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Myostatin propeptide expands primitive CML cells
Statistical analysis
All bar graphs show mean values and standard deviation. Differences between groups were assessed by unpaired Student’s t-test using Prism 6 software (GraphPad Software, USA).
Results
Cytokine screening identifies MSTNpp as a positive regulator of CD34+CD38low chronic myeloid leukemia cells To identify positive regulators of primitive CML cells,
we established a high-content cytokine screen using 313 unique human cytokines (Online Supplementary Table S2). CD34+CD38low chronic phase CML PB samples with high
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leukemic burden (Online Supplementary Figure S1) were sorted into 384-well plates with one cytokine condition per well. After a 7-day culture, the number of live cells was enumerated with automized fluorescence microscopy (Figure 1A).
In total, we identified 11 cytokines that increased cell numbers at least 2-fold compared to a no cytokine control (Figure 1B). Of these, the cytokines IL-3,12,13 IL-1a/b,8 GM-CSF,14 IL-6,15,16 and IFN-γ17 have previously been described as positive regulators of CML stem and progen- itor cells, thus validating the robustness of the screen. Notably, five cytokines not previously described as posi- tive regulators of CML stem and progenitor cells were identified: MSTNpp, soluble CD14 (sCD14), Interleukin 21 (IL-21), Interleukin 13 variant (IL-13v), and chemokine
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Figure 1. Cytokine screening identifies MSTNpp as a positive regulator of CD34+CD38low chronic myeloid leukemia (CML) cells. (A) Schematic illustration showing the arrayed cytokine screen with 500 sorted CD34+CD38low chronic phase CML peripheral blood (PB) cells per well, performed in a 384-well plate. A cytokine library of 313 human cytokines was used, each at a concentration of 100 ng/mL. Cell numbers were determined using automated fluorescent microscopy after seven days of culture. (B) Screening results showing cytokines with the ability to expand CD34+CD38low chronic phase CML PB at least 2-fold compared to no cytokine control. Three individual patient samples were used. (C) Validations of the top ranked cytokines identified in the original screen, using 2,000 CD34+CD38low chronic phase CML bone marrow (BM) cells in a 96-well format. Cell number was determined using flow cytometry after seven days of culture. Three individual patient samples were used. (D) Bar graph showing absolute cell numbers of normal CD34+CD38low BM cells cultured under the same conditions as in (C). Cells from two normal donors were used.
haematologica | 2020; 105(8)
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