AAV tropism in kidney organoids derived from human pluripotent stem cells.

Experiment: AAV tropism in kidney organoids derived from human pluripotent stem cells.

PI: Ryuji Morizane, MD, PhD

Description: Human kidney organoids generated from human ES and iPS cells are used to evaluate tropism of AAV2, AAV8, and AAV9 that express eGFP under CMV promoter. Organoids are exposed to AAVs at MOI 10^5 from differentiaiton day 21 to 28. Immunohistochemistry is performed to visualize transduced cells with staining for podocytes (PODXL), proximal tubules (LTL), loops of Henle and distal nephrons (CDH1), interstitial stromal cells (PDGFRb), and endothelia (CD31). AAV2/2 shows the highest transduction efficiency among three serotypes evaluated, and the GFP expression is highest in proximal tubular cells.

Delivery Assays: Transduction efficiency was calculated by counting fluorescent cells (GFP) among LTL+ Proximal Tubule, CDH1+ Loop of Henle/Distal Tubule, PDGFR-B+ Interstitial, CD31+ Vascular, and PODXL+ podocyte cells.

Parent Project: Vascularized kidney organoids on chip for efficacy and toxicity testing of somatic genome editing

Other experiments in this project: 6

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Delivery Systems

AAV

Models

Human kidney organoid (stem cell-derived)

Vectors

AAV2/2CMVeGFP

AAV2/8CMVeGFP

AAV2/9CMVeGFP

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Results

AAV_tropism_in_kidney_organoids_derived_from_human_pluripotent_stem_cells.
					Delivery Efficiency
Record Id	Condition	Model	Delivery	Vector	Dosage	Delivery Efficiency % GFP positivity in LTL+ cells	Delivery Efficiency % GFP positivity in CDH1+ cells	Delivery Efficiency % GFP positivity in PDGFR-B+ cells	Delivery Efficiency % GFP positivity in CD31+ cells	Delivery Efficiency % GFP positivity in PODXL+ cells	Image
15000003399	Control	Human kidney organoid (stem cell-derived)			control	0; 0; 0; 0; 0; 0;	0; 0; 0; 0; 0; 0;	0; 0; 0; 0; 0; 0;	0; 0; 0; 0; 0; 0;	0; 0; 0; 0; 0; 0;
15000003400	AAV2/2	Human kidney organoid (stem cell-derived)	AAV	AAV2/2CMVeGFP	MOI=10^5	38.3; 45.6; 69.1; 40.49; 44.89; 38.95;	77.777; 60; 80.39; 25; 9.6; 21.9;	11.11; 20.89; 17.02; 15.38; 10.83; 12.57;	0; 9.0909; 0; 0; 7.14; 5.88;	0; 2.4; 1.6; 1.74; 1.95; 2.97;
15000003401	AAV2/8	Human kidney organoid (stem cell-derived)	AAV	AAV2/8CMVeGFP	MOI=10^5	0; 0.68; 0.74; 2.07; 0.99; 4.8;	4.444; 3.17; 0; 1.7; 0; 0.99;	7.14; 2.72; 3.17; 1.4; 0; 1.02;	7.69; 0; 0; 0; 0; 0;	0; 0; 0; 0; 0.65; 1.05;
15000003402	AAV2/9	Human kidney organoid (stem cell-derived)	AAV	AAV2/9CMVeGFP	MOI=10^5	4.3; 2; 4; 4.16; 5.62; 8.28;	0; 4.5; 0; 0.71; 2.49; 1.14;	4.17; 2.67; 0; 2.53; 1.68; 0;	0; 0; 0; 0; 0; 0;	0; 4.5; 0; 0.71; 2.49; 1.14;

AAV tropism in kidney organoids derived from human pluripotent stem cells. (A) Immunostaining images of kidney organoids exposed to AAV2/2, 2/8, or 2/9 at MOI 10^5 for 1 week. 5 cell types in kidney organoids are visualized by staining for LTL (proximal tubules), PODXL (podocytes), CDH1 (loops of Henle~distal nephrons), PDGFRb (interstitial stromal cells), and CD31 (endothelia). (B) Graphs showing percentages of AAV transduced cells in each cell type in kidney organoids.

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Experiment: AAV tropism in kidney organoids derived from human pluripotent stem cells.

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Results