Megakaryocytic Progenitors Can Be Generated Ex Vivo and Safely Administered to Autologous Peripheral Blood Progenitor Cell Transplant Recipients

Abstract

We evaluated different culture conditions to obtain a lineage-selected proliferation of clonogenic megakaryocytic progenitors (MP). In low-density (LD) or CD34+ cell cultures, the best results were obtained in serum-free medium in the presence of megakaryocyte growth and development factor, stem cell factor, interleukin-3 (IL-3), IL-6, IL-11, FLT-ligand, and macrophage inflammatory protein-1α. In paired studies, expansion of LD cells was less effective than expansion of CD34+ cells, and pre-enrichment of CD34+ cells using negative depletion of lineage-positive cells produced significantly larger quantities of MP than pre-enrichment using positive selection. MP proliferation peaked on day 7 in culture, and an 8- ± 5-fold expansion of CD34+/CD61+ cells, a 17- ± 5-fold expansion of colony-forming units-megakaryocytes, and a 58- ± 14-fold expansion of the total number of CD61+ cells was obtained. In a feasibility clinical study, 10 cancer patients (8 with breast cancer and 2 with non-Hodgkin's lymphoma) undergoing autologous peripheral blood progenitor cell (PBPC) transplant received MP generated ex vivo (range, 1 to 21 × 105/kg CD61+ cells) together with unmanipulated PBPC. Eight patients received a single allogeneic platelet transfusion, whereas platelet transfusion support was not needed in 2 of the 4 patients receiving the highest doses of cultured MP. This result compares favorably with a retrospective control group of 14 patients, all requiring platelet transfusion support. Adverse reactions or bacterial contamination of cell cultures have not been observed. In conclusion, MP can be expanded ex vivo and safely administered to autologous transplant recipients. Further clinical trials will indicate the reinfusion schedule able to consistently abrogate the need for allogeneic platelet transfusion support in autologous transplantation.

A combination of megakaryocyte growth and development factor and interleukin‐1 is sufficient to culture large numbers of megakaryocytic progenitors and megakaryocytes for transfusion purposes

“…Addition of IL-6, IL-11 or SCF to PEG-rHuMGDF and IL-1 did not result in signi®cantly higher numbers of megakaryocytes and megakaryocytic progenitors. In accordance with previous reports (Birkmann et al, 1997;Bertolini et al, 1997), our culture conditions did not lead to the formation of erythroid or lymphoid cells. Besides megakaryocytes, megakaryocytic progenitors and CD34 /lineage marker negative cells (CD14 À , CD15 À , CD41 À ), some myeloid cells (CD15 ) cells and monocytes (CD14 CD36 ) were observed.…”

“…We obtained a 12´5-fold increase in CFU-Meg numbers after 8 d of liquid culture in the presence of PEG-rHuMGDF and IL-1. This is comparable to the increase in CFU-Meg numbers that Bertolini et al (1997) and Ratajczak et al (1998) described after 7 d of liquid culture in serum-free medium. Furthermore, we observed a strong correlation between the number of CFU-Meg and CD34 CD41 cells; 1´8 CD34 CD41 cells correlates with the presence of one CFU-Meg.…”

“…In the past we and others have shown that the number of CD34 CD41 cells or CFU-Meg in a stem cell transplant is positively related with the time of platelet recovery (Dercksen et al, 1995;Feng et al, 1998;Takamatsu et al, 1995;Leibundgut et al, 1995). Bertolini et al (1997) reported that administration of autologous ex vivo expanded megakaryocyte progenitors in combination with autologous stem cell transplantation was well tolerated and that it may reduce the need of platelet transfusions if high enough numbers of megakaryocytic cells are reinfused. Therefore it would seem worthwhile to investigate whether the addition of megakaryocytic cells to a peripheral blood stem cell transplant will accelerate platelet recovery in vivo.…”

“…A broad range of cytokines, i.e. TPO (pegylated megakaryocyte growth and development factor (PEGrHuMGDF), a truncated, pegylated mpl ligand related to TPO), stem cell factor (SCF), interleukin 1 (IL-1), IL-3, IL-6, IL-11, erythropoietin, granulocyte macrophage-colony stimulating factor (GM-CSF),¯t3-ligand, macrophage in¯ammatory protein-1a and G-CSF, have been tested, and different cytokine cocktails have been shown to be able to stimulate megakaryocytopoiesis in vitro (Gehling et al, 1997;Guerriero et al, 1995;Birkmann et al, 1997;Dolzhanskiy et al, 1997;Debili et al, 1995;Bertolini et al, 1997;Catani et al, 1998;Ohmizono et al, 1997;Williams et al, 1998). However, inclusion of large numbers of cytokines in an ex vivo expansion protocol would be too expensive for routine clinical use.…”

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

“…In the present experiments, the two-step culture system was used for selective expansion of granulocytic cells, and the results show that the culture conditions of group B are preferable to those of group A to obtain sufficient numbers of mature neutrophils with normal functions in the resting state, but not in the activated state. We suggest that these mature neutrophils expanded ex vivo, like precursor cells expanded ex vivo (Williams et al, 1996;Bertolini et al, 1997), could be used for possible autologous neutrophil transfusion to prevent bacterial infections during severe neutropenia after cytotoxic chemotherapy or PBSCT.…”