CD133 isolation, expansion, and differentiation

1. Renal progenitor cells were obtained from the normal portion of cortex obtained from surgically removed kidneys.
2. After dissection and passage through a graded series of meshes, CD133⁺ cells were isolated from the tubular fraction by magnetic cell sorting, using the MACS system.
3. CD133⁺ cells were plated onto fibronectin in the presence of an expansion medium, consisting of 60% DMEM LG, 40% MCDB-201, with 1× insulin-transferrin-selenium, 1× linoleic acid 2-phosphate, 10−9 mol/L dexamethasone, 10−4 ascorbic acid 2-phosphate, 100 U penicillin, 1000 U streptomycin, 10 ng/ml epidermal growth factor, and 10 ng/ml platelet-derived growth factor-BB and 2% fetal calf serum.
4. For cell cloning, single cells were deposited in 96-well plates in the presence of the expansion medium.
5. Epithelial differentiation was obtained in the presence of fibroblast growth factor-4 (10 ng/ml) and hepatocyte growth factor (20 ng/ml).
6. Endothelial differentiations were obtained by culturing the cells in EBM medium with vascular endothelial growth factor (10 ng/ml) and 10% fetal calf serum on endothelial cell attachment factor.
7. CD133+ cells were also isolated from the blood of granulocyte-colony stimulating factor mobilized patients using the MACS system.
8. Mesenchymal cells were obtained from the bone marrow of healthy donors and cultured in α-minimal essential medium supplemented with 10% fetal calf serum and 10% horse serum.
9. The nonadherent cells were removed by medium change at 48 hours and every 4 days thereafter.
10. Tube formation on Matrigel was performed as described.

Reference
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