Figure 4.

Lentiviral Gene Transduction into Human iPSC-Derived Intestinal Organoids

(A) Schematic representation of gene transduction into organoids. TkDN4-M-derived organoids cultured in Matrigel were disrupted by a 29G needle, seeded in collagen I-coated plates, and cultured for 4 days. After 2D cultures from organoids were infected by lentiviruses, cells (including IESCs) were trypsinized, harvested, and re-embedded in Matrigel to reform organoids.

(B) The proportions of Venus⁺ organoids per microscopic bright field after 7 days of infection with IRES-Venus lentivirus. “3D” and “2D” indicate lentiviral infection into organoids harvested from Matrigel and 2D-cultured cells in collagen I-coated plates, respectively. The assay was performed in three independent biological replicates. Data are presented as mean ± SEM. ^∗p < 0.05, Student's t test.

(C) Bright-field and fluorescent images of TkDN4-M-derived organoids as in (B). Scale bars, 200 μm.

(D) Regenerated organoid numbers per microscopic bright field followed by 2D cultures after 7 days of infection with or without IRES-Venus lentivirus. The assay was performed in three independent biological replicates. Data are presented as mean ± SEM.

(E) Bright-field and fluorescent images of TkDN4-M-derived organoids 3 weeks after infection with Wnt3a-IRES-Venus lentivirus in 2D cultures. Scale bars, 200 μm.

(F) Relative mRNA levels of exogenous Wnt3a and Wnt target genes (LGR5, AXIN2, and EPHB3) in TkDN4-M-derived organoids infected with lentiviruses as in (E). The relative mRNA levels were determined by qPCR and normalized to GAPDH. The assay was performed in three independent biological replicates. Expression levels presented as mean ± SEM. ^∗p < 0.05, Student's t test. n.d., not detected.