Identification of Philadelphia-negative granulocyte-macrophage colony-forming units generated by stroma-adherent cells from chronic myelogenous leukemia patients

Carmelo Carlo-Stella, Lina Mangoni, Giovanna Piovani, Daniela Garau, Camillo Almici, Vittorio Rizzoli

Research output: Contribution to journalArticlepeer-review


Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by the coexistence of Philadelphia-negative (Ph-) with Ph+ progenitors. CML progenitor cells have been shown to be defective in adherence to marrow stroma. The present study investigated at the cytogenetic level marrow-derived CML clonogenic cells generated from the stroma-adherent cell fraction. On direct cytogenetic analysis, the overall mean (±SEM) percentage of Ph- metaphases was 3% ± 1%. Mononuclear marrow cells from CML patients (n = 18) were incubated with mafosfamide (100 μg/mL) or control medium, seeded onto marrow stromal layers and allowed to adhere (2 hours, 37°C). After a short-term (3-day) liquid culture, the cells were harvested, incorporated in methylcellulose, and individual colonies were analyzed by single colony karyotyping. The mean (±SEM) percentage of Ph- colonies generated from the stroma-adherent fraction was 35% ± 6%. As compared with marrow colony-forming unit granulocyte-macrophage plated before any manipulation, the mean (±SEM) percentage of Ph- clones was significantly increased by stroma adherence (35% ± 6% v 15% ± 4%, P ≤ .005) and mafosfamide (10 μg/mL) incubation of marrow cells before stroma adherence (58% ± 9% v 35% ± 6%, P ≤ .005). An additive effect was observed by combining mafosfamide treatment and stroma adherence. Single-colony transfer experiments showed that 50% ± 4% stroma-adherent and 70% ± 4% stroma-adherent mafosfamide-treated progenitors gave rise to secondary colonies. To further characterize the stroma-adherent fraction, experiments were performed in which CD34+ marrow cells were used. The mean (±SEM) output of progenitors generated by 10,000 CD34+, stroma-adherent cells was 888 ± 188 and 570 ± 258 for untreated and mafosfamide-treated cells, respectively. Individual colonies were analyzed by single-colony karyotyping and fluorescent in situ hybridization using a biotinylated cosmid DNA probe that hybridize to abl oncogene. The CD34+, stroma-adherent fraction contained 38% ± 14% (untreated) and 56% ± 18% (mafosfamide-treated) (P ≤ .025) Ph- progenitors. In conclusion, the present data show the possibility to select Ph- clones that (1) have a maintained capability of stroma adherence, (2) are mafosfamide resistant, (3) are derived from the CD34+ fraction, and (4) have high-replating potential.

Original languageEnglish
Pages (from-to)1373-1380
Number of pages8
Issue number5
Publication statusPublished - Mar 1 1994

ASJC Scopus subject areas

  • Hematology


Dive into the research topics of 'Identification of Philadelphia-negative granulocyte-macrophage colony-forming units generated by stroma-adherent cells from chronic myelogenous leukemia patients'. Together they form a unique fingerprint.

Cite this