Design and strategy for manufacturing kidney organoids

Despite a continuing increase in the number of patients suffering from chronic kidney disease,currently available treatments for these patients,such as dialysis and kidney transplantation,are imperfect.The kidney is also a critical target organ vulnerable to the toxicity of various new drugs,and the lack of a reliable in vitro culture model imposes a severe limitation on drug discovery.Although the development of induced pluripotent stem cells(iPSCs)revolutionized strategies in biomedical fields,the complexity of the kidney imposed additional challenge to the application of this technology in kidney regeneration.Nonetheless,the recent advancement in our understanding on the developmental origin of kidney progenitor cells and the mechanisms of their reciprocal induction and self-organization has boosted research in kidney regeneration.Research since then has demonstrated that kidney organoids derived from iPSCs can serve as a useful model for drug discovery and toxicity screening,as well as for disease modeling,especially in combination with gene editing techniques.Moreover,attempts at kidney organoid implantation in animals have suggested their potential as an alternative source of kidney transplantation.In this review,we summarize recent progress on the generation of kidney organoids,as well as the obstacles that remain.

Growth and differentiation of human induced pluripotent stem cell(hiPSC)-derived kidney organoids using fully synthetic peptide hydrogels

Human induced pluripotent stem cell(hiPSC)-derived kidney organoids have prospective applications ranging from basic disease modelling to personalised medicine.However,there remains a necessity to refine the bio-physical and biochemical parameters that govern kidney organoid formation.Differentiation within fully-controllable and physiologically relevant 3D growth environments will be critical to improving organoid reproducibility and maturation.Here,we matured hiPSC-derived kidney organoids within fully synthetic self-assembling peptide hydrogels(SAPHs)of variable stiffness(storage modulus,G′).The resulting organoids con-tained complex structures comparable to those differentiated within the animal-derived matrix,Matrigel.Single-cell RNA sequencing(scRNA-seq)was then used to compare organoids matured within SAPHs to those grown within Matrigel or at the air-liquid interface.A total of 13,179 cells were analysed,revealing 14 distinct clusters.Organoid compositional analysis revealed a larger proportion of nephron cell types within Transwell-derived organoids,while SAPH-derived organoids were enriched for stromal-associated cell populations.Notably,dif-ferentiation within a higher G’SAPH generated podocytes with more mature gene expression profiles.Addi-tionally,maturation within a 3D microenvironment significantly reduced the derivation of off-target cell types,which are a known limitation of current kidney organoid protocols.This work demonstrates the utility of syn-thetic peptide-based hydrogels with a defined stiffness,as a minimally complex microenvironment for the selected differentiation of kidney organoids.

Kidney organoids as a promising tool in nephrology

Kidney disease has become a global public health problem affecting over 750 million people worldwide and imposing a heavy economic burden on patients.The complex architecture of the human kidney makes it very difficult to study the pathophysiology of renal diseases in vitro and to develop effective therapeutic options for patients.Even though cell lines and animal models have enriched our understanding,they fail to recapitulate key aspects of human kidney development and renal disease at cellular and functional levels.Organoids can be derived from either pluripotent stem cells or adult stem cells by strictly regulating key signalling pathways.Today,these self-differentiated organoids represent a promising technology to further understand the human kidney,one of the most complex organs,in an unprecedented way.The newly established protocols improved by organ-on-chip and coculture with immune cells will push kidney organoids towards the next generation.Herein,we focus on recent achievements in the application of kidney organoids in disease modelling,nephrotoxic testing,precision medicine,biobanking,and regenerative therapy,followed by discussions of novel strategies to improve their utility for biomedical research.The applications we discuss may help to provide new ideas in clinical fields.

High-throughput“read-on-ski”automated imaging and label-free detection system for toxicity screening of compounds using personalised human kidney organoids

Organoid models are used to study kidney physiology,such as the assessment of nephrotoxicity and underlying disease processes.Personalized human pluripotent stem cell-derived kidney organoids are ideal models for compound toxicity studies,but there is a need to accelerate basic and translational research in the field.Here,we developed an automated continuous imaging setup with the“read-on-ski”law of control to maximize temporal resolution with minimum culture plate vibration.High-accuracy performance was achieved:organoid screening and imaging were performed at a spatial resolution of 1.1µm for the entire multi-well plate under 3 min.We used the in-house developed multi-well spinning device and cisplatin-induced nephrotoxicity model to evaluate the toxicity in kidney organoids using this system.The acquired images were processed via machine learning-based classification and segmentation algorithms,and the toxicity in kidney organoids was determined with 95%accuracy.The results obtained by the automated“read-on-ski”imaging device,combined with label-free and non-invasive algorithms for detection,were verified using conventional biological procedures.Taking advantage of the close-to-in vivo-kidney organoid model,this new development opens the door for further application of scaled-up screening using organoids in basic research and drug discovery.