Page 108 - 2021_10-Haematologica-web

S. Romero Marquez et al.
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Figure 1. Single cell cultures in serum-free medium and mouse embryonic fibroblast-conditioned medium. (A) Experimental design: single CD34- SLAM cells from 129Ly5.1 mice were sorted into a 96-well round-bottomed plate with SFM and MEF-CM, supplemented with either murine SCF (100 ng/mL) and murine IL-11 (20 ng/mL) alone (2GF) or with additional human NGF (250 ng/mL) and Col1 (100 ng/mL) (4GF). Cells were microscopically inspected and counted every 24 h for 5 days. (B) Heat maps of the number of calculated cell divisions per well for each CD34- SLAM cell at each day of culture. Left heat map: culture in SFM 2GF; right heat map: culture in SFM 4GF. Each row represents cell divisions of a single cell in a single well. (C) Heat maps of the number of calculated cell divisions per well for each CD34- SLAM cell at each day of culture. Left heat map: culture in MEF-CM 4GF; right heat map: culture in MEF-CM 2GF. Again, each row represents cell divisions of a single cell in a single well. (D) Percentage of wells with detectable cells per day from each plate cultured in SFM 4GF, MEF-CM 4GF, and MEF-CM 2GF. (E) Percentage of wells with divided cells (≥2 cells) from each plate cultured in SFM 4GF, MEF-CM 4GF, and MEF-CM 2GF. (F) Mean hours of time to first division of cells cultured in SFM 4GF, MEF-CM 4GF, and MEF-CM 2GF. (G) Mean clone size per day from each plate cultured in SFM 4GF, MEF-CM 4GF, and MEF-CM 2GF. Representative examples of two plates are shown in (B) and (C). Panels (D-G) show the results of at least four independent experiments with separate donor mice totalling: n=11 for SFM 4GF, n=15 for MEF-CM 4GF, and n=11 for MEF-CM 2GF. In all graphs, black dots represent results for SFM 4GF, blue dots represent results for MEF-CM 4GF and red dots represent results for MEF-CM 2GF. *P<0.05 using the non-parametric Mann-Whitney U-test of comparisons between SFM 4GF and either MEF-CM 4GF or MEF-CM 2GF. MEF: mouse embryonic fibro- blasts; SFM: serum-free medium; CM: conditioned medium; 2GF: two growth factors (i.e., stem cell factor [SCF] and interleukin-11 [IL-11]); 4GF: four growth factors (i.e., SCF, IL-11, nerve growth factor [NGF] and collagen 1 [Col1]); SLAM: CD48- CD150+ Lineage- SCA1+ KIT+; FACS: fluorescence activated cell sorting.
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haematologica | 2021; 106(10)