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. 2021 Sep 27;2(4):100846.
doi: 10.1016/j.xpro.2021.100846. eCollection 2021 Dec 17.

Isolation of human and murine hematopoietic stem cells for DNA damage and DNA repair assays

Alfredo Rodríguez  1   2 , Jessica Filiatrault  1 , Patricia Flores-Guzmán  3 , Héctor Mayani  3 , Kalindi Parmar  1 , Alan D D'Andrea  1
Affiliations

Isolation of human and murine hematopoietic stem cells for DNA damage and DNA repair assays

Alfredo Rodríguez et al. STAR Protoc. .

Abstract

Hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow and supply blood cells. Efficient methods for isolation of HSPCs are required. Here, we present protocols for the isolation of human and murine HSPCs using manual and FACS-assisted techniques. Isolated HSPCs can be used for downstream applications, including colony forming unit assays and DNA damage and repair assays. For complete details on the use and execution of this protocol, please refer to Rodríguez et al. (2021a) and (2021b).

Keywords: Cell Biology; Cell isolation; Cell-based Assays; Flow Cytometry/Mass Cytometry; Genetics; Microscopy; Stem Cells.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Strategies for enrichment of HSPC (A) Human Lin- cells can be enriched using a magnetic stand. Left. Cells are incubated with the progenitor cell enrichment cocktail followed by incubation with magnetic particles and incubation in the magnetic stand. Right. Lin+ cells bound by the antibodies of the enrichment cocktail and by the magnetic particles are attracted to the tube wall by the magnetic field. Lin- cells remain in suspension and can be recovered by simply pouring off the liquid into a new tube. The Lin+ cells remain in the original tube. (B) Mouse and human HSPCs can be enriched using columns for magnetic cell separation. Cells are incubated with cell enrichment cocktail followed by incubation with magnetic beads. Left. Enrichment of mouse Lin- cells is performed with the LS Miltenyi columns; enrichment of human CD34+ cells is performed with the MS Miltenyi columns. The column is mounted into the MidiMACS separator on the MACS MultiStand and prepared by adding column buffer. Center. The cell suspension is added to the top of the column. Right. The cell suspension is left to flow through the column via gravity flow. Cells bound by the magnetic particles remain bound to the column in the magnetic field, whereas unbound cells are recovered into a clean tube. (C) Mouse LT-HSCs can be purified from the enriched population of Lin- cells using FACS and a 5-flurochrome strategy based on the SLAM markers. Left. Detection of Lin- cells. Center. LSK cells within the Lin- population are recognized by being c-Kit+ Sca+. Right. LT-HSCs within the LSK population are recognized by being CD150+ CD48-
Figure 2
Figure 2
Types of hematopoietic colonies recognized in the CFU assay (A) Hematopoietic cells from human bone marrow were cultured in methylcellulose semisolid medium supplemented with recombinant hematopoietic cytokines and cultured for 14 days at 37°C and 5% CO2. Image shown corresponds to a CFU assay as seen in the STEMvision system. Left panel: negative image. Right panel: standard image. (B) Representative hematopoietic colonies from human bone marrow scored at day 14 of culture (10×). (C) Morphological appearance of a CFU-GEMM. Left panel: 10×. Right panel: 100×. Scale bar represents the size of the colonies in mm.
Figure 3
Figure 3
Immunofluorescence for γH2AX foci and the comet assay can be used for DNA damage assessment in HSPCs (A) Representative pictures of an immunofluorescence for detection of γH2AX foci in sorted mouse LT-HSCs treated in vitro with 5 Gy of IR. Green arrowhead points a cell negative for γH2AX foci, pink arrowhead points a cell with multiple γH2AX foci. Scale bar represents 20 μm. (B) Picture demonstrating the correct alignment of the comet assay slides with respect to the black cathode of the electrophoretic chamber. (C) Representative pictures of a comet assay in sorted mouse LT-HSCs untreated (left) or treated in vitro with 5 Gy of IR (right). Scale bar represents 20 μm.
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