In Vitro Generation of Murine Dendritic Cells from Hoxb8-Immortalized Hematopoietic Progenitors.
In Vitro Generation of Murine Dendritic Cells from Hoxb8-Immortalized Hematopoietic Progenitors.
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从 Hoxb8 永生化造血祖细胞体外生成鼠树突状细胞。
DOI：
10.1007/978-1-0716-2938-3_7
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Häcker,Hans
中科院分区：
文献类型：
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作者：
Häcker,Hans
小鼠树突状细胞（DCs）通常是基于从骨髓（BM）中分离的细胞产生的，并在支持DC发育的生长因子存在下培养，如fms样酪氨酸激酶3配体（FLT3L）和粒细胞-巨噬细胞集落刺激因子（GM-CSF） （Guo等，J Immunol Methods 432:24 - 29, 2016）。在这些生长因子的作用下，DC祖细胞扩增和分化，而其他类型的细胞在体外培养期间死亡，最终导致相对同质的DC群体。本章将详细讨论另一种方法，即利用雌激素调节形式的Hoxb8 (ERHBD-Hoxb8)，在体外条件下使具有DC潜能的祖细胞永生化。这种祖细胞是通过用表达ERHBD-Hoxb8的逆转录病毒载体将大部分未分离的BM细胞转导而建立的。用雌激素处理表达erhbd -Hoxb8的祖细胞会导致Hoxb8活化，从而阻止细胞分化，并允许在FLT3L存在下同质祖细胞群的扩增。这些细胞，被称为Hoxb8-FL细胞，保留淋巴细胞和髓系的谱系潜力，包括DC谱系。在去除雌激素（Hoxb8失活）后，Hoxb8- fl细胞在GM-CSF或FLT3L存在下分化为高度同质的DC群体，类似于内源性的同类细胞。由于这些细胞具有无限的增殖能力和基因操作的适应性，例如，通过CRISPR/Cas9，这些细胞为研究DC生物学提供了大量的选择。在这里，我描述了从小鼠BM中建立Hoxb8-FL细胞的方法，以及使用慢病毒递送CRISPR/Cas9产生DC和基因缺失的程序。
Mouse dendritic cells (DCs) are routinely generated based on cells isolated form the bone marrow (BM) and cultured in the presence of growth factors that support DC development, such as FMS-like tyrosine kinase 3 ligand (FLT3L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Guo et al., J Immunol Methods 432:24–29, 2016). In response to these growth factors, DC progenitors expand and differentiate, while other cell types die during the in vitro culture period, ultimately leading to relatively homogenous DC populations. An alternative method, which is discussed in detail in this chapter, relies on conditional immortalization of progenitor cells with DC potential in vitro using an estrogen-regulated form of Hoxb8 (ERHBD-Hoxb8). Such progenitors are established by retroviral transduction of largely unseparated BM cells with a retroviral vector expressing ERHBD-Hoxb8. Treatment of ERHBD-Hoxb8-expressing progenitors with estrogen results in Hoxb8 activation, which blocks cell differentiation and allows for expansion of homogenous progenitor cell populations in the presence of FLT3L. These cells, referred to as Hoxb8-FL cells, retain lineage potential for lymphocyte and myeloid lineages, including the DC lineage. Upon removal of estrogen (inactivation of Hoxb8), Hoxb8-FL cells differentiate into highly homogenous DC populations in the presence of GM-CSF or FLT3L akin to their endogenous counterparts. Given their unlimited proliferative capacity and amenability for genetic manipulation, for example, by CRISPR/Cas9, these cells provide a large number of options to investigate DC biology. Here, I am describing the method to establish Hoxb8-FL cells from mouse BM, as well as procedures for DC generation and gene deletion using lentivirally delivered CRISPR/Cas9.