Figure 4

Endothelial differentiation of the human Isl1⁺ progenitors can be achieved by repeated transfections of the VEGF-A modRNA. (A) FACS result showing expression of mCherry in the day-7 purified human Isl1-cre eGFP⁺ cells 24 h post transfection of the mCherry modRNA (1 μg/10⁶ cells). (B) Quantification of mCherry expression by FACS showing percentage of mCherry⁺ Isl1-cre eGFP⁺ cells following 0-150 h post transfection. (C) ELISA analyses using VEGF-A-containing supernatant (refreshed 6 h before collection) cultured with the day-7 purified human Isl1-cre eGFP⁺ cells following 0-72 h post transfection of different concentrations of the VEGF-A modRNA. (D) Trypan blue staining showing the survival percentage of the day-7 purified human Isl1-cre eGFP⁺ cells following 24 h post transfection of different concentrations of the VEGF-A modRNA. (E) ELISA analyses showing the rate of VEGF-A secreted by the day-7 purified human Isl1-cre eGFP⁺ cells (supernatant refreshed 6 h before collection) following two rounds of daily transfections of 1 μg/10⁶ cells with VEGF-A modRNA. (F) FACS analyses showing the efficiency of CD144⁺CD31⁺ EC differentiation by one transfection or repeated transfections (following medium change) of the VEGF-A modRNA compared to the use of VEGF-A protein 3 or 7 days post treatment.