Heterogeneity of non-cycling and cycling synchronized murine hematopoietic stem/progenitor cells

Abstract

Purified long-term multilineage repopulating marrow stem cells have been considered to be homogenous, but functionally these cells are heterogeneous. Many investigators urge clonal studies to define stem cells but, if stem cells are truly heterogeneous, clonal studies can only define heterogeneity. We have determined the colony growth and differentiation of individual lineage negative, rhodamine low, Hoechst low (LRH) stem cells at various times in cytokine culture, corresponding to specific cell cycle stages. These highly purified and cycle synchronized (98% in S phase at 40 h of culture) stem cells were exposed to two cytokine cocktails for 0, 18, 32, or 40 h and clonal differentiation assessed 14 days later. Total heterogeneity as to gross colony morphology and differentiation stage was demonstrated. This heterogeneity showed patterns of differentiation at different cycle times. These data hearken to previous suggestions that stem cells might be similar to radioactive isotopes; decay rate of a population of radioisotopes being highly predictable, while the decay of individual nuclei is heterogeneous and unpredictable (Till et al., 1964). Marrow stem cells may be most adequately defined on a population basis; stem cells existing in a continuum of reversible change rather than a hierarchy.

Fig. 1

Experimental design. LRH marrow cells are separated by fluorescent activated cell sorting (FACS), cultured in primary cytokines for 0–40 h, then deposited as single cells into microtiter wells with different secondary cytokine combinations. An evaluation of colony number, morphology, and differentiation then occurs 14 days later.

Fig. 2

Cell cycle status of LRH cells at different points in cell cycle transit after stimulation with thrombopoietin (TPO), FLT-3, and steel factor for different time periods. Cells were stained with propidium iodide and analyzed by FACS. (Reproduced courtesy of Colvin et al., 2007.)

Fig. 3

A–D: Gross colony morphology. LRH marrow cells were cultured in steel factor, FLT-3, and thrombopoietin for different times; 0, 18, 32, and 40 h, and differentiation stimulated with G-CSF, GM-CSF, and steel factor at level C. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 4

Box plots for the total cell count expression for different experimental conditions. Subparts (B–D) represent different levels of the cytokine cocktails (see Experimental Procedures Section). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 5

A: Individual colony numbers. This graph illustrates the expression of proliferative granulocytes (PG), non-proliferative granulocytes (NPG), and macrophages (Mac) at different time points with the cytokine cocktail TFSGGM at level B. All colonies for this specific experimental condition are displayed. B: Proliferative granulocytes. This figure provides a closer look at the expression of proliferative granulocytes at different timepoints, utilizing the GGMS cytokine cocktail at level B. Both graphs again demonstrate the large variability in the expression of the different cell types, even when cultured under identical conditions.

Fig. 6

Patterns of differentiation. PG, proliferative granulocytes; NPG, non-proliferative granulocytes; MCC, macrophages; megs, megakaryocytes.

Fig. 7

An array graphic. This graphic is plotted in software R by the function called image, and the high expression of each experiment is indicated in bright red and light green indicates low expression. PG, proliferative granulocytes; NPG, non-proliferative granulocytes; MCC, macrophages; meg, megakaryocytes.