Flow cytometry analysis of murine hematopoietic stem cells

Abstract

Hematopoietic stem cells (HSCs) remain the most well-characterized adult stem cell population both in terms of markers for purification and assays to assess functional potential. However, despite over 40 years of research, working with HSCs in the mouse remains challenging because of the relative abundance (or lack thereof) of these cells in the bone marrow. The frequency of HSCs in murine bone marrow is about 0.01% of total nucleated cells and ∼5,000 can be isolated from an individual mouse depending on the age, sex, and strain of mice as well as purification scheme utilized. Adding to the challenge is the continual reporting of new markers for HSC purification, which makes it difficult for the uninitiated in the field to know which purification strategies yield the highest proportion of long-term, multilineage HSCs. In this updated version of our review from 2009, we review different strategies for hematopoietic stem and progenitor cell identification and purification. We will also discuss methods for rapid flow cytometric analysis of peripheral blood cell types, and novel strategies for working with rare cell populations such as HSCs in the analysis of cell cycle status by BrdU, Ki-67, and Pyronin Y staining. The purpose of updating this review is to provide insight into some of the recent experimental and technical advances in mouse hematopoietic stem cell biology.

Figure 1

Side population staining to identify HSCs. Hoechst-stained cells are displayed for red and blue fluorescence simultaneously, and SP cells are identified as a small population of cells (~0.02% of WT 4-month old C57Bl/6 whole mouse bone marrow) off to the side, as indicated by the gate. These cells can be gated to the lineage profile, where most of them will be negative if SP staining is done properly and the cytometer is properly adjusted. This SP and Lineage-negative gate is then displayed for c-Kit and Sca-1 staining, where most of the cells should appear positive. These SP-KLS cells will then be largely CD34^−/low, Flk-2⁻, and CD48⁻, but heterogeneous for CD150 staining. With aging (24 months), the frequency of SP cells steadily increases and the percentage of CD150+ cells also increases among the SP^KSL population.

Figure 2

Comparison of different HSC isolation schemes demonstrating their highly overlapping nature. (A) The standard SP gating scheme with sequential gates applied. About 40% of the SP-KSL cells are CD150+. (B) Starting with whole bone marrow, around 8% of whole bone marrow is considered “lineage-marker negative (Lin⁻)”. When these Lin⁻ cells are displayed for c-Kit and Sca-1, about 4-5% are double positive. This is the so-called “KSL” population that is enriched for stem and progenitor cells. About 7% of these KSL cells are CD150+ and CD48⁻ (hallmarks of the long-term HSC), but almost all of those cells are SP cells. (C) Starting again from the KSL scheme, around 10% of KSL are Flk2-negative and CD34-negative (hallmarks of the long-term HSC). Again, almost all of these cells display the SP phenptype.

Figure 3

Isolation of fetal liver HSCs. Lineage depleted (not including Mac1) fetal liver cells that have been gated through FSC/SSC and PI for live-dead discrimination are then gated on a lineage plot to exclude any remaining lineage positive cells, then to a c-Kit vs. Sca-1 plot with the double positives being further gated for CD48-/CD150+, and finally EPCR+.

Figure 4

Separation of hematopoietic progenitor populations by flow cytometry. (A) Gating scheme for the LT-HSC, ST-HSC and multipotent progenitors (MPP) (B) Gating scheme for the common myeloid progenitor (CMP), megakaryocyte-erythrocyte progenitors (MEPs) and granulocyte-macrophage progenitors (GMPs). (C) Gating scheme for the common lymphoid progenitor (CLP). (D) Gating scheme for lymphoid myeloid progenitors (LMPP).

Figure 5

Rapid flow cytometric analysis of peripheral blood of mice transplanted with HSCs transduced with MSCV-GFP retrovirus. For this analysis, red blood cells are depleted or lysed, and the remainder gated out on a FSC/SSC plot. Then viable white blood cells are gated and displayed on a CD45.2-APC versus GFP dot-plot. Progeny of donor HSC (CD45.2) can be discriminated from recipient cells (CD45.1) by CD45 alleles, and the donor HSCs that were successfully transduced to over-express a test gene are GFP⁺. The CD45.2⁺ / GFP⁺ population can then be gated to a PeCy7 versus Pacific Blue dot-plot to analyze distribution of the blood lineages. Myeloid cells are labeled with Gr1 and Mac-1 conjugated to PeCy7. T-cells are labeled with CD4 and CD8 antibodies conjugated to Pacific Blue (Pac-Blue). By labeling B cells with both B220-PeCy7 and B220-Pac-Blue, all major hematopoietic lineages can be displayed simultaneously on the same plot. The B cells are the double positive population, the myeloid cells (Gr-1⁺, Mac-1⁺) are the PeCy7⁺PacBlue⁻ population, while the T cells (CD4⁺, CD8⁺) are the PeCy7⁻ PacBlue⁺ population. If we were analyzing transplanted mice in which the GFP was not used to follow retroviral marking, we typically use CD45.1-FITC to track the recipient cells in addition to the donor cells.

Figure 6

Analysis of HSCs turnover by BrdU labeling using flow cytometry. (A) HSCs from mice injected with BrdU are purified from Sca-1-enriched bone marrow (increasing the proportion of SP cells 10-fold) and then fixed and permeabilized overnight. (B) Reanalysis of the sorted cells following intracellular staining for BrdU. A PE versus PeCy5 dot-plot allows for discrimination between sorted HSCs and carrier B cells (the majority of carrier cells are lined up against the x-axis). The HSCs can then be gated to either a stem cells marker versus BrdU dot-plot or to a histogram to determine BrdU incorporation.

Figure 7

Cell cycle reanalysis of purified SP^KLS cells by Ki-67 and Pyronin Y. (A) Using the carrier cell technique, SP^KLS cells were purified then fixed and permeabilized for Ki-67 staining. On the reanalysis, HSCs (Sca-1⁺B220⁻) were easily identified from carrier cells (Sca-1⁻B220⁺) and gated to show a distribution of Ki-67 versus DNA content using propidium iodide. This plot allows discrimination of the various stages of cell cycle of the HSC population. (B) Analysis of HSC cell cycle status by Pyronin Y staining. As above, SP^KLS were sorted into carrier cells and then both populations were stained for Pyronin Y analysis. On reanalysis, HSCs are gated away from carrier cells to a Hoechst versus Pyronin Y plot which shows the different stages of cell cycle. The use of this assay is clearly demonstrated when comparing normal HSCs to those which have been stimulated with the chemotherapeutic agent 5-flurouracil (5-FU) which brings them out of quiescence and into cell activation programs. In normal HSCs, the vast majority are resting in the G0 stage, while six days after 5-FU stimulation a much higher proportion are actively engaged in the cell cycle in S/G2-M.