Single MEPs isolated from Gata1^low mice generate with high frequency cells with extensive proliferation capacity under BMMC conditions. (A) Experimental design used to measure the differentiation and proliferation potential of single Gata1^low MEPs. MEPs isolated from the marrow of Gata1^low mice were diluted at a concentration of 3 cells/mL, and 100 μL of this solution cultured in each well of a 96-multiwell plate (Table S4). At this cell concentration, 30% of the wells will contain, on average, one cell and the probability that one of them will contain 2 cells is less than 9%. Seven days later, the wells were scored for sign of proliferation. Number and morphology of the cells present in 10 randomly selected wells were analyzed. Fourteen days later, the cells contained in 10 additional randomly selected wells, were harvested, counted, and cloned under limiting conditions in secondary single-cell cultures. This procedure was repeated again after 21 (tertiary cultures) and 28 (quaternary cultures) days, as indicated. MCPs purified from wild-type mice were processed in parallel, as control. (B) Cloning efficiency over time (ie, number of wells in which cell growth was detected) observed in primary cultures seeded with single wild-type MCPs (▪) and Gata1^low MEP (•). Results are presented as mean (± SD) of those obtained in 4 independent experiments per animal group (see also Table S4). (C) May-Grünwald staining of the progeny observed over time in cultures of a representative wild-type MCP and a representative Gata1^low MEP, as indicated. At day 7, the progeny of wild-type MCP have an homogeneous mastocytic morphology, whereas megakaryocytes (Mk), erythroblasts (Ery), and mast cells (MC) are present among the progeny of Gata1^low MEPs. From 14 days on, only cells with mast cell morphology were detectable in the cultures. The results are representative of those obtained with at least 10 individual wells per experimental point; b.d. = below detectable levels. Original magnification, × 40. Images were obtained using a Zeiss Axiostat Plus microscope equipped with a Zeiss CP-ACHORMAT 40×/0.65 objective lens (Zeiss, Arese, Italy). Photographs were taken with an Olympus Camedia c-5060 digital camera and were acquired with Camedia Master 4.1 software (Opera Zerbo, Milan, Italy). Subsequent image processing was performed with Adobe Photoshop 6.0 software. (D) Total cell number per well and replating efficiency (ie, number of single cells that proliferated in following passages) in single-cell cultures of the progeny of wild-type MCPs (▪) and Gata1^low MEPs (•). Results are presented as mean (± SD) of those obtained in 3 independent experiments per animal group. Values statistically different (*P < .01, **P < .001) between cultures of wild-type and Gata1^low cells (see also Table S4).