Figure 4. LPA dependency and reversibility of apical-out organization.

(A) Dose-response series for LPA for whole organoids.

(B) Higher magnification views from the panels in (A) highlight the intermediate effect at 3 nM, the most consistent apical-out structure from 30 to 300 nM, and obvious loss of ZO1 tight-junctions in some regions with 3,000 nM LPA treatment.

(C) Images of time series with vehicle (BSA) or LPA treatment at 0–48 h.

(D) Quantification of dose response data presented in (A and B) with four to eight organoids per group across two independent experiments. An agonist curve (black) was fitted to the data from 0 to 300 nM LPA. A quadratic equation was fitted to the data from 3 to 3,000 nM (blue) to show the inverted U. The mean LPA concentration and SD from human CSF are also shown as vertical dashed and dotted lines, respectively.

(E) Quantification of time series resented in (C) with three or four organoids per group from one experiment. An exponential plateau curve was fit to the data.

(F) At 96 h, the polarity persists based on the treatment; however, at 48 h, some organoids were switched from LPA-into BSA vehicle-containing media or from BSA into LPA with the resulting polarity (apical-in or apical-out) determined by the final treatment condition.

Organoids are day 7 of differentiation. Scale bars, 100 μm. Each data point is a single organoid.