MKs were differentiated from human iPSCs (HPSI1113i-qolg_3 HipSci cell line, wild-type and ZFPM2 knockout) using the protocol described in Moreau et al.32 (link). Briefly, cells were trypsinized and seeded with 100,000 cells per well. The next day, cells were infected with lentiviruses encoding the GATA1, FLI1 and TAL1 transcription factors (Vectalis). The first two days after infection, cells were cultured with BMP4 (BioTechne) and FGF2 (Wellcome—MRC Cambridge Stem Cell Institute, Tissue Culture facility) to induce mesoderm. Eighteen days were allowed for the differentiation and maturation of MKs, during which flow-cytometry experiments were used to monitor the expression of CD41a (BD, cat 559777) and CD42b (BD, 555473) as measures of maturation. Three technical replicates were nested within each of three biological replicates: two knockouts and one wild-type.
Akbari P., Vuckovic D., Stefanucci L., Jiang T., Kundu K., Kreuzhuber R., Bao E.L., Collins J.H., Downes K., Grassi L., Guerrero J.A., Kaptoge S., Knight J.C., Meacham S., Sambrook J., Seyres D., Stegle O., Verboon J.M., Walter K., Watkins N.A., Danesh J., Roberts D.J., Di Angelantonio E., Sankaran V.G., Frontini M., Burgess S., Kuijpers T., Peters J.E., Butterworth A.S., Ouwehand W.H., Soranzo N, & Astle W.J. (2023). A genome-wide association study of blood cell morphology identifies cellular proteins implicated in disease aetiology. Nature Communications, 14, 5023.
Other organizations :
British Heart Foundation, University of Cambridge, Wellcome Sanger Institute, Boston Children's Hospital, Harvard University, National Institute for Health Research, University of Oxford, Wellcome Centre for Human Genetics, European Bioinformatics Institute, Wellcome Trust, National Health Service, MRC Biostatistics Unit, Medical Research Council, Health Data Research UK
Genetic modification (wild-type vs. ZFPM2 knockout)
dependent variables
Expression of CD41a and CD42b (as measures of MK maturation)
control variables
Cell line (HPSI1113i-qolg_3 HipSci)
Cell seeding density (100,000 cells per well)
Infection with lentiviruses encoding GATA1, FLI1 and TAL1 transcription factors
Use of BMP4 and FGF2 to induce mesoderm
Duration of differentiation and maturation (18 days)
controls
Positive control: Not explicitly mentioned
Negative control: Wild-type (non-knockout) cell line
Annotations
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