| 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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| Monocyte-MSC Co-cultures
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Author:
Date:
2015-01-20
[Abstract] To assess the effect of multipotent stromal cells (MSC) on monocytes, 3-day cultures were performed of freshly isolated monocytes in MSC-conditioned medium (CM). As a control condition, monocytes were stimulated with low dose macrophage colony-stimulating factor (M-CSF). Monocytes were isolated from peripheral blood mononuclear cell (PBMC) populations by magnetic activated cell sorting (MACS) using CD14 microbeads.
[摘要] 为了评估多潜能基质细胞（MSC）对单核细胞的作用，在MSC条件培养基（CM）中对新鲜分离的单核细胞进行3天培养。 作为对照条件，用低剂量巨噬细胞集落刺激因子（M-CSF）刺激单核细胞。 通过磁激活细胞分选（MACS）使用CD14微珠从外周血单核细胞（PBMC）群体中分离单核细胞。
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| Purification of Human Monocytes and Lymphocyte Populations by Counter Current Elutriation – A Short Protocol
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Author:
Date:
2013-12-05
[Abstract] Investigations of the activation processes involved in human monocytes and monocyte-derived macrophages and dendritic cells often required large numbers of cells that have not been possibly altered or activated by adherence to surfaces, by binding of antibodies to surface antigens during positive selection, or by release of activators by platelets or other non myeloid cells during isolation or co-culture. Human peripheral blood monocytes as well as lymphocytes from the same blood donor can be isolated by counterflow elutriation using a modification of the technique of Lionetti et al. (1980) as described previously (Bobak et al., 1986). From a unit of blood drawn into anticoagulant, 60-120 million monocytes can be obtained. These cells are not activated and have been ...
[摘要] 涉及人单核细胞和单核细胞衍生的巨噬细胞和树突细胞的激活过程的研究通常需要大量的细胞，这些细胞不可能通过附着于表面而改变或活化，通过在阳性选择期间抗体与表面抗原的结合，或通过释放 的血小板或其他非骨髓细胞在分离或共培养期间的活化剂。 人类外周血单核细胞以及来自相同血液供体的淋巴细胞可以通过逆流淘析使用Lionetti等人（1980）的技术的修改来分离，如前所述（Bobak等人 。，1986）。 从吸入抗凝血剂的单位，可以获得60-120,000个单核细胞。 这些细胞没有被活化并且已经显示在多个研究中适当地能够差别激活。
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