Abstract

Mass cytometry is a recently introduced technology that utilizes transition element isotope-tagged antibodies for protein detection on a single-cell basis. By circumventing the limitations of emission spectral overlap associated with fluorochromes utilized in traditional flow cytometry, mass cytometry currently allows measurement of up to 40 parameters per cell. Recently, a comprehensive mass cytometry analysis was described for the hematopoietic differentiation program in human bone marrow from a healthy donor. The present study describes approaches to delineate cell cycle stages utilizing 5-iodo-2- deoxyuridine (IdU) to mark cells in S phase, simultaneously with antibodies against cyclin B1, cyclin A, and phosphorylated histone H3 (S28) that characterize the other cell cycle phases. Protocols were developed in which an antibody against phosphorylated retinoblastoma protein (Rb) at serines 807 and 811 was used to separate cells in G0 and G1 phases of the cell cycle. This mass cytometry method yielded cell cycle distributions of both normal and cancer cell populations that were equivalent to those obtained by traditional fluorescence cytometry techniques. We applied this to map the cell cycle phases of cells spanning the hematopoietic hierarchy in healthy human bone marrow as a prelude to later studies with cancers and other disorders of this lineage.

Experiment Overview

Purpose:
The goal of this study was to develop a methodology to allow measurement of cell cycle state without the need for fluorescent dyes traditionally used in fluorescent flow cytometry.
Surface and intracellular markers measured
Mass cytometry: CD3, CD45, CD45RA, CD133, CD19, CD71, CD11b, CD4, CD8, CD20, CD34, CD90, CD117, CD123, CD235, HLA-DR, Cyclin A, Cyclin B1, CD33, CD38, CD14, CD7, CD15, p-pRb (S807/811), Cyclin D1/2/3, Ki-67, CD13, p-CDK1(Y15), CD56, cleaved-PARP(D214), p-S6(S235/36), CD10, CD16, p-Histone H3(S28)
Fluorescent cytometry: CD3, p-pRb(S807/811), BrdU, Cyclin B1, pHH3(S28)
Cytometer Used:
DVS Sciences, Inc. CyTOF™ Mass Cytometer BD Biosciences, Inc. LSRII Cytometer

Figure 1A: IdU incorporation identifies S phase cells

Mass cytometric measurement of IdU incorporation specifically identifies S phase cells

Figure 1A: U937 cells were incubated with 10 μM IdU for the indicated times, fixed, and analyzed by mass cytometry; S-phase gate is indicated. Cells not incubated with IdU have no significant iodine signal.

Figure 1B: IdU incorporation identifies S phase cells

Mass cytometric measurement of IdU incorporation specifically identifies S phase cells

Figure 1B: Treatment with 1.5 μM hydroxyurea for 22 hours blocked IdU incorporation; IdU incorporation was restored 24 hours after release from the hydroxyurea block.

Figure 2 A-C: Phospho-Rb discriminates G0 from G1

Measurement of phosphorylated Rb on serines 807 and 811 discriminates of G0 from G1 cells by fluorescent cytometry.

Figure 2A: Identification of G0 and G1 cells in stimulated T cells using Hoechst 3342 and pyronin Y staining (left) and pyronin Y staining vs. antibody detection of IdU incorporation (right).
Figure 2B: The p-Rb(S807/811) staining pattern is similar to that of pyronin Y and defines similar G0 and G1 populations.
Figure 2C: A plot of Hoechst 3342 vs. pyronin Y colored for staining by p-Rb(S807/811), Alexa 647.

Figure 2D: Phospho-Rb discriminates G0 from G1

Measurement of phosphorylated Rb on serines 807 and 811 discriminates of G0 from G1 cells by mass cytometry.

Figure 2D: Analysis of the same cell sample by mass cytometry using p-Rb(S807/811) labeled on Ho165 vs. iridium intercalator (left) or IdU incorporation

Figure 3: Total cyclin levels

Total cyclin levels can be detected by mass cytometry and used to sub-divide the cell cycle

Cyclin A, and cyclin B1 were measured relative to Iridium intercalator (Figure 3A) or IdU incorporation (Figure 3B).

Figure 4 - Human T Cells: Method for analysis of all phases

Cell cycle analysis by mass cytometry yields results equivalent to fluorescence cytometry methods

Figure 4A: A plot of IdU vs. p-Rb(S807/811) allows for gating of G0 and G1 phase populations.
Figure 4B: A plot of IdU incorporation vs. cyclin B1 allows gating of G1, S, and G2/M populations as shown.
Figure 4C: p-HH3(S28) defines an M-phase population.

Figure 4 - U937 Cells: Method for analysis of all phases

Cell cycle analysis by mass cytometry yields results equivalent to fluorescence cytometry methods

Figure 4A: A plot of IdU vs. p-Rb(S807/811) allows for gating of G0 and G1 phase populations.
Figure 4B: A plot of IdU incorporation vs. cyclin B1 allows gating of G1, S, and G2/M populations as shown.
Figure 4C: p-HH3(S28) defines an M-phase population.

Figure 4 - HL60 Cells: Method for analysis of all phases

Cell cycle analysis by mass cytometry yields results equivalent to fluorescence cytometry methods

Figure 4A: A plot of IdU vs. p-Rb(S807/811) allows for gating of G0 and G1 phase populations.
Figure 4B: A plot of IdU incorporation vs. cyclin B1 allows gating of G1, S, and G2/M populations as shown.
Figure 4C: p-HH3(S28) defines an M-phase population.

Figure 4 - NALM6 Cells: Method for analysis of all phases

Cell cycle analysis by mass cytometry yields results equivalent to fluorescence cytometry methods

Figure 4A: A plot of IdU vs. p-Rb(S807/811) allows for gating of G0 and G1 phase populations.
Figure 4B: A plot of IdU incorporation vs. cyclin B1 allows gating of G1, S, and G2/M populations as shown.
Figure 4C: p-HH3(S28) defines an M-phase population.

Figure 5A: Validation with stimulated T cells

Figure 5A: Fluorescent cytometry cell cycle assessment. Left, measurement of cells in G0/G1, S, and G2/M using Hoechst and anti-IdU antibody (detected by FITC secondary antibody) at indicated timepoints following PMA and ionomycin stimulation. Right, discrimination of G0 and G1 cells using Hoechst and pyronin Y.

Figure 5B: Validation with stimulated T cells

Validation of mass cytometric cell cycle measurement during stimulation of human peripheral blood T cells by PMA and ionomycin

Figure 5B: Mass cytometry cell cycle assessment. Left, measurement of cells in G0/G1, S, and G2/M using cyclin B and direct IdU measurement at indicated timepoints following stimulation. Right, discrimination of G0 and G1 cells using p-Rb(S807/811) and IdU detection.

Figure 6: Immunophenotype of normal marrow

Immunophenotypic analysis of healthy human bone marrow

Figure 6: A minimum spanning tree was constructed using SPADE analysis based on 25 cell surface markers. The size of each circle in the tree approximates the relative frequency of cells that fall within boundaries of surface marker expression that define each node. Node color is scaled to the median intensity of expression of the indicated markers. Putative cell populations were annotated manually based on previous studies. Eight of the SPADE tree clusters could not be definitively identified on the basis of the surface markers present in this antibody panel. (Minimum and maximum node size was constrained to allow visualization of marker intensity.

Figure 7A: Cell cycle across hematopoiesis

Mass cytometric measurement of the cell cycle distribution across normal human hematopoiesis

Figure 7A: Cell cycle distribution across B cell development. Left, “branch” of B cell development from hematopoietic tree shown in Figure 6. Center, population annotations and expression levels of population defining surface markers for the B cell “branch” (node color and node sizing scaling as described in Figure 6). Right, distribution of cells in each phase of the cell cycle in the B cell “branch” of the tree. The number of cells within each node is indicated by the size of the gray circle, while the number of cells in the labeled cell cycle phase is indicated by the size of the red circle. In the left column, the size of each circle is directly correlated with the number of cells within each node and there is no maximum or minimum size constraint. The red circle size is scaled such that a completely filled circle represents 67% of cells within the node in the indicated cell cycle phase.

Figure 7B: Cell cycle across hematopoiesis

Mass cytometric measurement of the cell cycle distribution across normal human hematopoiesis

Figure 7B: Cell cycle distribution across erythroid development. Left, “branch” of erythroid development from hematopoietic tree shown in Figure 6. Center, population annotations and expression levels of population defining surface markers for the erythroid “branch” (node color and node sizing scaling as described in Figure 6). Right, distribution of cells in each phase of the cell cycle in the erythroid “branch” of the tree. Node sizing as described in (A).

Figure 7C: Cell cycle across hematopoiesis

Mass cytometric measurement of the cell cycle distribution across normal human hematopoiesis

Figure 7C: Cell cycle distribution across granulocytic development. Left, “branch” of granulocytic development from hematopoietic tree shown in Figure 6. Center, population annotations and expression levels of population defining surface markers for the myeloid “branch” (node color and node sizing scaling as described in Figure 6). Right, distribution of cells in each phase of the cell cycle in the granulocytic “branch” of the tree. Node sizing as described in (A).

Figure 7D: Cell cycle across hematopoiesis

Mass cytometric measurement of the cell cycle distribution across normal human hematopoiesis

Figure 7D: Cell cycle distribution across monocyte development. Left, “branch” of monocyte development from hematopoietic tree shown in Figure 6. Center, population annotations and expression levels of population defining surface markers for the monocyte “branch” (node color and node sizing scaling as described in Figure 6). Right, distribution of cells in each phase of the cell cycle in the monocyte “branch” of the tree. Node sizing as described in (A).

Figure 7E: Cell cycle across hematopoiesis

Mass cytometric measurement of the cell cycle distribution across normal human hematopoiesis

Figure 7E: The amount of IdU incorporation varies across S phase cells of different developmental lineages. IdU signal versus cyclin B1 signal in the indicated cell subpopulations (as described in Figures 6).

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