Fig. 3

Development and in vitro characterization of transgenic hiPSCs. A The coding sequences of several of the described nBios were cloned into piggyBac transposon vectors in which nBio expression is driven by the constitutive CAG promoter and linked to a puromycin-resistance gene. Vectors were separately transfected into FS-hiPSCs and clonal cells expressing the nBio transgenes were isolated by puromycin selection. TR, terminal repeat; IRES, internal ribosome entry site; pA, polyadenylation. B Quantification of nBios secreted into the culture supernatant by clonal transgenic hiPSCs by anti-human Fc ELISA. Each dot represents a separately generated clone expressing the same nBio format, according to its color. Bars represent the mean clonal secretion of nBio formats ± SEM of two independent experiments. The differences between average secretion levels between nBio formats is not significant. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. Wt, wild-type; ND, not detected. C Neutralization of SARS-CoV-2 pseudovirus by hiPSC-derived nBios on hACE2-overexpressing HEK293T target cells. Inhibition of infection was measured as a function of nBio concentration in the supernatant of the highest expressing hiPSC clone of each format. Two independent experiments were performed with similar results. Curves were fit by nonlinear regression. Error bars represent SD