Ex vivo expansion and maturation of peripheral blood CD34+ cells into the myeloid lineage

Abstract

Hematopoietic reconstitution (HR) after peripheral blood stem cell transplantation is characterized by a delay of 8 and 12 days for recovery to safe levels of neutrophils and platelets even in patients with the most rapid engraftment. We postulate that a further enhancement in the rate of HR may be achieved by transplanting with an expanded postprogenitor cell population that can provide mature functional cells within days of infusion. In this study we investigated the ability of combinations of hematopoietic growth factors (HGF) to generate nascent granulocyte-macrophage colony-forming units (CFU-GM) in a 7-day suspension culture of peripheral blood CD34+ cells. A combination of 6 HGF, ie, interleukin-1 beta (IL-1), IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage- CSF (GM-CSF), and stem cell factor (SCF), was identified as the most potent combination of those tested. Subsequently, large volume suspension cultures of CD34+ cells from the same patients using the same 6-factor combination were established and monitored for 21 days. An exponential rate of nucleated cell production (mean 1,324-fold increase) occurred during culture. CFU-GM production paralleled nucleated cell production until day 10, peaked at day 14 (mean 66-fold increase), and was then maintained until day 21. Cells produced in culture were predominantly neutrophil precursors and developed normally as assessed by morphology, immunophenotype, and superoxide generation. This stroma-free, cytokine-driven culture system can achieve a degree of amplification, which suggests the feasibility of ex vivo culture of hematopoietic progenitor cells as an adjunct to hematopoietic stem cell transplantation.

AC133+ umbilical cord blood progenitors demonstrate rapid self‐renewal and low apoptosis

“…Our results emphasized the importance of selecting a population enriched for immature HSPC as starting material for ex vivo expansion (Haylock et al, 1992). AC133 + cells were obtained through direct immunomagnetic selection.…”

Flow cytometric and functional characterization of AC133+ cells from human umbilical cord blood

“…However, the experimental conditions employed have rarely been encouraging in obtaining an effective increase in stem and early progenitor cells (Piacibello et al, 1997). This failure has been ascribed to the lack of accessory cells, to the predominant effect of induction of differentiation by the cytokines added (Haylock et al, 1992) or, alternatively, to the presence of very immature, deeply quiescent cells, which proliferate after a more prolonged time and/or do not respond to known cytokines (Berardi et al, 1995;Hao et al, 1996). In our stroma-free culture system, the simple combination of two early acting cytokines, i.e.…”

Ex vivo expansion of mature human neutrophils with normal functions from purified peripheral blood CD34+ haematopoietic progenitor cells

“…Peripheral blood mononuclear cells were collected by leucapheresis using a Fenwal CS-3000 plus (Baxter Fenwal Division, Deerfield, IL, USA) during the recovery phase of haematopoiesis. CD34 1 cells were further purified by positive selection, using immunomagnetic beads (Isolex), as described previously (Law et al, 1993;Hasuike et al, 1997). The expression of cell-surface antigens was analysed by flow cytometry using a Cytron (Ortho, Westwood, MA, USA).…”

“…One possible way to reduce further the duration of severe neutropenia after cytotoxic chemotherapy may be the transfusion of mature neutrophils expanded ex vivo from autologous haematopoietic progenitor cells. Haematopoietic progenitor cells proliferate and differentiate in vitro into mature neutrophils when cultivated in the presence of appropriate haematopoietic growth factors (Haylock et al, 1992;Mayani et al, 1993;Smith et al, 1993;Berliner et al, 1995;Hasuike et al, 1997;Neildez-Nguyen et al, 1998). However, the functions of mature neutrophils differentiated in vitro in the presence of various haematopoietic growth factors have never been well defined.…”