Efficient discrimination of functional hematopoietic stem cell progenitors for transplantation by combining alkaline phosphatase activity and CD34⁺ immunophenotyping.

4区生物学 Q4 Biochemistry, Genetics and Molecular Biology

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2023-09-20 DOI:10.1016/bs.mcb.2023.08.003

Laura G Rico, Jordi Juncà, Roser Salvia, Michael D Ward, Jolene A Bradford, Jordi Petriz

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引用次数: 0

Abstract

Alkaline phosphatase (ALP) is a membrane-associated hydrolase enzyme with dimeric structure that catalyzes phosphate esters, optimally at alkaline pH. ALP has a focus of interest, since this enzyme is highly expressed in primitive stem cells, such as progenitor cells, non-differentiating cells, and primordial cells. We previously adapted a fluorescent microscopy-based assay for quantifying ALP^high and ALP^low cells by flow cytometry in combination with immunophenotyping. Our method uses a minimal sample perturbation approach, avoiding the use of erythrocyte lysing solutions and washing steps, and offering opportunities to combine live cell response and functional assessment with cell immunophenotyping, while minimizing sample preparation effects on the cell biology. Here we provide a detailed experiment protocol to determine alkaline phosphatase activity in CD34⁺ hematopoietic stem cells from blood and apheresis products obtained from patients involved in a stem cell mobilization process for allo- or auto-transplant. This study may provide the early detection of progenitors at different levels of differentiation and therefore, relate this information to long-term engraftment in hematopoietic stem cell transplants.

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碱性磷酸酶（ALP）是一种膜相关水解酶，具有二聚体结构，能催化磷酸酯，在碱性pH值下发挥最佳作用。由于 ALP 在原始干细胞（如祖细胞、未分化细胞和原始细胞）中高表达，因此成为人们关注的焦点。我们之前改良了一种基于荧光显微镜的检测方法，通过流式细胞术结合免疫分型来量化ALPhigh和ALPlow细胞。我们的方法采用最小样本扰动法，避免了红细胞裂解液的使用和洗涤步骤，提供了将活细胞反应和功能评估与细胞免疫分型相结合的机会，同时将样本制备对细胞生物学的影响降至最低。在此，我们提供了一个详细的实验方案，用于测定从参与异体或自身移植干细胞动员过程的患者血液和血液制品中提取的 CD34+ 造血干细胞的碱性磷酸酶活性。这项研究可及早检测处于不同分化水平的祖细胞，从而将这一信息与造血干细胞移植的长期移植联系起来。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Methods in cell biology 生物-细胞生物学

CiteScore

3.10

自引率

0.00%

发文量

125

审稿时长

3 months

期刊介绍： For over fifty years, Methods in Cell Biology has helped researchers answer the question "What method should I use to study this cell biology problem?" Edited by leaders in the field, each thematic volume provides proven, state-of-art techniques, along with relevant historical background and theory, to aid researchers in efficient design and effective implementation of experimental methodologies. Over its many years of publication, Methods in Cell Biology has built up a deep library of biological methods to study model developmental organisms, organelles and cell systems, as well as comprehensive coverage of microscopy and other analytical approaches.