| Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs) into Osteoclasts
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Author:
Date:
2020-12-20
[Abstract] Defects in bone resorption by osteoclasts result in numerous rare genetic bone disorders as well as in some common diseases such as osteoporosis or osteopetrosis. The use of hiPSC-differentiated osteoclasts opens new avenues in this research field by providing an unlimited cell source and overcoming obstacles such as unavailability of human specimens and suitable animal models. Generation of hiPSCs is well established but efficient differentiation of hiPSCs into osteoclasts has been challenging. Published hiPSC-osteoclast differentiation protocols use a hiPSC-OP9 co-culture system or hiPSC-derived embryoid bodies (EBs) with multiple cytokines. Our three-stage protocol consists of 1) EB mesoderm differentiation, 2) expansion of myelomonocytic cells and 3) maturation of hiPSC-osteoclasts. ...
[摘要] [摘要]破骨细胞引起的骨吸收缺陷导致许多罕见的遗传性骨疾病以及某些常见的疾病，例如骨质疏松症或骨质疏松症。采用的hiPSC -分化破骨细胞通过提供无限的细胞来源和克服障碍，如人体标本和合适的动物模型的可用性打开了该领域的新途径。hiPSC的生成已被公认，但是将hiPSC高效分化为破骨细胞一直具有挑战性。发布的hiPSC -osteoclast分化协议使用的hiPSC-OP9共培养体系或hiPSC细胞来源的胚状 具有多种细胞因子的机体（EB）。我们的三阶段协议包含：1）中胚层EB分化，2）的扩张骨髓单核细胞和3）的成熟的hiPSC -osteoclasts。我们通过在Nunclon Sphera微孔板上培养Accutase分离的hiPSCs来产生大小均一的EB，并在4天的细胞因子混合物中促进EB中胚层分化。对于第2阶段，将EBs转移至明胶包被的平板中，并用hM -CSF和hIL-3培养，以扩增骨髓单核细胞群。通过与维生素d，补充hTGF β，HM -CSF和hRANKL ，在第2阶段结束时收集的细胞的diff erentiated成成熟破骨细胞（第3阶段）。与其他技术相比，我们的协议不需要共培养系统。诱导EBs分化为中胚层 均匀的方式; 使用较少的细胞因子进行分化；只需要很短的时间就可以使破骨细胞成熟，并产生足够数量的破骨细胞用于后续的分子分析。
图形摘要： ...
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| Differentiation of Human Induced Pluripotent Stem Cells (iPS Cells) and Embryonic Stem Cells (ES Cells) into Dendritic Cell (DC) Subsets
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Author:
Date:
2017-08-05
[Abstract] Induced pluripotent stem cells (iPS cells) are engineered stem cells, which exhibit properties very similar to embryonic stem cells (ES cells; Takahashi and Yamanaka, 2016). Both iPS cells and ES cells have an extraordinary self-renewal capacity and can differentiate into all cell types of our body, including hematopoietic stem/progenitor cells and dendritic cells (DC) derived thereof. This makes iPS cells particularly well suited for studying molecular mechanisms of diseases, drug discovery and regenerative therapy (Grskovic et al., 2011; Bellin et al., 2012; Robinton and Daley, 2012).
DC are the major antigen presenting cells of the immune system and thus they are key players in modulating and directing immune responses (Merad et al., 2013). DC ...
[摘要] 诱导的多能干细胞（iPS细胞）是工程干细胞，其表现出与胚胎干细胞（ES细胞，Takahashi和Yamanaka，2016）非常相似的性质。 iPS细胞和ES细胞都具有非凡的自我更新能力，可以分化成我们身体的所有细胞类型，包括造血干细胞/祖细胞和源自其的树突状细胞（DC）。这使得iPS细胞特别适用于研究疾病，药物发现和再生治疗的分子机制（Grskovic等人，2011; Bellin等人，2012; Robinton和Daley，2012）。
＆NBSP; DC是免疫系统的主要抗原呈递细胞，因此它们是调节和引导免疫应答的关键参与者（Merad等人，2013）。 DC巡逻外周和界面组织（例如，肺，肠和皮肤）以检测入侵的病原体，并且在激活时，它们迁移到淋巴结以激活和引发淋巴细胞。
＆NBSP; DC包含具有功能专门子集的表型异质家族（Schlitzer和Ginhoux，2014）。通常，经典DC（cDC）和浆细胞样DC（pDC）是分别表现出典型的和等离子体细胞样的DC形态。 cDC识别许多病原体并在激活后分泌促炎细胞因子，而pDC专门检测细胞内病原体并分泌I型干扰素（Merad等，2013; Schlitzer和Ginhoux，2014）。在被称为CD141 Clec9a + cDC1和CD1c + ...
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| Pit Assay to Measure the Bone Resorptive Activity of Bone Marrow-derived Osteoclasts
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Author:
Date:
2016-06-20
[Abstract] Although it is possible to use a tartrate-resistant acid phosphatase (TRAP) stain to assist in identifying osteoclasts, a separate method is needed to determine the bone resorption activity of osteoclasts. Since osteoclasts leave “pits” after bone matrix resorption (Charles et al., 2014), it is possible to stain pits as a method of measuring osteoclast bone resorption activity. The pit assay protocol enables researchers to stain bony slices that were co-cultured with osteoclasts with toluidine blue in order to allow the visualization, capture, and analysis of osteoclast resorptive activity based on the number, size and depth of pits (Zhou et al., 2015). The pit assay protocol is separated into three sequential stages: Preparation of bone slices (1); preparation of ...
[摘要] 尽管可以使用抗酒石酸盐的酸性磷酸酶（TRAP）染色来辅助鉴定破骨细胞，但是需要单独的方法来确定破骨细胞的骨吸收活性。 由于破骨细胞在骨基质吸收之后离开"凹陷"（Charles等人，2014），因此可以将斑点染色作为测量破骨细胞骨吸收活性的方法。 坑测定方案使研究人员能够将与破骨细胞与甲苯胺蓝共培养的骨切片染色，以便基于坑的数量，尺寸和深度可视化，捕获和分析破骨细胞再吸收活性（Zhou等人， et al。，2015）。 坑测定方案分为三个连续阶段：骨切片的制备（1）; 制备破骨细胞前体（Ross等人，2006; Teitelbaum等人，2000）（2）和骨吸收坑测定（3）。
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