| Generation of Human iPSC-derived Neural Progenitor Cells (NPCs) as Drug Discovery Model for Neurological and Mitochondrial Disorders
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Author:
Date:
2021-03-05
[Abstract] The high attrition rate in drug development processes calls for additional human-based model systems. However, in the context of brain disorders, sampling live neuronal cells for compound testing is not applicable. The use of human induced pluripotent stem cells (iPSCs) has revolutionized the field of neuronal disease modeling and drug discovery. Thanks to the development of iPSC-based neuronal differentiation protocols, including tridimensional cerebral organoids, it is now possible to molecularly dissect human neuronal development and human brain disease pathogenesis in a dish. These approaches may allow dissecting patient-specific treatment efficacy in a disease-relevant cellular context. For drug discovery approaches, however, a highly reproducible and cost-effective cell model is ...
[摘要] [摘要]药物开发过程中的高流失率要求使用其他基于人的模型系统。但是，在脑部疾病的情况下，不适合对活的神经元细胞进行采样以进行化合物测试。人类诱导的多能干细胞（iPSC ）的使用彻底改变了神经元疾病建模和药物发现领域。由于基于iPSC的神经元分化方案（包括三维脑类器官）的发展，现在可以在一个碟子中分子解剖人神经元发育和人脑疾病的发病机理。这些方法可以允许在与疾病相关的细胞环境中解剖患者特异性的治疗功效。但是，对于药物发现方法，需要高度可复制且具有成本效益的细胞模型。在这里，我们描述了一种一步-步骤，用于从人产生健壮和可膨胀的神经祖细胞（NPC）工艺的iPSC 。用此协议生成的NPC是同质的且高度增殖。这些功能使NPC适合开发用于药物发现的高通量化合物筛选。人iPSC衍生的NPC示出了代谢依赖于线粒体活性，因此可也用于研究神经病症，其中线粒体功能受到影响。该协议涵盖了制备，培养和表征人iPSC来源的NPC所需的所有步骤。
图形摘要：
示意性的协议的所述发电机密封的离子人类源自iPSC的的NPC
[背景技术]近年来，目标为中心的药物发现的缺点已经用于寻址的神经系统疾病的方案变得明显，特别是（保罗等人，2010） ...
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| Double Labeling of PDGFR-β and α-SMA in Swine Models of Acute Kidney Injury to Detect Pericyte-to-Myofibroblast Transdifferentation as Early Marker of Fibrosis
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Author:
Date:
2020-10-05
[Abstract] Growing evidences suggest that peritubular capillaries pericytes are the main source of scar-forming myofibroblasts during chronic kidney disease (CKD), as well as early phases of acute kidney injury (AKI). In a swine model of sepsis and I/R (Ischemia Reperfusion) injury-induced AKI we demonstrated that renal pericytes are able to transdifferentiate toward α-SMA+ myofibroblasts leading to interstitial fibrosis. Even if precise pericytes identification requires transmission electron microscopy and the co-immunostaining of several markers (i.e., Gli, NG2 chondroitin sulphate proteoglycan, CD146, desmin or CD73) and emerging new markers (CD248 or TEM1, endosialin), previous studies suggested that PDGFR-β could be used as marker for renal pericytes characterization. ...
[摘要] [摘要]越来越多的证据表明，肾小管周围的毛细血管周细胞是慢性肾脏病（CKD）以及急性肾损伤（AKI）早期形成疤痕的成纤维细胞的主要来源。在败血症和I / R（缺血再灌注）损伤诱导的AKI的猪模型中，我们证明了肾周细胞能够向α- SMA +肌成纤维细胞转分化，从而导致间质纤维化。即使精确周细胞识别需要透射电子显微镜和几个标志物联合免疫（即。，的Gli ，NG2硫酸软骨素蛋白聚糖，CD146，结蛋白或CD73）和新兴的新的标志物（CD248或TEM1，唾液酸蛋白），以往的研究表明，PDGFR-β可用作肾周细胞表征的标志物。最近，对PDGFR-β和α-SMA进行了双重免疫荧光染色，以鉴定在纤维化发展的早期受损激活的周细胞（PDGFR-β + /α-SMA +细胞）。我们的数据强调了肾周细胞在败血症和I / R相关性AKI的生理病理中的关键作用。在该协议中，我们描述了猪福尔马林固定石蜡包埋（FFPE）肾脏活检中PDGFR-β和α-SMA双重免疫荧光染色的程序以及图像分析和定量方法。
[背景】肾脏纤维化被认为是主要负责肾脏疾病的进展，其与肾的损伤后的容量有限，再生有关。进行性肾脏疾病中间质纤维化的主要来源（Simone等人，2014 ; ...
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| GC/MS-based Analysis of Volatile Metabolic Profile Along in vitro Differentiation of Human Induced Pluripotent Stem Cells
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Author:
Date:
2017-12-05
[Abstract] Human induced pluripotent stem cells (hiPSCs) are a promising tool in cell-based therapies for degenerative diseases. A safe application of hiPSCs in vivo, requires the detection of the presence of residual undifferentiated pluripotent cells that can potentially cause the insurgence of teratomas. Several studies point out that metabolic products may provide an alternative method to identify the different steps of cells differentiation. In particular, the analysis of volatile organic compounds (VOCs) is gaining a growing interest in this context, thanks to its inherent noninvasiveness. Here, a protocol for VOCs analysis from human induced pluripotent stem cells (hiPSCs) is illustrated. It is based on Solid-Phase Microextraction (SPME) technique coupled with gas chromatography-mass ...
[摘要] 人诱导的多能干细胞（hiPSC）是用于退化性疾病的基于细胞的疗法中的有前景的工具。 hiPSCs在体内的安全应用需要检测残留未分化多能细胞的存在，这可能会导致畸胎瘤的爆发。几项研究指出，代谢产物可能提供了另一种方法来确定细胞分化的不同步骤。特别是挥发性有机化合物（VOCs）的分析由于其固有的非侵入性而在这方面越来越受到关注。在这里，说明了从人诱导的多能干细胞（hiPSC）分析VOC的方案。它基于固相微萃取（SPME）技术与气相色谱 - 质谱联用（GC / MS）。该方法用于测量从绒毛膜样品（CVS）到hiPSC的细胞重编程期间和沿着hiPSC体外分化成早期神经祖细胞（NP）的细胞顶空中的挥发性代谢物修饰，穿过胚状体机构（EBs）的形成。
【背景】提出细胞代谢作为在分化的各个步骤期间研究干细胞的替代物。事实上，假设干细胞从多能性向完全分化的转变可能引起代谢产物的剧烈变化是合理的。在诱导的多能干细胞，亲本成纤维细胞和胚胎干细胞之间观察到了这种假设的第一个证据（Meissen等人，2012）。
在代谢产物中，挥发性有机化合物（VOC）吸引了人们对其收集的简单性，内在的非侵入性和广泛的分析方法的广泛关注（Boots et。，2015 ...
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