Fresh and ex-vivo expanded cord blood CD34+ cell megakaryocyte reconstitution in NOD/SCID mice

S. Bruno, S. Bruno, A. Dane

Research output: Contribution to journalArticlepeer-review

Abstract

Cord blood (CB) is used as an alternative source of reconstituting hematopoietic stem cells in young patients treated with high-dose chemotherapy. Compared to BM or MPB, CB transplantation is characterized by delayed engraftment, in particular a slow platel et recovery. Transfusion of ex-vivo expanded human megakaryocytic (MK) progenitor cells, could be a potential therapy to shorten the period of thrombocitopenia. CB CD34 cells can be expanded in a liquid culture system in the presence of FL, TPO and SCF and maintain their in vivo "populating capacity. A central issue remains whether CD34 ex-vivo expanded cells, still retain proliferation and differentation potential towards megakaryocytic lineage. Proliferation and differentation capacity toward the Mk lineage, as well as the Mk reconstitution in NOD/SCID mice of unmanipulated CB CD34 cells and those recovered after extensive expansion in stroma-free cultures grown in the presence of FL, TPO and SCF were compared. Unmanipulated and expanded + immunoselected CB CD34+ cells were grown in suspension cultures + 10% FCS in the presence of : 1 ) TPO, 2) TPO+SCF, 3) IL3+SCF+IL-6, 4) IL-3+SCF+1L-6+TPO. On day 7, 14, 21 and 28 cell production, immunophenotype (CD41+ and CD34VCD41+) and clonogenic ability were evaluated. Fresh and expanded CB CD34+ cells were injected into sublethally NOD/SCID mice to evaluate the megakaryocyte reconstitution. TPO alone triggered maximum Mk production (up to 500-fold) in fresh but not in expanded cells. The combination TPO+SCF was found to be sufficient to generate mature and more primitive Mk progenitors from fresh CB CD34+ cells. CD41+cell production was maximal between 7 to 14 days, Mk production was over 200-fold the initial number and represented up to 60% of the total cell population. In contrast only the cytokine combination TPO+SCF+IL-6+IL-3 induced a significant increase of Mk in expanded cells. CD41+ cell production was maximal after 21 days of culture; Mk production was over 55-fold the initial number. Mk generation from expanded cells was much slower and necessitated all of the 4 growth factors. Injection in NOD/SCID mice showed that there was no difference in Mk repopulation between unmanipulated and expanded CB CD34+ cells. Expanded and immunoselected CD34 were studied in the same settings above described. In the presence of SCF+TPO after 14 days of culture 50-60%% of the CD34+ cells were CD41+. Absolute counts showed a 25-fold expansion, thus demonstrating that expanded CD34 cells are capable of mature Mk differentiation and are likely to play a substantial role in early Mk engraftment.

Original languageEnglish
JournalBlood
Volume96
Issue number11 PART II
Publication statusPublished - 2000

ASJC Scopus subject areas

  • Hematology

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