An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, including the elucidation of rare disease pathologies, the development of new medicines, and regenerative medicine aiming to restore damaged organs. Despite this, the social implementation of hiPSCs is still limited. This is partly because of the difficulty of reproducing differentiation in culture, even with advanced knowledge and sophisticated technical skills, due to the high sensitivity of iPSCs to minute environmental changes. The application of an automated culture system can solve this issue. Experiments with high reproducibility independent of a researcher's skill can be expected according to a shared procedure across various institutes. Although several automated culture systems that can maintain iPSC cultures and induce differentiation have been developed previously, these systems are heavy, large, and costly because they make use of humanized, multi-articulated robotic arms. To improve on the above issues, we developed a new system using a simple x-y-z axis slide rail system, allowing it to be more compact, lighter, and cheaper. Furthermore, the user can easily modify parameters in the new system to develop new handling tasks. Once a task is established, all the user needs to do is prepare the iPSC, supply the reagents and consumables needed for the desired task in advance, select the task number, and specify the time. We confirmed that the system could maintain iPSCs in an undifferentiated state through several passages without feeder cells and differentiate into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. The system will enable highly reproducible experiments across institutions without the need for skilled researchers and will facilitate the social implementation of hiPSCs in a wider range of research fields by diminishing the obstacles for new entries.

Compact automated culture machine for human induced pluripotent stem cell maintenance and differentiation.

The technologies used to generate human induced pluripotent stem cell (iPSC) from somatic cells potentially enable the wide application of iPSC-derived differentiated cells in industrial research fields as a replacement for animals. However, as highly trained individuals are required to obtain reproducible results, this approach has limited social implementation. In the research field of iPSC, it is believed that documentable information is not enough for reproducing the quality of the differentiated cells. Therefore, automated culture machines for cell processing should make the starting of iPSC-using researches easier. We developed a programmable all-in-one automated culture machine, with dense and compact constitution that fits within a normal biosafety cabinet (200 mm wide, 233 mm height, and 110 mm depth). This instrument was fabricated using novel x-y-z-axes-rail-system, such as an overhead traveling crane, in a factory, which served as the main handling machinery. This machine enabled stable and efficient expansion of human iPSC under the feeder-free condition, without karyotype alterations, and simultaneously differentiated the cells into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. Overall, this machine would facilitate the social implementation of human pluripotent stem cells and contribute to the accumulation of sharable knowledge for the standardization of the entire handling processes of iPSC in pharmaceutical, food, and cosmetic research.

Differential influence of lesion length on fractional flow reserve in intermediate coronary lesions between each coronary artery.

OBJECTIVES: This study evaluated whether the influence of lesion length on functional significance is similar between each target artery. BACKGROUND: In the presence of a similar moderate degree of stenosis, the fractional flow reserve (FFR) in the left anterior descending coronary artery (LAD) is more often <0.80 than in the other arteries. METHODS: A total of 221 lesions with intermediate stenosis on coronary angiography that underwent FFR measurement for the evaluation of myocardial ischemia were enrolled. Quantitative coronary angiographic analysis including percent diameter stenosis and lesion length was performed. The area under the receiver operating characteristics (ROC) curve was estimated for the best cutoff value as a predictor of FFR value of ≤0.80 for each coronary artery. RESULTS: Although lesion length was similar among the lesions with an FFR >0.80 at different locations, the mean lesion length was significantly longer for lesions in the right coronary artery (RCA) with an FFR ≤0.80 than for those in the LAD and left circumflex artery (13.4 ± 3.4 vs. 8.6 ± 3.1 vs. 12.0 ± 3.7 mm, p < .001). ROC analysis demonstrated that the optimal cutoff value of lesion length for predicting an FFR ≤0.80 was 10.0 mm in the LAD (0.56 area under the curve [AUC], 48% sensitivity, and 76% specificity), whereas 13.1 mm in the RCA (0.84 AUC, 67% sensitivity, and 93% specificity). CONCLUSIONS: The impact of lesion length on myocardial ischemia is different for each coronary artery. A longer lesion length is required in the RCA than in the LAD to achieve an FFR ≤0.80.

IgA Vasculitis with Simultaneous Cardiopulmonary Involvement.

A 60-year-old man with a history of hypertension, type 2 diabetes, and reflux esophagitis was admitted to our hospital with hemoptysis, dyspnea, and leg edema. We diagnosed him with adult IgA vasculitis based on the presence of purpura, elevated serum IgA fibronectin complexes, pathophysiological findings, a skin biopsy showing leukocytoclastic vasculitis, and immunofluorescence studies demonstrating granular IgA and C3 deposits in the blood vessel wall. He showed concurrent cardiopulmonary involvement without involvement of the gastrointestinal system and kidneys, which are commonly affected in IgA vasculitis patients. Following treatment with prednisolone, the patient recovered with improvement in cardiopulmonary manifestations.

Angioscopic observation of extremely late arterial repair after intracoronary implantation of the first-generation sirolimus-eluting stents.

BACKGROUND: Arterial repair delays after intracoronary implantation of Cypher sirolimus-eluting stents (SES), a representative first-generation drug-eluting stent. It remains unclear whether this delay would catch up with bare metal stents (BMS) during extremely long observation. The aim of this study was angioscopic observation of extremely late arterial repair after Cypher SES implantation. METHODS: Thirty-seven SES and 17 BMS were implanted into 22 and 9 patients with angina pectoris, respectively. Duration after implantation (DAI) ranged from 3 to 10.5years in both stents. Coronary angioscopy revealed neointimal stent coverage (NSC), presence of in-stent yellow plaque (YP), and mural thrombi (MT). NSC was semi-quantified into 4 grades (grade 0, no coverage; grade 1, thin coverage; grade 2, thick coverage; grade 3, fully embedded into neointima). RESULTS: In the BMS-implanted lesions (BMSL), NSC was either grade 1 (24%) or grade 3 (74%), with rare YP and no MT. In the SES-implanted lesions (SESL), NSC was various, i.e. grade 0 (5%), grade 1 (59%), grade 2 (22%), and grade 3 (14%). YP and MT were observed in 27 and 24% of in SESL, respectively. In SESL with DAI>8years (n=5), NSC was either grade 1 (40%) or grade 3 (60%), although YP and MT were more frequently observed (60 and 40%, respectively). CONCLUSIONS: Arterial repair after SES implantation caught up with BMS at around 8years with regards to NSC, although prevalence of YP and MT remained still greater in SESL than BMSL at extremely late phase.

Distinct binding of amyloid imaging ligands to unique amyloid-ß deposited in the presubiculum of Alzheimer's disease.

Non-invasive determination of amyloid-ß peptide (Aß) deposition with radioligands serves for the early diagnosis and clarification of pathogenetic mechanisms of Alzheimer's disease (AD). The polymorphic binding site on multimeric Aß for current radioligands, however, is little understood. In this study, we investigated the binding of several radioligands including (11)C-Pittsburgh Compound B ((11)C-PiB), (3)H-AZD2184, and two recently developed compounds, (125)I-DRM106 and (125)I-DRK092, with unique presubicular Aß deposits lacking interaction with the commonly used amyloid dyes FSB. (11)C-PiB, (3)H-AZD2184, and (125)I-DRK092 showed overt binding to presubicular Aß deposits, while (125)I-DRM106 barely bound to these aggregates despite its strong binding in the hippocampal CA1 sector. Unlike neuritic plaques in the CA1, Aß lesions in the presubiculum were not accompanied by inflammatory gliosis enriched with 18-kDa translocator protein (TSPO). Thus, there are at least two different components in Aß aggregates providing distinct binding sites for the current amyloid radioligands, and one of these binding components is distinctly present in the presubicular Aß deposits. Amyloid radioligands lacking affinity for this component, such as (125)I-DRM106, may selectively capture Aß deposits tightly associated with TSPO neuroinflammation and neurodegeneration as exemplified by CA1 neuritic plaques. Hence, comparative autoradiographic assessments of radioligand binding in CA1 and presubiculum could serve for the development of an amyloid PET imaging agent visualizing neurotoxicity-related Aß pathologies. Non-invasive determination of amyloid-ß peptide (Aß) serves for the early diagnosis and clarification of pathogenetic mechanisms of Alzheimer's disease (AD). We found that there are at least two different amyloid components in hippocampal CA1 and presubiculum providing distinct binding sites for the current amyloid radioligands. Comparative analysis for radioligand binding in these two regions could serve for developing novel imaging agents selectively visualizing neurotoxicity-related Aß pathologies.

In vivo SPECT imaging of amyloid-ß deposition with radioiodinated imidazo[1,2-a]pyridine derivative DRM106 in a mouse model of Alzheimer's disease.

UNLABELLED: Noninvasive determination of amyloid-ß peptide (Aß) deposition has important significance for early diagnosis and medical intervention for Alzheimer's disease (AD). In the present study, we investigated the availability of radiolabeled DRM106 ((123/125)I-DRM106 [6-iodo-2-[4-(1H-3-pyrazolyl)phenyl]imidazo[1,2-a]pyridine]), a compound with sufficient affinity for the synthesis of human Aß fibrils and satisfactory metabolic stability, as a SPECT ligand in living brains. METHOD: The sensitivity of (125)I-DRM106 for detecting Aß deposition was compared with that of (125)I-IMPY (2-(4'-dimethylaminophenyl)-6-iodo-imidazo[1,2-a]pyridine), a well-known amyloid SPECT ligand, by ex vivo autoradiographic analyses in 18-mo-old amyloid precursor protein transgenic mice. To verify the sensitivity and quantitation of radiolabeled DRM106 for in vivo imaging, we compared the detectability of Aß plaques with (123)I-DRM106 and a well-known amyloid PET agent, (11)C-labeled Pittsburgh compound B ((11)C-PiB), in 29-mo-old transgenic mice and age-matched nontransgenic littermates. Additionally, we compared the binding characteristics of (125)I-DRM106 with those of (11)C-PiB and (11)C-PBB3, which selectively bind to Aß plaques and preferentially to tau aggregates, respectively, in postmortem AD brain sections. RESULTS: Ex vivo autoradiographic analysis showed that measurement with (125)I-DRM106 has a higher sensitivity for detecting Aß accumulation than with (125)I-IMPY in transgenic mice. SPECT imaging with (123)I-DRM106 also successfully detected Aß deposition in living aged transgenic mice and showed strong correlation (R = 0.95, P < 0.01) in quantitative analysis for Aß plaque detection by PET imaging with (11)C-PiB, implying that sensitivity and quantitation of SPECT imaging with (123)I-DRM106 are almost as good as (11)C-PiB PET for the detectability of Aß deposition. Further, the addition of nonradiolabeled DRM106 fully blocked the binding of (125)I-DRM106 and (11)C-PiB, but not (11)C-PBB3, to AD brain sections, and (125)I-DRM106 showed a lower binding ratio of the diffuse plaque-rich lateral temporal cortex to the dense-cored/neuritic plaque-rich hippocampal CA1 area, compared with (11)C-PiB. CONCLUSION: All of these data demonstrated the high potential of (123)I-DRM106 for amyloid imaging in preclinical and clinical application, and it might more preferentially detect dense-cored/neuritic amyloid deposition, which is expected to be closely associated with neuropathologic changes of AD.

Biological evaluation of the radioiodinated imidazo[1,2-a]pyridine derivative DRK092 for amyloid-ß imaging in mouse model of Alzheimer's disease.

Non-invasive determination of amyloid-ß peptide (Aß) deposition has important significance for early diagnosis and medical intervention in Alzheimer's disease (AD). In this study, we investigated the availability of a radioiodinated imidazo[1,2-a]pyridine derivative, termed (125)I-DRK092, as single photon emission computed tomography (SPECT) ligand for in vivo detection of Aß deposition. DRK092 showed high binding affinity for either synthetic human Aß fibrils or brain homogenates from amyloid precursor protein transgenic (Tg) mouse (PS1-ki/JU-Tg2576) and AD patient with a dissociation constant (Kd) of one-digit nM, and excellent brain permeability (peak value of uptake: approximately 0.9% of injection dose/g rat brain). Ex vivo autoradiographic analysis showed that measurement with (125)I-DRK092 has higher sensibility for detecting Aß accumulation than with (125)I-IMPY, a well-known amyloid SPECT ligand, in Tg mice. In vitro autoradiography with (125)I-DRK092 also confirmed higher accumulation of radioactivity in the cortical area, enriched with Aß plaques, of Tg mouse and AD patient brains, as compared with the corresponding areas in non-Tg mouse and healthy control brains. All the data presented above lead us to draw the conclusion that radioiodinated DRK092 is a potential SPECT ligand for amyloid imaging in AD.

Synthesis and biological evaluation of novel radioiodinated imidazopyridine derivatives for amyloid-ß imaging in Alzheimer's disease.

Non-invasive detection for amyloid-ß peptide (Aß) deposition has important significance for the early diagnosis and medical intervention for Alzheimer's disease (AD). In this study, we developed a series of imidazopyridine derivatives as potential imaging agents for single-photon emission computed tomography (SPECT). Two of them, compounds DRK092 and DRM106, showed higher affinity for synthetic human Aß 1-40 fibrils than did the well-known amyloid-imaging agent IMPY. A metabolite analysis revealed brain-permeable radioactive metabolites of (125)I-labeled DRK092 and IMPY; no radioactive metabolites from (125)I-labeled DRM106 ([(125)I]DRM106) were detected. In addition, in vitro autoradiography clearly demonstrated specific binding of [(125)I]DRM106 in the hippocampal region of AD enriched with Aß plaques. Thus, our results strongly suggested that compound DRM106 can be used as an imaging agent for SPECT to detect Aß deposition in AD brain.