Ex vivo expansion of murine hematopoietic progenitor cells generates classes of expanded cells possessing different levels of bone marrow repopulating potential

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The objective of ex vivo expansion of primitive hematopoietic progenitor cells (HPC) is to increase the number of progeny cells possessing hematopoietic potential similar to the original HPC. In the context of bone marrow (BM) transplantation in mice, this implies that expanding a number of HPC sufficient for long-term rescue of one lethally irradiated animal should generate enough cells to rescue more than one lethally irradiated recipient. In the present study, Sca-1+Lin- cells from male C57B1/6 mice were expanded in vitro with stem cell factor (SCF), interleukin-1α (IL-1α), IL-3, and IL-6 and used to transplant lethally irradiated syngeneic female recipients. Expanded cells were tracked in vitro with the fluorescent membrane dye PKH2, which becomes evenly distributed among dividing daughter cells, and fractionated on day 7 into Sca-1+ cells which did not divide (Sca-1+PKH2(bright)), those which had divided 1 to 2 times (Sca-1+PKH2(moderate)), or those which had divided four or more times (Sca-1+PKH2(dim)). Grafts of expanded cells consisted of either the same number of fresh cells proven to rescue lethally irradiated animals [3 x 103 cells; referred to as one repopulating dose (1 RD)] or the expansion equivalent (EE) of these cells. One EE of cells represented 3 x 103 multiplied by the fold increase in the number of cultured cells on day 7. All animals transplanted with 3 x 103 freshly isolated Sca-1+Lin- cells survived long-term. Only 53% of animals receiving 1 EE of all cultured day-7 cells survived. One RD from all three PKH2 fractions (bright, moderate, and dim) of day-7 cultured Sca-1+ cells failed to rescue more than 30% of lethally irradiated recipients. Comparable survival rates were obtained when 1 EE of Sca-1+PKH2(dim) or only 4 RD of Sca-1+PKH2(bright) cells were used as grafts, suggesting that a larger frequency of long-term repopulating cells may have been retained within the fraction of Sca-1+ cells undergoing minimal or no proliferation in culture. Engraftment of male ex vivo expanded cells in recipients was confirmed by polymerase chain reaction (PCR) analysis with Y chromosome-specific primers. When analyzed for their cell cycle status, Sca-1+PKH2(bright) cells were mostly quiescent, whereas a higher percentage of Sca-1+PKH2(dim) cells were in active phases of cell cycle. These data suggest that ex vivo expansion does not augment the number of BM repopulating HPC and that ex vivo expansion generates classes of progenitor cells with different BM repopulating potentials depending on their proliferative history. These studies also suggest that the cell cycle status of graft cells may affect the ability of these cells to engraft in myeloablated hosts.

Original languageEnglish (US)
Pages (from-to)299-306
Number of pages8
JournalExperimental Hematology
Issue number2
StatePublished - Mar 20 1996


  • bone marrow transplantation
  • ex vivo expansion
  • murine hematopoietic stem/progenitor cells

ASJC Scopus subject areas

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

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