In vitro Differentiation of Human iPSC-derived Cardiovascular Progenitor Cells (iPSC-CVPCs)
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Author:
Date:
2020-09-20
[Abstract] Induced pluripotent stem cell derived cardiovascular progenitor cells (iPSC-CVPCs) provide an unprecedented platform for examining the molecular underpinnings of cardiac development and disease etiology, but also have great potential to play pivotal roles in the future of regenerative medicine and pharmacogenomic studies. Biobanks like iPSCORE ( Stacey et al., 2013; Panopoulos et al., 2017), which contain iPSCs generated from hundreds of genetically and ethnically diverse individuals, are an invaluable resource for conducting these studies. Here, we present an optimized, cost-effective and highly standardized protocol for large-scale derivation of human iPSC-CVPCs using small molecules and purification using metabolic selection. We have successfully applied this protocol ...
[摘要] [摘要 ] 诱导性多能干细胞衍生的心血管祖细胞(iPSC-CVPCs)为检查心脏发育和疾病病因的分子基础提供了前所未有的平台,但在再生医学和药物基因组学的未来中也具有重要作用。像iPSCORE这样的生物库(Stacey 等,2013 ;Panopoulos 等,2017), 其中包含由数百个遗传和种族不同的个体产生的iPSC,是进行这些研究的宝贵资源。在这里,我们为小分子大规模衍生人iPSC-CVPCs和代谢选择纯化提供了一种优化,具有成本效益和高度标准化的方案。我们已经成功地应用了该协议,从154种不同的iPSCORE iPSC品系中获得了iPSC-CVPC,从而获得了大量的高纯度心脏细胞。一个重要的我们的协议的组成部分是Ç ELL Ç onfluency 估计S(ccEstimate ),用于估计当iPSC集单层将达到80%汇合,这是用于发起的iPSC-CVPC推导最佳的时间的自动方法,并且使得协议为易于在具有不同增长率的iPSC系列中使用。此外,我们发现跨iPSC-CVPC的细胞异质性是由于两种截然不同的心脏细胞类型(心肌细胞(CMs)和心外膜衍生细胞(EPDCs))的比例不同导致的,这两种细胞在心脏再生中均具有关键作用。该协议消除了iPSC线到线优化的需要,并且可以轻松地进行调整和扩展,以进行高通量研究或生成大量适用于再生医学应用的细胞。
[背景 ] ...
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Implantation of Human Peripheral Corneal Spheres into Cadaveric Human Corneal Tissue
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Author:
Date:
2017-07-20
[Abstract] Stem and progenitor cells isolated from human limbal tissue can be cultured in vitro as spheres. These spheres have potential for use as transplantable elements for the repopulation of corneal tissue (Mathan et al., 2016). Herein we describe the detailed protocol for the implantation of human corneal spheres into cadaveric human corneal tissue. This protocol describes the procedure for sphere formation and culture, preparation of tissue for sphere implantation, corneal limbus microsurgery and sphere implantation.
[摘要] 可以将从人角膜缘组织分离的茎细胞和祖细胞作为球体体外培养。 这些球体有可能用作角膜组织再造的可移植元件(Mathan等人,2016)。 这里我们描述了将人角膜球植入尸体人角膜组织的详细方案。 该协议描述了球形成和培养的过程,球体植入组织的制备,角膜缘显微外科手术和球体植入。 【背景】以前的研究集中在隔离角膜缘细胞,这些细胞完全是上皮细胞(角膜缘干细胞)或基质(角质细胞祖细胞),以破译他们在角膜内稳态和伤口修复中的各自角色。 该方案旨在通过其在培养物中形成球体的功能能力来分离角膜缘细胞,并且其本质将包括上皮和间质的多种细胞,其有助于角膜缘的形成。 在分离这些球体之后,我们正在调查其在植入后角膜修复中的潜在用途。 在这里,我们描述了一种用于将这些球体植入人角膜组织和下游分析的体外手术方案。
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Isolation and Flow-cytometric Analysis of Mouse Intestinal Crypt Cells
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Author:
Date:
2015-11-05
[Abstract] The intestinal epithelial layer forms tubular invaginations into the underlying connective tissue of the lamina propria. These structures, termed crypts, are the basic functional unit of the intestine. Colon crypts and the surrounding lamina propria house different cell types, including epithelial cells, stem cells, enterocytes, goblet cells, as well as cells of the innate and adaptive immune systems (Clevers, 2013; Mowat and Agace, 2014). Here we describe a technique for the isolation of mouse intestinal crypt cells as well as their characterization by flow cytometry analysis (FACS) (Del Reino et al., 2012).
[摘要] 肠上皮层形成管状内陷到固有层的下面的结缔组织。 这些结构,称为隐窝,是肠的基本功能单位。 结肠隐窝和周围固有层存在不同的细胞类型,包括上皮细胞,干细胞,肠细胞,杯状细胞,以及先天和适应性免疫系统的细胞(Clevers,2013; Mowat和Agace,2014)。 在这里我们描述了用于分离小鼠肠道隐窝细胞的技术以及它们通过流式细胞术分析(FACS)的表征(Del Reino等人,2012)。
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