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Preprint

Erythroid differentiation

KM Kumarasamypet M Mohankumar
NR Nithin Sam R

Last updated date: Jan 14, 2023 Views: 445 Forks: 0

original An abbreviated version of this protocol was published in eLife in Feb, 2022
Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin
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CD34+ expansion media composition:

ComponentsFromStock Conc.Working Conc.
StemSpan™ SFEM IISTEMCELL Technologies  
SCFPeprotech(100ng/µl)(240ng/ml)
FLT3Peprotech(100ng/µl)(240ng/ml)
IL6Peprotech(100ng/µl)(40ng/ml)
TPOPeprotech(50ng/µl)(75ng/ml)
Pen/StrepGibco(100X)(1X)
  • 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:

ComponentsFromStock Conc.Working Conc.
IMDM glutamaxGibco  
AB serumMP 5%
Insulin (5ml)Sigma9.5-11.5 mg/mL1ml/1000ml
Heparin 100U/mgSigma1200U/ml2U/ml
EPOZyrop(2U/µl)(3U/µl)
Holo-TransferrinBB sol330mg/ml330µg/ml
SCFPeprotech100ng/µl100ng/ml
IL3Peprotech5ng/µl2.5ng/ml
Pen/StrepGibco(100X)(1X)
HydrocortisoneSigma 1µM

 

  • 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).

 

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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Mohankumar, K and R, N(2023). Erythroid differentiation. Bio-protocol Preprint. bio-protocol.org/prep2117.
  2. Ravi, N. S., Wienert, B., Wyman, S. K., Bell, H. W., George, A., Mahalingam, G., Vu, J. T., Prasad, K., Bandlamudi, B. P., Devaraju, N., Rajendiran, V., Syedbasha, N., Pai, A. A., Nakamura, Y., Kurita, R., Narayanasamy, M., Balasubramanian, P., Thangavel, S., Marepally, S., Velayudhan, S. R., Srivastava, A., DeWitt, M. A., Crossley, M., Corn, J. E. and Mohankumar, K. M.(2022). Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin. eLife. DOI: 10.7554/eLife.65421

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