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 later.
10. Tube formation on Matrigel was performed as described.
Reference
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2. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002; 418: 41–49.
3. Rafii S, Lyden D, Benezra R, Hattori K, Heissig B. Vascular and haematopoietic stem cells: novel targets for anti-angiogenesis therapy? Nat Rev Cancer. 2002; 2: 826–835.
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