Martina Peterková

Proliferation and differentiation potential of CD133+ and CD34+populations from the bone marrow and mobilized peripheral blood

CD34 is the most frequently used marker for the selection of cells for bone marrow (BM) transplantation. The use of CD133 as an alternative marker is an open research topic. The goal of this study was to evaluate the proliferation and differentiation potential for hematopoiesis (short and long term) of CD133+ and CD34+ populations from bone marrow and mobilized peripheral blood. Eight cell populations were compared: CD34+ and CD133+ cells from both the BM (CML Ph−, CML Ph+, and healthy volunteers) and mobilized peripheral blood cells. Multicolor flow cytometry and cultivation experiments were used to measure expression and differentiation of the individual populations. It was observed that the CD133+ BM population showed higher cell expansion. Another finding is that during a 6-day cultivation with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE), more cells remained in division D0 (non-dividing cells). There was a higher percentage of CD38− cells observed on the CD133+ BM population. It was also observed that the studied populations contained very similar but not the same pools of progenitors: erythroid, lymphoid, and myeloid. This was confirmed by CFU-GM and CFU-E experiments. The VEGFR antigen was used to monitor subpopulations of endothelial sinusoidal progenitors. The CD133+ BM population contained significantly more VEGFR+ cells. Our findings suggest that the CD133+ population from the BM shows better proliferation activity and a higher distribution of primitive progenitors than any other studied population.

BCR–ABL activity measured by 50% inhibitory concentration for imatinib, p-CrkL/CrkL ratio or p-CrkL ratio in CD34+ cells of patients with chronic myeloid leukemia does not predict treatment response

In this study, we assessed BCR-ABL kinase activity by measuring the protein levels of CrkL and its phosphorylated form (p-CrkL) in order to predict the clinical outcome of newly diagnosed chronic myeloid leukemia patients. CD34+ cells from these patients were collected before the start of imatinib therapy and treated in vitro with imatinib. The reduction of p-CrkL and CrkL protein levels was then measured by flow cytometry. The data were processed using three independent approaches: an assessment of a) IC50imatinib, b) p-CrkL/CrkL ratio, and c) p-CrkL ratio. The results were subsequently correlated with the clinical response of patients after 3 and 6 months of imatinib therapy. None of the three p-CrkL parameters measured in CD34+ cells was found to be predictive of clinical outcome.