Fig. 5: Periportal assembloids as tools to investigate disease mechanisms.
From: Mouse liver assembloids model periportal architecture and biliary fibrosis
a–d, Short interfering RNA (siRNA)-mediated knockdown experiments in assembloids. Mesenchyme cells (green nuclei and magenta membrane) were transfected with targeting or non-targeting siRNAs before assembly. a, Experimental design, including schematic of the DETECT distance ratio metric (d1/d0). b, Live imaging analysis of assembloids formed with mesenchyme cells transfected with non-targeting (left) or Cdh11 (right) siRNA. Cell boundaries are indicated by SiR-actin. The white dotted line indicates segmentation of the organoid border. Scale bars, 50 µm. c, Segmented assembloids from b were used as input for the DETECT algorithm and to calculate the DETECT distance metric. Violin plots show median and quartiles of the DETECT distance ratio for the non-targeting and Cdh11-knockdown (KD) group. Mann–Whitney test, two-tailed. Dots represent individual assembloids (n = 16 from 2 independent experiments). d, SHG images of assembloids showing fibrillar collagen deposition in non-targeting control and Cdh11-knockdown groups. PDGFRα–H2B–GFP marks portal fibroblasts. n = 2 independent experiments. Scale bars, 25 µm. e, Left, schematic of Mdr2+/+ and Mdr2−/− HepOrgs. Right, immunofluorescence images of HepOrgs derived from wild-type (top) or Mdr2−/− (bottom) livers show dilated bile canaliculi on Mdr2−/− organoids. CD13 (green) marks bile canaliculi. Phalloidin marks cell borders. n = 3 independent experiments with n = 3 biological replicates. Scale bars: 20 µm (main images), 10 µm (zoom views). f–h, Chimeric assembloids were formed by Mdr2−/− HepOrgs (bright field, grey), wild-type cholangiocytes (nTom-Chol) and wild-type portal mesenchyme (PDGFRa–H2B–GFP). f, Schematic of experimental design. g, Still images from live imaging experiments (n = 2 independent experiments) of assembloids formed with Mdr2+/+ or Mdr2−/− HepOrgs. Scale bars, 50 µm. h, Change in cholangiocyte numbers between day 0 and day 5 in assembloids formed with Mdr2+/+ or Mdr2−/− HepOrgs. Data are mean ± s.e.m. of 2 biological replicates from n = 2 independent experiments with n = 23 (Mdr2+/+) and n = 22 (Mdr2−/−) assembloids. Dots represent individual assembloids. Mann–Whitney test, two-tailed.
