Long-term hematopoietic culture-initiating cells are more abundant in mobilized peripheral blood grafts than in bone marrow but have a more limited ex vivo expansion potential

Edward Srour, Marco Bregni, Christie Orschell, Bibiana Agüero, Steven T. Kosak, Ronald Hoffman, Salvatore Siena, A. Massimo Gianni

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Mobilized peripheral blood hematopoietic progenitor cells obtained from cancer patients treated with high-dose cyclophosphamide (7g/m2) followed by G-CSF, GM-CSF, IL-3, PIXY321, or combinations of these cytokines have been successfully used for autologous stem cell transplantation. We investigated the ability of hematopoietic progenitor cells (HPC) derived from mobilized peripheral blood (PB) to undergo ex vivo expansion in short term cultures by enumerating numbers of-de novo generated CD34+ cells, assayable progenitor cells, and the frequency of long-term hematopoietic culture-initiating cells (LTHC-IC). These parameters were examined in CD34+ cells generated in culture through the use of cell tracking with the membrane dye PKH2. Fresh isolated mobilized CD34+ cells contained 0.49 ± 0.36% LTHC-IC. However, due to the high number of total CD34 cells in mobilized PB, the absolute number of LTHC-IC was higher than that contained in a bone marrow (BM) harvest. Mobilized CD34+ cells were stained with PKH2 and incubated with SCF, IL-3, and IL-6. After 5 to 6 days, numbers of total CD34+ cells and clonogenic progenitors increased 1.4- and 2.2-fold, respectively. Numbers of total progenitors continued to increase such that 10 to 12 days after the initiation of cultures a 6.4-fold increase was demonstrable. However, between days 5 and 7 of culture, the frequency of LTHC-IC in CD34+PKH2(bright) cells (cells which did not divide) was less than 50% of that determined for fresh cells, while the frequency among CD34+PKH2(dim) cells (cells that had divided) was very low or undetectable. However, moderately higher frequencies of LTHC-IC were detected following expansion for 48 hours only. In similar assays, both BM and cord blood cells were capable of generating LTHC-IC in CD34+PKH2(dim) cells but not to expand the overall number of these progenitors. These observations suggest that although mobilized PB CD34+ cells contain large numbers of LTHC-IC, these cells might not be capable of further ex vivo expansion and generation of additional LTHC-IC in vitro. Furthermore, these data indicate that mobilized PB CD34+ cells may have undergone maximal 'in vivo expansion' such that additional ex vivo expansion of primitive progenitor cells may not be possible.

Original languageEnglish
Pages (from-to)68-81
Number of pages14
JournalBlood Cells, Molecules and Diseases
Volume22
Issue number1
DOIs
StatePublished - Apr 1996

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Cell Culture Techniques
Bone Marrow
Transplants
Blood Cells
Stem Cells
Interleukin-3
Granulocyte-Macrophage Colony-Stimulating Factor
Hematopoietic Stem Cells
Cell Tracking
Stem Cell Transplantation
Granulocyte Colony-Stimulating Factor
Fetal Blood
Cyclophosphamide
Interleukin-6
Coloring Agents
Cytokines
Membranes

Keywords

  • Ex vivo expansion
  • Mobilized peripheral blood CD34

ASJC Scopus subject areas

  • Molecular Biology
  • Molecular Medicine
  • Hematology

Cite this

Long-term hematopoietic culture-initiating cells are more abundant in mobilized peripheral blood grafts than in bone marrow but have a more limited ex vivo expansion potential. / Srour, Edward; Bregni, Marco; Orschell, Christie; Agüero, Bibiana; Kosak, Steven T.; Hoffman, Ronald; Siena, Salvatore; Gianni, A. Massimo.

In: Blood Cells, Molecules and Diseases, Vol. 22, No. 1, 04.1996, p. 68-81.

Research output: Contribution to journalArticle

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abstract = "Mobilized peripheral blood hematopoietic progenitor cells obtained from cancer patients treated with high-dose cyclophosphamide (7g/m2) followed by G-CSF, GM-CSF, IL-3, PIXY321, or combinations of these cytokines have been successfully used for autologous stem cell transplantation. We investigated the ability of hematopoietic progenitor cells (HPC) derived from mobilized peripheral blood (PB) to undergo ex vivo expansion in short term cultures by enumerating numbers of-de novo generated CD34+ cells, assayable progenitor cells, and the frequency of long-term hematopoietic culture-initiating cells (LTHC-IC). These parameters were examined in CD34+ cells generated in culture through the use of cell tracking with the membrane dye PKH2. Fresh isolated mobilized CD34+ cells contained 0.49 ± 0.36{\%} LTHC-IC. However, due to the high number of total CD34 cells in mobilized PB, the absolute number of LTHC-IC was higher than that contained in a bone marrow (BM) harvest. Mobilized CD34+ cells were stained with PKH2 and incubated with SCF, IL-3, and IL-6. After 5 to 6 days, numbers of total CD34+ cells and clonogenic progenitors increased 1.4- and 2.2-fold, respectively. Numbers of total progenitors continued to increase such that 10 to 12 days after the initiation of cultures a 6.4-fold increase was demonstrable. However, between days 5 and 7 of culture, the frequency of LTHC-IC in CD34+PKH2(bright) cells (cells which did not divide) was less than 50{\%} of that determined for fresh cells, while the frequency among CD34+PKH2(dim) cells (cells that had divided) was very low or undetectable. However, moderately higher frequencies of LTHC-IC were detected following expansion for 48 hours only. In similar assays, both BM and cord blood cells were capable of generating LTHC-IC in CD34+PKH2(dim) cells but not to expand the overall number of these progenitors. These observations suggest that although mobilized PB CD34+ cells contain large numbers of LTHC-IC, these cells might not be capable of further ex vivo expansion and generation of additional LTHC-IC in vitro. Furthermore, these data indicate that mobilized PB CD34+ cells may have undergone maximal 'in vivo expansion' such that additional ex vivo expansion of primitive progenitor cells may not be possible.",
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AU - Kosak, Steven T.

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AU - Gianni, A. Massimo

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