Erythroid differentiation

CD34+ expansion media composition:

• Once the CD34+ cells are isolated from the PBMNCs, the cells can be resuspended in the expansion media (day 0).
• Seeding density: 0.5–0.7 × 10⁶ cells/ml.
• On day 2 of expansion perform base editing and maintain the cells in the expansion media till day 6, with a media change on day 4.
• Base editing is carried out with 1 million CD34+ cells + 100pm synthego gRNA + 5µg of ABE 8e mRNA. 
• Seeding density after editing: 0.5 × 10⁶ cells/ml.
• On day 6 we can set for erythroid differentiation

CD34+ erythroid differentiation media:

• The composition given above is for Phase 1 media (media change on day 0 and day 3).
• Without IL3 and hydrocortisone is Phase 2 media (media change on day 6 and day 9).
• Without IL3, SCF and hydrocortisone is Phase 3 media (media change on day 12, 15 and 18).
• Seeding density: 0.8× 10⁶ cells/ml.
• We start differentiation in 65mm dish (Day0) and then change to 10cm dish during differentiation (day 3, 6, 9, 12, 15, 18).