|
Author:
Date:
2018-05-05
[Abstract] Bone Marrow Hematopoietic Stem Cells (HSCs) require bone marrow microenvironment for their maintenance and proliferation. Culture of Bone Marrow Mesenchymal Stromal Cells (MSCs) provides appropriate environmental signals for HSCs survival in vitro. Here, we provide a detailed protocol that describes culture conditions for MSCs, flow cytometric isolation of HSCs from mouse bone marrow, and perform co-culture of MSCs and HSCs known as Cobblestone area-forming cell (CAFC) assay. Altogether, CAFC assays can be used as a high-throughput in vitro screening model where efforts are made to understand and develop therapies for complex bone marrow diseases. This protocol needs 3 to 4 weeks starting from culturing MSCs, isolating LSK cells (HSCs), and to performing limited dilution ...
[摘要] 骨髓造血干细胞（HSC）需要骨髓微环境来维持和增殖。 骨髓间充质基质细胞（MSC）的培养为体外HSC存活提供适当的环境信号。 在这里，我们提供了描述MSCs培养条件的详细方案，从小鼠骨髓中流式细胞术分离HSCs，并进行称为鹅卵石区域形成细胞（CAFC）分析的MSC和HSC的共培养。 总而言之，CAFC分析可用作高通量体外筛选模型，其中努力了解和开发复杂骨髓疾病的治疗方法。 该方案需要培养MSC，分离LSK细胞（HSC）和执行有限稀释CAFC测定3至4周。
【背景】HSC的增殖，存活和分化潜力非常依赖于其微环境，也被称为小生境。骨髓MSC支持HSC以使其在骨髓龛中保持静止状态。由生态位接收的内在和外在信号有助于将HSC分化为也称为造血的成熟血细胞谱系，而不诱导异常扩增（Yoshihara等人，2007; Spindler等人 ，2014; Hu等人，2016）。鹅卵石区域形成细胞试验（CAFC试验）是长期骨髓HSC和MSC的体外共培养试验。当培养MSC在组织培养皿中完成融合时，将HSC铺在MSC上（de Haan和Ploemacher，2002）。 CAFC测定与骨髓的体内研究相当，并且可用作快速筛选测定以测试HSC的干细胞活性和MSC的支持活性（Ploemacher等人， ...
|