Kidney Organoid Generation at the Air-Liquid Interface

Summary

This protocol describes asynchronous mixing of human embryonic stem cells derived kidney progenitors at the air-liquid interface to efficiently generate kidney organoids.

Abstract

The prevalence of kidney diseases continues to increase worldwide, driving the need to develop transplantable renal tissues. The kidney develops from four major renal progenitor populations: nephron epithelial, ureteric epithelial, interstitial and endothelial progenitors. Methods have been developed to generate kidney organoids but few or dispersed tubular clusters in the organoids hamper its use in regenerative applications. Here, we describe a detailed protocol of asynchronous mixing of kidney progenitors using organotypic culture conditions to generate kidney organoids tightly packed with tubular clusters and major renal structures including endothelial network and functional proximal tubules. This protocol provides guidance in the culture of human embryonic stem cells and their directed differentiation to kidney organoids. Our 18-day protocol provides a rapid method to generate kidney organoids that facilitate the study of different nephrological events including in-vitro tissue development, disease modeling and chemical screening. However, further studies are required to optimize the protocol to generate additional renal-specific cells types, interconnected nephron segments and physiologically functional renal tissues.

Introduction

Chronic kidney disease (CKD) is a worldwide healthcare problem with 13.4% global estimated prevalence¹. Approximately 10% of the adult population of the United States suffers from CKD². There is no curative treatment available for patients with CKD except renal transplantation. The lack of availability of transplantable organs warrants research into technologies to understand how new kidney tissues can be generated. In recent years, procedures have been reported to generate kidney organoids^3,4 from human embryonic stem cells (hESCs) and human induced pluripotent ....

Protocol

Cell line WA09 (H9) was approved by the National Institutes of Health (registration number 0062) and was tested negative for mycoplasma infection.

1. Medium and plate preparation for hESCs culture

1. Dilute matrix (reduced growth factor basement membrane e.g., Geltrex) in DMEM/F12 (1:100) and add 1 mL/well in 2 wells of a 6 well plate (reagents utilized in the manuscript are summarized in the Table of Materials).
2. Incubate coated plate undisturbed at 37 °.......

Discussion

Asynchronous mixing of progenitors at the air-liquid interface (Figure 1) presents an efficient method to generate kidney organoids from hESCs. This work describes stepwise protocols for thaw and culture of hESCs, directed differentiation to kidney progenitors, making cell aggregates at the air-liquid interface, asynchronous mixing of progenitors to generate kidney organoids tightly packed with tubular clusters and major renal structures including endothelial network and functional proximal .......

Materials

Name	Company	Catalog Number	Comments
15 mL falcon tube	VWR	62406-200
24 well plate	VWR	29443-952
40 micron strainer	VWR	21008-949
50 mL falcon tube	VWR	21008-940
6 well plate	VWR	29442-042
96 well Clear Round Bottom Ultra-Low Attachment Microplate (U bottom)	Corning	7007
Accutase	STEMCELL Technologies	7920	Store at -20 °C
Activin A	R&D systems	338-AC-010	Aliquot and store at -20 °C
Advanced RPMI 1640	ThermoFisher Scientific	12633-012	Store at 4 °C
APEL2	STEMCELL Technologies	5270	Store at -20 °C
BMP7	R&D systems	354-BP-010	Aliquot and store at -20 °C
CHIR99021 IN SOLUTION	Reprocell	04-0004-10	Aliquot and store at -20 °C
Confocal microscope	Leica Microsystems	SP8
Dextran, AF 488, 10,000 MW	Thermo fisher Scientific	D22910	Store at -20 °C
DMEM/F12	Thermo fisher Scientific	11330-032	Store at 4 °C
DPBS	VWR	45000-434
FBS	Atlanta Biologicals	S11550	Store at -20 °C
FGF2	R&D systems	234-FSE-025	Aliquot and store at -20 °C
FGF9	R&D systems	273-F9-025	Aliquot and store at -20 °C
Forceps	Roboz	RS-5040
Geltrex	Thermo fisher Scientific	A1413301	Aliquot and Store at -20 °C
Glutamax	Thermo fisher Scientific	35050061
H9 cells	WiCell	WA09	Store in liquid Nitrogen
Heparin	Sigma	H3393-25KU
Isopore membrane	EMD Millipore	VCTP01300
Paraformaldehyde	P6148-500G	P6148-500G
PFHM II	Thermo fisher Scientific	12040077	Store at 4 °C
Rock inhibitor Y-27632	EMD Millipore	688002-1mg	Aliquot and store at -20 °C
StemFit Medium	amsbio	SFB-500	Store at -20 °C
Triton X-100	Sigma	X100-100ML
TrypLe express	Thermo fisher Scientific	12563029