Separation of nod-scid mouse repopulating cells by cell cycle fractionation of human cd34+ cells with hoechst 33342 and pyronin

Y. A. Gqthot, C. Fink, J. C.M. Van Per Loo, D. W. Clapp, E. F. Srour

Research output: Contribution to journalArticle

Abstract

We have previously described a flow cytometric cell sorting technique with which human CD34+ cells could be identified and isolated in subcompartments of the cell cycle using simultaneous DNA staining with Hoechst 33342 and RNA staining with Pyronin Y. Diploid CD34+ cells with a low RNA content are in GO (GOCD34+), while cells accumulating RNA are in Gl phase of cell cycle (GICD34+). Compared to G1CD34+ cells, GOCD34+ cells are characterized by a delayed onset of proliferation in response to in vitro cytokine stimulation and are enriched for long-term hematopoietic culture-initiating cells. Here we report the use of cell cycle fractionation to separate NOD-SCID mouse repopulating cells (SRC). Mobilized peripheral blood (MPB) CD34+ cells isolated from G-CSF-treated normal volunteers were fractionated into GO and Gl subcompartments and intravenously injected into sublethally irradiated NOD-SCID mice, at a dose of 2x10 cells/mouse. Mice were not supplemented with human cytokines before or after transplantation. At 7 weeks post-transplant, the extent of human cell engraftment was determined by phenorypic analysis of murine bone marrow cells and progenitor cell assays. Mice transplanted with GOCD34+ cells had a higher level of human engraftment (76.8 ±0.3 % CD45+ cells) than those injected with GICD34+ cells (6.8 ±5.7 % CD45+ cells). Mathematical estimation of the total number of CD34+ cells/mouse revealed a 3.5 fold-expansion in total CD34+ cell number relatively to input number in GOCD34+ cell-transplanted mice, and a 78% decrease in G1CD34+ cell-transplanted mice. Multilineage differentiation was evident in GOCD34+ cell-transplanted mice, since 65.5 ±1.1 % of CD34+ were CD19+ and 13.85 ±0.45 % were CD33+. Human colony-forming units (CPU) were recovered from murine BM at the frequency of 83.0 ±12.9 and 21.75 ±15.9 per 5xl04 BM cells in GO and GICD34+ cell-injected mice, respectively. Human origin of CPU was confirmed by PCR amplification of human -globin gene. These studies demonstrate that MPB CD34+ cells isolated in GO are enriched for SRC and that cell cycle status has an important effect on the engraftment capability of human CD34+ cells in the NOD-SCID mouse model. Further studies aimed at determining whether mitotic quiescence' is an intrinsic property of SRC or whether cell cycle dormancy indirectly affects homing properties of CD34+ cells are now underway.

Original languageEnglish (US)
Number of pages1
JournalExperimental Hematology
Volume25
Issue number8
StatePublished - Dec 1 1997

ASJC Scopus subject areas

  • Molecular Biology
  • Hematology
  • Genetics
  • Cell Biology
  • Cancer Research

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