Pesticide-Exposed BM-MSCs and CD34+ Hematopoietic Progenitor Assay

BM-MSCs were exposed to pesticides only during the 21 days of expansion culture before co-cultures with normal CD34⁺ cells, and CAFC assays were performed as previously described and as detailed in supplementary methods [22 (link)]. Briefly, CD34⁺ cells were thus co-cultured with the pesticide-exposed BM-MSCs for 7 days (37 °C, 5% CO₂), seeded at 6000 CD34⁺/cm² (ratio CD34⁺ cells/BM-MSCs = 1/3) in Myelocult™ H5100 medium (StemCell Technologies, Vancouver, Canada). At the end of coculture, CD34⁺ cells were sorted (BD FACSMelody^TM cell sorter, Becton-Dickinson) and cultured on MS5 stromal cells [25 (link)] for 5 additional weeks in 96-well plates (37 °C, 5% CO₂) at 6 different dilutions (range 1–100 CD34⁺ cells/well) in 0.2 mL of Myelocult™ H5100 medium. After 5 weeks of co-culture, each well was examined by inverted phase contrast microscopy (DMI 3000 B, Leica) to identify cobblestone areas. The percentage of wells with at least one CAFC was quantified and the frequency of CAFC in the initial CD34⁺ cell population was calculated as previously described [26 (link)]. The CAFC proliferative capacity was determined by subculturing the wells containing one cobblestone area in semi-solid medium (MethoCult^TM H4434, StemCell Technologies) to determine the mean total number of clonogenic progenitors (including CFU-GM, BFU-E and CFU-M) generated by each CAFC.