Kidney Week

Abstract: TH-OR043

Highly Efficient Organoid Cystogenesis Reveals a Critical Role for Physical Microenvironment in Human Polycystic Kidney Disease

Session Information

• Cystic Kidney Diseases: Genes, Mechanisms, Interventions
  November 02, 2017 | Location: Room 390, Morial Convention Center
  Abstract Time: 04:54 PM - 05:06 PM

Category: Genetic Diseases of the Kidney

• 801 Cystic Kidney Diseases

Authors

• Cruz, Nelly M., University of Washington, Seattle, Washington, United States

Nelly M. Cruz, PhD



Nelly M. Cruz holds a Ph.D. degree in Biology from the Massachusetts Institute of Technology (MIT). She also completed a Postdoctoral Research Fellowship at the Department of Ophthalmology, Harvard Medical School and served as the Instructor of undergraduate laboratory courses at the MIT Biology Department. She is a Research Scientist in Dr. Benjamin Freedman Laboratory at the University of Washington, where she continues to mentor undergraduate students and investigates polycystic kidney disease using pluripotent stem cell-derived kidney organoids.

• Song, Xuewen, University Health Network and University of Toronto, Toronto, Ontario, Canada

Xuewen Song,

• Pei, York P., University Health Network and University of Toronto, Toronto, Ontario, Canada

York P. Pei,

• Freedman, Benjamin S., University of Washington, Seattle, Washington, United States

Benjamin S. Freedman,

Background

Polycystic kidney disease (PKD) is a life-threatening disorder in which tubular epithelia form fluid-filled cysts, disrupting organ architecture. A major barrier to understanding the pathophysiology of PKD is the absence of human cellular models that accurately and efficiently recapitulate cystogenesis. Previously, a genetic model of PKD has been generated using human pluripotent stem cells (hPSCs) and derived kidney organoids. Here we show that systematic substitution of physical components in this system can be used to dramatically increase or decrease cyst formation, unveiling a critical role for microenvironment in PKD.

Methods

Genome-modified PKD1^-/-, PKD2^-/-, or isogenic control hPSCs were differentiated in vitro into kidney organoids and subsequently cultured in the presence or absence of extracellular matrix and stroma. To directly test the effect of PKD mutations on the matrix microenvironment, individual organoids were embedded into larger collagen droplets. Structure and composition of organoid cysts were analyzed by immunofluorescence, microarray and immunoblot, and compared histologically to disease tissue from PKD patients.

Results

Removal of adherent cues increased cystogenesis 10-fold, producing cysts that phenotypically resembled human PKD cysts and expanded massively to 1-centimeter diameters. Non-PKD control organoids of identical genetic background did not form cysts. Cysts derived from hyperproliferative kidney tubular epithelial cells and showed upregulation of known PKD signaling pathways. Removal of stroma enabled proliferation and outgrowth of PKD cell lines, which revealed PC1 deficiency as a common molecular endpoint. Collagen droplets implanted with organoids furthermore contracted dramatically over a period of 2 weeks in a PKD-dependent manner.

Conclusion

Culture conditions have a significant impact on rates of cystogenesis in PKD organoids, enabling us to establish a highly efficient model of PKD cystogenesis. Collagen contraction could not be explained by differences in proliferation alone. Our findings directly implicate the microenvironment as a critical component at the earliest stages of PKD, with PC1 likely functioning as an adhesive or signaling molecule to control tissue contractility.

Funding

• NIDDK Support – Northwest Kidney Centers (unrestricted gift)