In Vivo Clonal Analysis of Aged Hematopoietic Stem Cells: Single-Cell Transplantation
Hematopoietic stem cells (HSCs) display heterogeneity in their characteristic features of undergoing self-renewal and multipotency within the blood system. While the same cell surface protein markers can be used to isolate HSCs from young and aged mice, r
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Introduction The production of blood throughout hematopoiesis is maintained by the unique ability of hematopoietic stem cells (HSCs) to undergo self-renewal and develop into all blood cell types. Despite their critical function in the hematopoietic system, HSCs are detected in less than 0.002% of the nucleated hematopoietic cell population in the bone marrow, where HSCs reside. Decades of extensive research have led to the identification of their distinct surface marker protein expression, which enables HSC isolation via flow cytometry techniques. Murine HSCs can be purified and are enriched within the CD150+ CD48 CD34 /low CD201+ c-Kit+ Sca-1+ lineage marker (KSL) population [1–3]. Upon isolation, transplantation experiments are conducted to assess the HSC potential of the transplanted cells. Though the same cell surface markers can be used to isolate young and aged murine HSCs, transplantation assays have revealed
Ce´sar Cobaleda and Isidro Sa´nchez-Garcı´a (eds.), Leukemia Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 2185, https://doi.org/10.1007/978-1-0716-0810-4_11, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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Kyomi J. Igarashi and Ryo Yamamoto
that aged HSCs have different functional capabilities. Recent studies have linked HSC aging to leukemia and cardiovascular disease [4, 5]. However, many of these observations have been made based on conventional transplantation assays, ignoring various issues that can complicate effective studies of aged HSC function. In this chapter, we incorporate two components that allow us to address these issues and gain a better understanding of aged HSC potential: (1) single-cell clonal assays and (2) a five-blood lineage tracing system. 1. Using Single-Cell Clonal Assays: Varying number of cells can be transplanted within a single mouse. However, cells within the designated HSC population are heterogeneous in HSC potential, suggesting that the greater number of cells transplanted, the more difficult it becomes to fully understand HSC function. Thus, investigating HSC multipotency requires transplantation studies at the single-cell level, also known as clonal assays. Unfortunately, these assays are rarely conducted owing to the experimental technicalities involved. 2. Using a Five-Blood Lineage Tracing System: The conventional Ly5-congenic transplantation system is often used to distinguish donor cells from recipient or competitor cells. The Ly5 antigen is expressed on all hematopoietic cells, with the exception of mature erythrocytes and platelets, complicating studies of HSC differentiation potential to these two lineages. However, we have previously developed a transgenic mouse line that can express the Kusabira-Orange (KuO) fluorescent protein in all blood cells, including platelets and erythrocytes [6]. We have also shown that this system successfully enables in vivo tracing of the five mature blood lineages, including neutrophils/monocytes, B lymphocytes, T lymphocytes, platelets, and erythrocytes [7, 8]. In
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