Expansion of blood CD34+ cells: committed precursor expansion does not affect immature hematopoietic progenitors.

The CD34 antigen is present at all differentiation stages of hematopoietic cells, from immature progenitor cells to committed precursor cells. In vivo, transplantation of CD34+ cells is sufficient to allow hematopoietic recovery after myeloablative chemotherapy, but a neutropenic period of 9-12 days still exists, even when hematopoietic growth factors are given posttransplantation. After ex vivo expansion cultures in the presence of cytokines, CD34+ cells can generate mature precursor cells in a stroma-free liquid culture system. This could lead to a shortening of the aplasia duration, but the persistence of primitive progenitor cells in the expanded CD34+ compartment remains to be demonstrated. In this study, CD34+ cells were isolated from eight peripheral blood (PB) and eight cord blood (CB) samples using either Isolex 50 (n = 6), Ceprate LC CD34 kit (n = 6), or Microcellector T-25 Stem Cell kit (n = 4). We have evaluated the functional potential of CD34+ cells after 7 days of ex vivo expansion culture in the presence of 500 UI/ml of interleukin-1 (IL-1), 10 ng/ml of IL-3, and 10 ng/ml of stem cell factor (SCF). The expansions of nucleated cells, granulocyte-macrophage colony-stimulating factor (GM-CSF)-responsive committed precursors, IL-1 + IL-3 + SCF + erythropoietin (EPO)-responsive multilineage progenitors, and 5-fluorouracil (5-FU)-resistant quiescent progenitor were 8-fold, 59-fold, 4.4-fold, and 2.2-fold, respectively. There was no significant difference in the amplification/expansion parameters between cultures initiated with CD34+ cells from PBSC or CB. Our data confirm that cytokine-mediated ex vivo expansion of blood CD34+ cells can produce large numbers of committed precursors and does not significantly affect the compartment containing more immature progenitors. Cytokine-mediated expansion could be of great interest in autologous transplantation to decrease the duration of marrow aplasia.

Effect of stem cell factor on leukemic progenitor cell growth and sensitivity to cytosine-arabinoside.

Recruitment of quiescent, clonogenic blasts from patients with acute myeloid leukemia (AML) by hematopoietic growth factors (HGFs) may improve the cytotoxic effects of cell-cycle-specific drugs like cytosine-arabinoside (Ara-C). Using the culture methods described by Nara and McCulloch and making a distinction between self-renewing and post-deterministic mitoses, we analyzed the effects of stem cell factor (SCF), a growth factor acting on early hematopoietic progenitor and stem cells. First, we demonstrated that SCF, used in combination with other HGFs included in fetal calf serum (FCS) and/or in 5637 cell line supernatant (5637-CM), stimulated both colony formation and self-renewal of blast progenitors from 10 patients, unlike SCF alone. We tested the effects of SCF on the recruitment of cells in the S-phase by using a bromodeoxyuridine/DNA (BrdUrd/DNA) staining method in flow cytometry (FCM). We showed that SCF stimulated proliferation of AML cells significantly in 9/18 patients with AML. Second, we tested the influence of SCF on the sensitivity to Ara-C of self-renewing leukemic cells from 18 patients with AML. We showed that SCF was efficient in increasing the toxicity of Ara-C on the self-renewing blast progenitors, especially with high concentrations of Ara-C. However, a large patient-to-patient heterogeneity was found and the activity of SCF was not correlated with its effect on the cell cycle. These data indicate that SCF can enhance sensitivity to Ara-C of some leukemic cells with self-renewing capacity.

Specific antisense oligomer anti Bcr-abl junctions in chronic myeloid leukemia: a cell cycle analysis and CFU-GM study.

Antisense oligonucleotides were used to determine the role of the BCR-ABL gene in the proliferation of chronic myeloid leukaemia (CML) clonogenic cells. Peripheral blood Philadelphia chromosome positive cells were obtained from eight CML patients at diagnosis (chronic phase = 7; accelerated phase = 1). Mononuclear cells were incubated with synthetic antisense 18-mer oligonucleotides complementary to the two different junctions b2a2 or b3a2. The type of junction (b2a2 or b3a2) was previously determined by RT-PCR techniques. Cells incubated for 12 to 14 hours with or without sense oligonucleotides served as controls. After incubation with oligonucleotides, the cell DNA synthesis was analysed by flow cytometry using the BrdUrd/DNA method and the cell plating efficiency in methylcellulose was determined. In six of the seven patients in chronic phase, there was a significant inhibition of CFU-GM production which was only 68.4 +/- 19%; (p < .01) of that found in controls. The S phase index, which depends upon the percentage of S phase cells as well as the fluorescence intensity, was 48 +/- 29% (p < .01) of the control values for the seven patients in chronic phase. Interestingly, for the only CML patient in accelerated phase, antisense oligomers had no inhibitory effect on either the production of CFU-GM or the number of S phase cells. In improving the specificity of oligomers, it might be useful for gene-targeted anti-leukemic therapy and/or bone marrow purging.

Development of an efficient serum-free semisolid culture system for the evaluation of hematopoietic progenitors.

Three different combinations of serum-free (SF) media proposed by Drouet et al., Ieki et al., and Wu et al. were tested to assess their ability to replace fetal calf serum (FCS) in a human hematopoietic progenitor semisolid culture system using human bone marrow and peripheral blood stem cells. This study confirmed that two (Drouet and Wu) of the three SF media tested were able to induce progenitor growth in the presence of either 5637-conditioned medium or a defined combination of growth factors: stem cell factor (SCF), interleukin-1 (IL-1), interleukin-3 (IL-3), and erythropoietin (EPO). The best results were obtained using the SF media described by Wu. The number of 5637-stimulated CFU-GM obtained with the Wu SF media was not different from that obtained with FCS-supplemented media when cultures were assessed on day 14 (79 +/- 14 versus 104 +/- 17), day 18 (64 +/- 11 versus 79 +/- 12), or day 21 (58 +/- 13 versus 62 +/- 12). Similar findings were obtained when the previously defined combination of growth factors was used to stimulate progenitor growth. Because the Wu medium was the most efficient SF medium to promote hematopoietic progenitor growth, we attempted to improve its efficacy by modifying the concentrations of the various components. A reduction in the concentration of bovine serum albumin (BSA) dramatically reduced the total number of clonogenic elements. Soybean lectin was not essential for colony proliferation; however, its presence had an favorable effect on the overall appearance of the colonies (greater number of cells per colony).(ABSTRACT TRUNCATED AT 250 WORDS)

Expansion of blood CD34 positive cells: committed precursors expansion does not affect immature hematopoietic progenitors.

CD34 positive (CD34+) cells contain all hematopoietic progenitors from stem cells to committed precursors. Therefore the transplantation of purified bone marrow or blood CD34+ cells is sufficient for hematopoietic recovery after a myeloablative radiochemotherapy. Using different techniques, CD34+ progenitors can be induced to undergo terminal differentiation in a stroma-free liquid culture system in the presence of cytokines. In the present study, we have evaluated the functional potential of CD34+ blood progenitors after ex-vivo expansion cultures. CD34+ cells were isolated from 16 samples (PBSC n = 8 and Cord Blood (CB) n = 8) using either ISOLEX 50 (n=6), CEPRATE LC CD34 kit (n = 6) or MICROCELLECTOR T-25 Stem Cell kit (n = 4). CD34+ cells were cultured for seven days in the presence of 500 UI/ML of IL-1, 10 ng/ml of IL-3 and 10 ng/ml of SCF. We obtained an 8-fold expansion of nucleated cells. We observed a 59-fold expansion of GM-CSF responsive committed precursors, a 4.4-fold expansion of IL-1+IL-3+SCF+Epo responsive multilineage progenitors and a 2.2-fold expansion of the 5-FU resistant quiescent progenitors. We did not observe any significant difference in the amplification/expansion parameters between cultures initiated with CD34+ cells from PBSC or CB. Our data show that cytokine mediated ex-vivo expansion of blood CD34+ cells can produce a large number of committed precursors without affecting the compartment of the most immature progenitors. These results suggest that cytokine-mediated amplification technology could be of great interest in the autologous transplantation setting.