Supplementary Methods
Isolation, stripping, reconstitution and functional analysis of FLM-erythroblast clusters
Clusters composed of FLMs and erythroblasts were isolated with a modified version of a published protocol1. Briefly, livers from individual E13.5 embryos were placed in prewarmed 0.05% collagenase (Sigma) and 0.002% DNase (Sigma) in RPMI and digested for 1 h at 37°C with gentle rotation. After tissue dissociation, cells were washed three times, suspended in 100 l of complete medium (RPMI plus 10% FBS) and allowed to attach to glass coverslips for 20 min at 37°C in 5% CO2. Cells were then flooded with medium and incubated further for 4 h. Adherent macrophages with attached erythroid clusters were obtained by dipping coverslips in RPMI to remove nonadherent cells (“native” clusters). To strip erythroblasts from macrophages, cells were incubated in PBS lacking calcium and magnesium for ten minutes and flushed five times in PBS to remove erythroblasts (“stripped” macrophages). Adherent macrophages were allowed to respread in complete medium for 2 h before receiving fresh erythroblasts (“reconstituted” clusters). For this purpose, collagenase-digested fetal livers from normal E14.5 embryos were used as the source of erythroblasts. Cells were pooled from multiple fetal livers and plated in 100-mm dishes for 4 h at 37°C to remove the attached macrophages. Non-adherent erythroid cells were recovered by flushing and washed in PBS. Typically, several million erythroblasts were isolated from the pooled fetal livers of one E14.5 litter. 106 cells in 50 l of medium were reincubated for 20 min with stripped macrophages derived from individual embryos. Finally, the reconstituted clusters were dipped to remove the unbound cells before being processed for double F4/80-TER119 immunofluorescence using standard procedures. The number of F4/80-positive macrophages and TER119-positive erythroblasts attached to adherent macrophages was determined by counting the total number of cells present in each native and reconstituted culture using a dual color Zeiss LSM 510 confocal fluorescence microscope. Statistical differences were determined by using an unpaired t test (Statview) and values are reported as the mean ± s.e.m.
Erythroid progenitors were obtained from the fetal liver of Rb+/+ and Rb-/- E12.5 embryos and cultured in serum-free medium containing 2 U/ml human recombinant erythropoietin (Epo, R&D Systems), 100 ng/ml murine recombinant stem cell factor (SCF, R&D Systems), 1 M dexamethasone (Sigma) and 40 ng/ml IGF-1 (Sigma) as described2. Erythroblasts were used for reconstitution experiments with FLMs after 7 days in culture. To analyze erythroid differentiation and enucleation after association with FLMs, wild type FLMs were stripped and reconstituted with 106Rb+/+ or Rb-/- erythroblasts and the reconstituted, heterologous erythroblastic islands were cultured on glass coverslips for 12 h in medium containing 30% FBS, 1 U/ml recombinant Epo, 40 ng/ml IGF-1, 300 g/ml iron-saturated transferrin (Sigma) and 1000 U/ml human recombinant CSF-1 (gift of Chiron, Emmeryville, CA). Control cultures included erythroblasts grown for 12 h in the same medium without FLMs and analyzed as cytospin. Cells were fixed and stained for F4/80 and TER119 and the presence or absence of nuclei was established by co-staining with DAPI. At least 500 erythroid cells were counted for each coverslip and the percentage of TER119-positive/DAPI-negative cells (enucleated red cells) was determined on a confocal fluorescence microscope.
References
1.Morris, L., Crocker, P. R. & Gordon, S. Murine fetal liver macrophages bind developing erythroblasts by a divalent cation-dependent hemagglutinin. J Cell Biol 106, 649-56 (1988).
2.von Lindern, M. et al. Leukemic transformation of normal murine erythroid progenitors: v- and c-ErbB act through signaling pathways activated by the EpoR and c-Kit in stress erythropoiesis. Oncogene 20, 3651-64 (2001).
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