Engraftment in Nonobese Diabetic Severe Combined Immunodeficient Mice of Human CD34+ Cord Blood Cells After Ex Vivo Expansion: Evidence for the Amplification and Self-Renewal of Repopulating Stem Cells

Abstract

Understanding the repopulating characteristics of human hematopoietic stem/progenitor cells is crucial for predicting their performance after transplant into patients receiving high-dose radiochemotherapy. We have previously reported that CD34+cord blood (CB) cells can be expanded in vitro for several months in serum containing culture conditions. The use of combinations of recombinant early acting growth factors and the absence of stroma was essential in determining this phenomenon. However, the effect of these manipulations on in vivo repopulating hematopoietic cells is not known. Recently, a new approach has been developed to establish an in vivo model for human primitive hematopoietic precursors by transplanting human hematopoietic cells into sublethally irradiated nonobese diabetic severe combined immunodeficient (NOD/SCID) mice. We have examined here the expansion of cells, CD34+ and CD34+38− subpopulations, colony-forming cells (CFC), long-term culture initiating cells (LTC-IC) and the maintenance or the expansion of SCID-repopulating cells (SRC) during stroma-free suspension cultures of human CD34+ CB cells for up to 12 weeks. Groups of sublethally irradiated NOD/SCID mice were injected with either 35,000, 20,000, and 10,000 unmanipulated CD34+ CB cells, which were cryopreserved at the start of cultures, or the cryopreserved cells expanded from 35,000, 20,000, or 10,000 CD34+ cells for 4, 8, and 12 weeks in the presence of a combination of early acting recombinant growth factors (flt 3/flk2 ligand [FL] + megakaryocyte growth and development factor [MGDF] ± stem cell factor [SCF] ± interleukin-6 [IL-6]). Mice that had been injected with ≥20,000 fresh or cryopreserved uncultured CD34+ cells did not show any sign or showed little engraftment in a limited number of animals. Conversely, cells that had been generated by the same number of initial CD34+ CB cells in 4 to 10 weeks of expansion cultures engrafted the vast majority of NOD/SCID mice. The level of engraftment, well above that usually observed when the same numbers of uncultured cells were injected in the same recipients (even in the presence of irradiated CD34− cells) suggested that primitive hematopoietic cells were maintained for up to 10 weeks of cultures. In addition, dilution experiments suggest that SRC are expanded more than 70-fold after 9 to 10 weeks of expansion. These results support and extend our previous findings that CD34+ CB stem cells (identified as LTC-IC) could indeed be grown and expanded in vitro for an extremely long period of time. Such information may be essential to design efficient stem cell expansion procedures for clinical use.

Prolonged ex vivo culture of cord blood CD34+ cells facilitates myeloid and megakaryocytic engraftment in the non‐obese diabetic severe combined immunodeficient mouse model

“…Prolongation of the cytokine exposure period to 14 d enhanced both the kinetics and level of human cells detected in the blood, BM and spleen of NOD±SCID mice. Our observations are in agreement with those of Piacibello et al (1999) who showed that CB CD34 1 cells could be expanded for up to 12 weeks with continual production of CD34 1 , CD34 1 CD38 2 cells, committed and multipotent progenitors without a reduction in self-renewal capacity, as measured in the NOD±SCID mouse model. The increased level of human engraftment may be directly related to greater numbers of haematopoietic precursors or indirectly related to the production of specific cell types that facilitate engraftment of haematopoietic precursors.…”

“…Cytokine-mediated expansion has been proposed as a means of increasing the cell dose available for transplantation and thereby facilitating the rate of engraftment. Clinical trials suggest that the rate of myeloid engraftment can be improved when unmanipulated and expanded cells are cotransplanted (Brugger et al, 1995;Williams et al, 1996;Bertolini et al, 1997;Kogler et al, 1999;Reiffers et al, 1999;McNiece et al, 2000). Indeed, Reiffers et al, 1999 have shown that patients transplanted for myeloma with mPB CD34 1 cells and CD34 1 cells that had been expanded for 10 d with stem cell factor (SCF) 1 granulocyte colonystimulating factor (G-CSF) 1 megakaryocyte growth development factor (MGDF) had a mean duration of neutropenia of 1´5 d. These studies suggest that infusion of ex vivo expanded CD34 1 cells can abrogate neutropenia and improve thrombocytopenia in patients receiving myeloablative therapy.…”

“…Ex vivo expansion of CD34 1 cells has been documented to facilitate the rate of engraftment following myeloablative therapy (Reiffers et al, 1999;McNiece et al, 2000). Cytokine combinations and the duration of cytokine exposure necessary for progenitor expansion have been empirically defined.…”

“…The principle reason for supplementing unmanipulated grafts with expanded grafts is to minimize the theoretical risk of stem cell exhaustion, which may manifest itself as myelodysplasia later in life. Recent clinical trials have retained unmanipulated cells in the transplant product (Kogler et al, 1999;Reiffers et al, 1999;McNiece et al, 2000). Extended periods of culture enable generation of greater numbers of mature post-progenitor cells and this is attractive to the clinician for rapid abrogation of neutropenia and thrombocytopenia.…”

Co‐administration of Flt‐3 ligand counteracts the actions of thrombopoietin in myelosuppressed rhesus monkeys

“…The in vivo observations contrast with in vitro studies, which show synergism of TPO, FL and SCF on stem cell expansion. CD34 + cord blood cells could be expanded in vitro for up to 10 weeks, with a retained repopulating capacity (Kobayashi et al, 1997;Ohmizono et al, 1997;Piacibello et al, 1999). Explanations for this apparent antagonism of TPO and FL in vivo might be extrapolated from other in vitro studies, which showed that the presence of the Flk-2/Flt-3 receptor in primitive haematopoietic cell populations resulted in preferential myeloid differentiation, whereas megakaryocytic CFU were found in the Flk-2/Flt-3-negative fraction (Banu et al, 1999).…”