Establishing long-term cultures with self-renewing acute myeloid leukemia stem/progenitor cells

Impaired Long-Term Expansion and Self-Renewal Potential of Pediatric Acute Myeloid Leukemia–Initiating Cells By PTK787/ZK 222584

“…After replating the cultured AML cells onto new MS5, secondary L-CAs were generated underneath the stroma and maintained self-renewing capacity for up to 10 weeks in 9 of the 13 (69%) pediatric AML samples. Our results are in agreement with data on adult LICs, which show that sorted CD34þ cells from adult patients with AML can be cultured on a stromal layer (25). In contrast with results from van Gosliga and colleagues, we were able to maintain a long-term culture up to 10 weeks in 2 samples from good risk patients (AML5 and AML11) although they were characterized by a slower expansion compared with other AML samples.…”

“…All drugs were added during demidepopulation. The fact that cocultures generated L-CAs after replating, a feature of self-renewal that has not been shown in normal cord blood CD34þ cells, confirms the leukemic origin of the expanding cells (25). In addition, with FLT3-ITD fragment analysis of the suspension cells in the LTC-IC cocultures at week 2, we showed for AML10 (FLT-ITD-positive sample) that the suspension cells harbor the heterozygous FLT3-ITD mutation (86%).…”

“…A total of 40-50 Â 10 3 sorted CD34þ cells (i.e., LICs) were plated in 12-well plates precoated with confluent layer of MS5 stromal cells. Cells were expanded in LTC medium [a-MEM supplemented with heat-inactivated 12.5% fetal calf-serum (FCS), heat-inactivated 12.5% horse serum (Sigma), penicillin and streptomycin, 2 mmol/L glutamine, 57.2 mmol/L b-mercaptoethanol (Sigma), and 1 mmol/L hydrocortisone (Sigma)] supplemented with 20 ng/mL IL-3, granulocyte colony-stimulating factor, and thrombopoietin as previously described (25). Cultures were kept at 37 C and 5% CO 2 .…”

“…To study the interaction between bone marrow-derived stromal cells and LICs, a previously described leukemic long-term culture-initiating cells (LTC-IC) assay has been used in which long-term leukemic expansion of LICs can be established using MS5 bone marrow stromal cells, thereby mimicking the stem cell niche (25). We cultured sorted CD34þ pediatric AML cells on stroma in the absence or presence of PTK/ZK.…”

Variability in responsiveness to lovastatin of the primitive CD34+ AML subfraction compared to normal CD34+ cells

“…Consequently, a role for these cholesterol synthesis inhibitors to improve standard antileukemic treatment has been suggested [4,[6][7][8][9][10]. In the present study, we focused on a subpopulation of AML cells (CD34 + ), i.e., cells that have more in common with the more primitive leukemic progenitor/stem cell compartment [11], and questioned whether this subpopulation of cells is especially prone for the effects of lovastatin and chemotherapeutic agents. Our data demonstrate a higher sensitivity of the primitive CD34 + subpopulation for lovastatin compared to the more mature CD34 − subpopulation of normal as well as AML cells.…”