Selection of BCR/ABL-negative stem cells from marrow or blood of patients with chronic myeloid leukemia.
Philadelphia (Ph) or BCR/ABL-negative cells with immature phenotype (CD34-positive, DR-negative) can be recovered from patients with chronic myeloid leukemia (CML) in chronic phase. We used the technique described by Berardi et al (Science 1995; 267: 104-108) to select stem cells from marrow or blood of CML patients at diagnosis or during treatment with alpha-interferon. Mononuclear cells (MNC), and in some experiments CD34+ cells, were maintained for 7 days in the presence of 5-fluorouracil (5-FU), stem cell factor and interleukin-3. The number of viable cells recovered after culture was between 7.4 and 70.2 for 10(6) cells plated. These cells exhibited the following phenotype: CD34+, CD117+, CD38-, lineage-, and were able to generate cobblestone areas and secondary colonies in long-term culture (LTC), with a frequency similar to that of cells selected from normal marrow. Study by fluorescence in situ hybridization of LTC cells or secondary colonies showed no evidence of BCR/ABL rearrangement. Reverse transcriptase polymerase chain reaction studies on pooled LTC cells or secondary colonies were also negative. By contrast, LTC cells or secondary colonies obtained from CML CD34+ cells without culture in the presence of 5-FU were always positive for BCR/ABL rearrangement. Finally, 5-FU selected cells were able to engraft NOD/SCID mouse, as human cells were detected in blood and marrow 10 weeks post transplantation, which were BCR/ABL negative by RT-PCR. This method of culture makes it possible to select constantly BCR/ABL-negative cells with capacities of development in LTC assay and of NOD/SCID mouse engraftment.
Laboratoire d'Hématologie Cellulaire et Moléculaire, Faculté de Médecine, St Etienne, France.
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Guyotat
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Wahbi
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Viallet
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Piselli
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Campos
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation; D005472:Fluorouracil / Q000627:therapeutic use; D016044:Fusion Proteins, bcr-abl / Q000032:analysis*; D006412:Hematopoietic Stem Cells / Q000737:chemistry*; D006801:Humans; D016130:Immunophenotyping; D017404:In Situ Hybridization, Fluorescence; D007377:Interleukin-3 / Q000627:therapeutic use; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D016133:Polymerase Chain Reaction; D019089:Stem Cell Factor / Q000627:therapeutic use; D014407:Tumor Cells, Cultured
D007377:Interleukin-3; D019089:Stem Cell Factor; D016044:Fusion Proteins, bcr-abl; D005472:Fluorouracil