Re-entry of CD34+ cells into mitotic quiescence following in vitro proliferation is not sufficient for the restoration of primitive hematopoietic potential

A. Gothof, R. Pvatf, J. McMahel, S. Grigsbv, Edward Srour

Research output: Contribution to journalArticle

Abstract

Since hematopotetic stem cells are mitotically quiesnt, this characteristic has been used to distinguish between mature and primitive hematopoietic progenitor cells (MPC). We have previously shown that cytokine nonresponsive(CNR) cells, which resist initial cytokine stimulation, are enriched for primitive HPC, while those undergoing proliferation appear to lose most of their hematopoietic potential. We hypothesized that it proliferating, cells were to maintain primitive functions, they should re-enter a mitotically quiescent state, namely the GO phase of cell cycle. Bone marrow IBM) and mobilized peripheral blood (MPB) CD34+ cells were cultured with SCF, ILS, and IL6. On day 7, CNR cells along with CD34+ cells which had divided once or more were distinguished and isolated by tracking with PKH2. Dividing cells were stained with Hoechst 33342 and Pyronin Y and cells residing in either GO, G1, or G2 + M were isolated by FACS. All groups of cells were assayed for their long-term hematopoietic culture-initiating cell (LTHC-IC) content in a limiting dilution analysis assay. Whereas frequencies of LTHC-IC among freshly isolated GO CD34+ cells were 3.5±3.3% and 9.85.9% lor BM and MPB, respectively, these frequencies for CD34+ cells re-entering GO following cell division were 1.80.3% and 1.9±1.0% for BM and MPB, respectively. Dividing cells traversing through G1 contained less than 55% of LTHC-IC detected within cells returning to GO. Cells which progressed from G1 into G2+M possessed nearly undetectable levels of LTHC-IC. Interestingly, LTHC-IC frequency of cells re-entering GO was tower than that detected among CNR cells, suggesting that maintenance of initial mitotic quiescence is essential for preservation of functional properties of primitive HPC. These data support the notion that activation of CD34+ cells in vitro is detrimental for primitive HPC function and suggest that re-acquisition of mitotic quiescence is insufficient for the restoration of these activities.

Original languageEnglish
Pages (from-to)1031
Number of pages1
JournalExperimental Hematology
Volume24
Issue number9
StatePublished - 1996

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Cell Culture Techniques
Cytokines
In Vitro Techniques
Pyronine
Hematopoietic Stem Cells
Cell Division
Interleukin-6
Blood Cells
Cell Cycle
Stem Cells
Bone Marrow
Maintenance

ASJC Scopus subject areas

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

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Re-entry of CD34+ cells into mitotic quiescence following in vitro proliferation is not sufficient for the restoration of primitive hematopoietic potential. / Gothof, A.; Pvatf, R.; McMahel, J.; Grigsbv, S.; Srour, Edward.

In: Experimental Hematology, Vol. 24, No. 9, 1996, p. 1031.

Research output: Contribution to journalArticle

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abstract = "Since hematopotetic stem cells are mitotically quiesnt, this characteristic has been used to distinguish between mature and primitive hematopoietic progenitor cells (MPC). We have previously shown that cytokine nonresponsive(CNR) cells, which resist initial cytokine stimulation, are enriched for primitive HPC, while those undergoing proliferation appear to lose most of their hematopoietic potential. We hypothesized that it proliferating, cells were to maintain primitive functions, they should re-enter a mitotically quiescent state, namely the GO phase of cell cycle. Bone marrow IBM) and mobilized peripheral blood (MPB) CD34+ cells were cultured with SCF, ILS, and IL6. On day 7, CNR cells along with CD34+ cells which had divided once or more were distinguished and isolated by tracking with PKH2. Dividing cells were stained with Hoechst 33342 and Pyronin Y and cells residing in either GO, G1, or G2 + M were isolated by FACS. All groups of cells were assayed for their long-term hematopoietic culture-initiating cell (LTHC-IC) content in a limiting dilution analysis assay. Whereas frequencies of LTHC-IC among freshly isolated GO CD34+ cells were 3.5±3.3{\%} and 9.85.9{\%} lor BM and MPB, respectively, these frequencies for CD34+ cells re-entering GO following cell division were 1.80.3{\%} and 1.9±1.0{\%} for BM and MPB, respectively. Dividing cells traversing through G1 contained less than 55{\%} of LTHC-IC detected within cells returning to GO. Cells which progressed from G1 into G2+M possessed nearly undetectable levels of LTHC-IC. Interestingly, LTHC-IC frequency of cells re-entering GO was tower than that detected among CNR cells, suggesting that maintenance of initial mitotic quiescence is essential for preservation of functional properties of primitive HPC. These data support the notion that activation of CD34+ cells in vitro is detrimental for primitive HPC function and suggest that re-acquisition of mitotic quiescence is insufficient for the restoration of these activities.",
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