Fig. 1: CRISPR-ETV2 knock-in line in RTT patient-derived MeCP2[R306C] iPS cells.

a Schematic illustration of engineering RTT’s MVN in the microfluidic device. The RTT patient-derived iPS cells that carry MECP2[R306C] mutations and isogenic controls were modified by knocking in the ETV2-gene cassette using CRISPR/Cas9 to accelerate endothelial differentiation. Then, iECs and fibroblasts were introduced to the microfluidic device to form MVNs. b, c, d ETV2 under Tet3G promoter cassettes were inserted into AAVS1 safe harbor loci, and cells were sorted by FACS based on the mCherry fluorescent marker to obtain a stable iPS cell line. iECs were obtained in 5 days from iPS cells in the presence of DOX. d Single cloned iPS cells (RTT patient-derived cells and controls) with ETV2 gene cassettes (mCherry) and representative images of the endothelial cell morphology 5 days after differentiation. e Endothelial differentiation was confirmed by flow cytometry according to CD31, CD34, VEGFR2, and UEA-1 expression.