and Spatial Transcriptional Fingerprints by Antipsychotic or Propsychotic Drugs in Mouse Brain

<div><p>Various types of antipsychotics have been developed for the treatment of schizophrenia since the accidental discovery of the antipsychotic activity of chlorpromazine. Although all clinically effective antipsychotic agents have common properties to interact with the dopamine D2 receptor (D2R) activation, their precise mechanisms of action remain elusive. Antipsychotics are well known to induce transcriptional changes of immediate early genes (IEGs), raising the possibility that gene expressions play an essential role to improve psychiatric symptoms. Here, we report that while different classes of antipsychotics have complex pharmacological profiles against D2R, they share common transcriptome fingerprint (TFP) profile of IEGs in the murine brain <i>in vivo</i> by quantitative real-time PCR (qPCR). Our data showed that various types of antipsychotics with a profound interaction of D2R including haloperidol (antagonist), olanzapine (antagonist), and aripiprazole (partial agonist) all share common spatial TFPs closely homologous to those of D2R antagonist sulpiride, and elicited greater transcriptional responses in the striatum than in the nucleus accumbens. Meanwhile, D2R agonist quinpirole and propsychotic NMDA antagonists such as MK-801 and phencyclidine (PCP) exhibited the contrasting TFP profiles. Clozapine and propsychotic drug methamphetamine (MAP) displayed peculiar TFPs that reflect their unique pharmacological property. Our results suggest that transcriptional responses are conserved across various types of antipsychotics clinically effective in positive symptoms of schizophrenia and also show that temporal and spatial TFPs may reflect the pharmacological features of the drugs. Thus, we propose that a TFP approach is beneficial to evaluate novel drug candidates for antipsychotic development.</p></div

D2R antagonist shows spatial TFPs homologous to those by antipsychotics, while D2 agonist and propsychotic agents exhibit their peculiar TFPs.

<p>TFPs of seven IEGs over time induced by D2R antagonist/agonist and propsychotic drugs in mouse brain; (a) Selective D2R antagonist sulpiride (100 mg/kg, i.p.) and agonist quinpirole (10 mg/kg, i.p.), and (b) Propsychotic agents MAP (3 mg/kg, s.c.), PCP (10 mg/kg, s.c.) and MK-801 (1 mg/kg, s.c.). The bar depicted in the bottom right corner represents the expression level of the gene searched in a TFP. Red represents high expression, green represents vehicle-control level expression, and blue represents low expression.</p

TFPs of seven IEGs by treatment of propsychotic agents showing dose-response pattern in mouse brain.

<p>Significant reduction of <i>Egr2</i> in nucleus accumbens, striatum and prefrontal cortex, and increases of <i>c-fos</i> and <i>Ccn1</i> in all tested regions are characteristic of NMDA antagonists, whilst MAP serves robust IEGs induction in all four regions studied here. The bar depicted in the bottom right corner represents the expression level of the gene searched in a TFP. Red represents high expression, green represents vehicle-control level expression, and blue represents low expression. Propsychotic agents; MAP (1 hr treatment, 1 mg/kg to 10 mg/kg, s.c.), PCP (1 hr treatment, 1 mg/kg to 10 mg/kg, s.c.), MK-801 (1hr treatment, 0.1 mg/kg to 3 mg/kg, s.c.).</p

Antipsychotics induce distinctive IEG expression changes in nucleus accumbens and striatum, mainly due to their prominent D2R antagonistic activity.

<p>(a) Time courses of seven IEG expressions induced by 0.3 mg/kg (p.o.) of haloperidol. Data are shown as mean± SEM (n = 5). Filled circles, p<0.05 in unpaired t-test with Welch’s correction, compound (n = 5) versus vehicle (n = 5) at the same time point; open circles, not significant. The changes of expression levels are shown as fold change over vehicle. (b) TFP of seven IEGs over time induced by antipsychotic agents in mouse brain. Antipsychotics; haloperidol (0.3 mg/kg, p.o.), aripiprazole (3 mg/kg, p.o.), olanzapine (10 mg/kg, p.o.) and clozapine (30 mg/kg, p.o.). The bar depicted in the bottom right corner represents the expression level of the gene searched in a TFP. Red represents high expression, green represents vehicle-control level expression, and blue represents low expression.</p