Colony forming cell (CFC) assay for human hematopoietic cells.
Colony forming cell (CFC) assay for human hematopoietic cells.
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DOI：
10.3791/2195
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发表时间：
2010-12-18
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影响因子：
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通讯作者：
Yaseen, Nabeel R
中科院分区：
文献类型：
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作者：
Sarma, Nayan J;Takeda, Akiko;Yaseen, Nabeel R
人类造血干/祖细胞通常从骨髓、脐带血或外周血中获得，用于研究造血和白血病发生。它们具有分化成淋巴系和骨髓系的能力。集落形成细胞 (CFC) 测定用于通过造血祖细胞在半固体培养基中形成集落的能力来研究造血祖细胞的增殖和分化模式。由固定数量的输入细胞形成的集落的数量和形态提供了有关祖细胞分化和增殖能力的初步信息。可以从单个集落或整个板中收获细胞，以使用流式细胞术和吉姆萨染色载玻片的形态学评估进一步评估其数量和分化状态。该测定可用于评估骨髓分化，但不适用于评估淋巴分化。本文中的术语“骨髓”以其更广泛的含义使用，包括粒细胞、单核细胞、红细胞和巨核细胞谱系。我们使用该测定来评估癌基因对源自外周血的原代人 CD34+ 细胞分化的影响。为此目的，用对照逆转录病毒构建体或表达感兴趣的癌基因的构建体（在本例中为NUP98-HOXA9）转导细胞。我们采用常用的逆转录病毒载体 MSCV-IRES-GFP，它表达产生感兴趣基因和 GFP 标记的双顺反子 mRNA。在逆转录病毒转导之前，细胞在细胞因子存在下生长两天来预激活。再过两天后，通过荧光激活细胞分选术 (FACS) 分离 GFP+ 细胞，并将其与补充有细胞因子的含甲基纤维素的半固体培养基混合，并孵育直至集落出现在表面，通常需要 14 天。记录菌落的数量和形态。然后将细胞从板中取出、洗涤、计数并进行流式细胞术和形态学检查。使用针对造血过程中表达的细胞表面标记物的特异性抗体进行流式细胞术提供有关谱系和成熟阶段的信息。赖特-吉姆萨染色后在显微镜下对单个细胞进行形态学研究提供了有关谱系和成熟的进一步信息。用对照空载体转导的细胞与用癌基因转导的细胞的比较揭示了癌基因对造血分化的影响。
Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages. We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.