Assessment of proliferative and colony-forming capacity after successive in vitro divisions of single human CD34+ cells initially isolated in G0

A. Gothot, R. Pyatt, J. McMahel, S. Rice, Edward Srour

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Exit of primitive hematopoietic progenitor cells (HPCs) from the G0 phase of the cell cycle in response to in vitro cytokine stimulation is a limiting step in successful ex vivo expansion. Simultaneous DNA/RNA staining with Hoechst 33342 and pyronin Y was used to separate human bone marrow CD34+ cells residing in G0 (G0CD34+) from those cycling in G1 and S/G2+M. Compared with CD34+ cells isolated in G1, G0CD34+ cells were characterized by a delayed response to cytokine stimulation and were enriched for long-term hematopoietic culture-initiating cells. We next compared the activation kinetics of individually sorted G0CD34+ cells stimulated with stem cell factor (SCF), flt3-ligand (FL), or interleukin-3 (IL-3) as single factors. In a novel clonal proliferation assay, the functional status of cells that had remained quiescent after an initial 7-day period and of those that had completed successive division cycles under each of these three factors was evaluated by assessment of subsequent proliferative capacity and maintenance of colony-forming cell precursor (pre-CFC) activity. All three cytokines were equally able to support the survival of primitive HPCs in the absence of cell division. Cells that did not respond to any cytokine stimulation for 7 days retained higher proliferative and pre-CFC activities than dividing cells. The hematopoietic function of cells that divided in response to SCF, FL, or IL-3 decreased after each division cycle. However, G0CD34+ cells displayed a heterogeneous response pattern to cytokine stimulation whereby SCF appeared to have a superior ability to promote the cycling of cells with high proliferative and pre-CFC activities. These results indicate that HPCs reside in opposing hierarchies of hematopoietic potential and responsiveness to cytokine stimulation. The data also begin to indicate relationships between cellular division in response to different stimuli and maintenance of hematopoietic function.

Original languageEnglish
Pages (from-to)562-570
Number of pages9
JournalExperimental Hematology
Volume26
Issue number7
StatePublished - 1998

Fingerprint

Cytokines
Stem Cell Factor
Hematopoietic Stem Cells
Interleukin-3
Pyronine
Maintenance
In Vitro Techniques
Cell Cycle Resting Phase
Bone Marrow Cells
Cell Division
Cell Cycle
Cell Culture Techniques
RNA
Staining and Labeling
DNA
flt3 ligand protein

Keywords

  • CD34
  • Cell cycle
  • Hematopoiesis
  • Hoechst 33342
  • Human
  • Pyronin Y

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Genetics
  • Hematology
  • Oncology
  • Transplantation

Cite this

Assessment of proliferative and colony-forming capacity after successive in vitro divisions of single human CD34+ cells initially isolated in G0. / Gothot, A.; Pyatt, R.; McMahel, J.; Rice, S.; Srour, Edward.

In: Experimental Hematology, Vol. 26, No. 7, 1998, p. 562-570.

Research output: Contribution to journalArticle

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