In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells - PubMed (original) (raw)

In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells

S H Cheshier et al. Proc Natl Acad Sci U S A. 1999.

Abstract

A rare set of hematopoietic stem cells (HSC) must undergo a massive expansion to produce mature blood cells. The phenotypic isolation of HSC from mice offers the opportunity to determine directly their proliferation kinetics. We analyzed the proliferation and cell cycle kinetics of long-term self-renewing HSC (LT-HSC) in normal adult mice. At any one time, approximately 5% of LT-HSC were in S/G2/M phases of the cell cycle and another 20% were in G1 phase. BrdUrd incorporation was used to determine the rate at which different cohorts of HSC entered the cell cycle over time. About 50% of LT-HSC incorporated BrdUrd by 6 days and >90% incorporated BrdUrd by 30 days. By 6 months, 99% of LT-HSC had incorporated BrdUrd. We calculated that approximately 8% of LT-HSC asynchronously entered the cell cycle per day. Nested reverse transcription-PCR analysis revealed cyclin D2 expression in a high proportion of LT-HSC. Although approximately 75% of LT-HSC are quiescent in G0 at any one time, all HSC are recruited into cycle regularly such that 99% of LT-HSC divide on average every 57 days.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Double-sorting is necessary to obtain pure KTSL−/lo and KTSL− cells. (Upper) c-kit (_y_-axis) and lineage (_x_-axis) density plots with outliers. Boxes indicate sort gates for KTSL−/lo (inner and outer boxes) and KTSL− (inner boxes only). (Lower) Sca-1 (_y_-axis) and Thy 1.1 (_x_-axis) density plots with outliers. Boxes indicate sort gates for KTSL−/lo and KTSL− cells. As seen in the second and third columns, significant numbers of cells outside the sort gates are present in a reanalysis of a single sorted sample. After the samples are double-sorted, the great majority of cells (≥95%) are within the sort gates that define KTSL−/lo (fourth column) and KTSL− (fifth column) HSC.

Figure 2

Figure 2

In vivo BrdUrd incorporation kinetics of KTSL−/lo and KTSL− cells. (A) BrdUrd incorporation rate of KTSL−/lo cells (□) and KTSL− cells (●). Each data point represents mean percentages of BrdUrd positive HSC from at least two separate experiments with a combined analysis of at least 500 cells. Some error bars are smaller than their associated symbol. (B) BrdUrd incorporation means plotted in semilogarithmic fashion as the proportion of BrdUrd-negative KTSL−/lo cells (□) and KTSL− cells (●) against time in days. The lines were generated by using least squares fit linear regression.

Figure 3

Figure 3

Cyclin D2 expression in five (A) and one (B) KTSL− cells as determined by using nested RT-PCR. No RT indicates lanes with RT-PCR reactions performed on KTSL− without the initial addition of reverse transcriptase to the RT reactions as a control.

Figure 4

Figure 4

Growth Fraction of KTSL− LT-HSC. FACS analysis of double-sorted KTSL− cells stained with PY (y axis) and Hoechst 33342 (x axis). The growth fraction of a population of cells is the fraction of actively dividing cells (G1/S/G2/M). On average, 23.5 ± 3.4% of KTSL− LT-HSC were in G1/S/G2/M at any one time.

Figure 5

Figure 5

Model of LT-HSC cell cycle regulation. LT-HSC are asynchronously dividing with a constant fraction of cells in the cell cycle and a constant fraction in G0. LT-HSC are continuously moving into and out of the cell cycle at rates indicated next to the arrows (p denotes probability of labeled event occurring) as determined from the BrdUrd incorporation kinetics of KTSL− cells. _T_c = apparent cell cycle time.

Similar articles

Cited by

References

    1. Metcalf D. The Molecular Control of Blood Cells. Cambridge, MA: Harvard Univ. Press; 1988.
    1. McCulloch E A, Till J E. Radiat Res. 1960;13:115. - PubMed
    1. Morrison S J, Weissman I L. Immunity. 1994;1:661–673. - PubMed
    1. Kay H E M. Lancet. 1965;ii:418–419. - PubMed
    1. Lemischka I R, Raulet D H, Mulligan R C. Cell. 1986;45:917–927. - PubMed

Publication types

MeSH terms

LinkOut - more resources