a-simplified-method-for-generating-kidney-organoids-from-human

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney ...

University of Auckland

Renal tubule epithelial cells can regenerate in response to acute injury. Although this process remains poorly understood, it appears to involve the reactivation of pathways that are operative during embryonic kidney formation. A better understanding of renal regeneration may lead to the development of new therapies that can attenuate acute kidney injury or expedite recovery. The zebrafish is being used as a model to understand renal regeneration. In this review, we summarize the current knowledge on zebrafish kidney formation, describe methods for inducing acute injury, and focus on the unique capacity of the zebrafish adult kidney to undergo de novo nephron formation in response to damage. 

Kidney injury and regeneration in zebrafish.

We report the development of a small fluorescent molecule, BDNCA3-D2, herein referred to as PT-Yellow. Soaking zebrafish embryos in PT-Yellow or intraperitoneal injection into adults results in non-toxic in vivo fluorescent labeling of the renal proximal tubules, the major site of blood filtrate reabsorption and a common target of injury in acute kidney injury. We demonstrate the applicability of this new compound as a rapid and simple readout for zebrafish kidney filtration and proximal tubule reabsorption function. 

The small molecule probe PT-Yellow labels the renal proximal tubules in zebrafish.

We describe the use of the fluorescent reporter compound CDr10b to label mid-intestinal structures in zebrafish larvae after simple immersion. CDr10b is deposited into the gut where it initially fills the lumen and is excreted. Using laser-mediated injury of the intestine, we show that CDr10b provides a useful readout of the integrity and repair of the epithelial cell barrier. In addition, CDr10b specifically labels the absorptive mid-intestine segment that is analogous to the mammalian small intestine. By perturbing retinoic acid signaling, which regulates the size of the mid-intestine segment, we show that CDr10b is a valuable tool to rapidly assess developmental malformations of the intestine in live animals.

The Vital Dye CDr10b Labels the Zebrafish Mid-Intestine and Lumen.

Kidney organoids made from pluripotent stem cells have the potential to revolutionize how kidney development, disease, and injury are studied. Current protocols are technically complex, suffer from poor reproducibility, and have high reagent costs that restrict scalability. To overcome some of these issues, we have established a simple, inexpensive, and robust method to grow kidney organoids in bulk from human induced pluripotent stem cells. Our organoids develop tubular structures by day 8 and show optimal tissue morphology at day 14. A comparison with fetal human kidneys suggests that day-14 organoid tissue most closely resembles late capillary loop stage nephrons. We show that deletion of HNF1B, a transcription factor linked to congenital kidney defects, interferes with tubulogenesis, validating our experimental system for studying renal developmental biology. Taken together, our protocol provides a fast, efficient, and cost-effective method for generating large quantities of human fetal kidney tissue, enabling the study of normal and aberrant kidney development.

A Simple Bioreactor-Based Method to Generate Kidney Organoids from Pluripotent Stem Cells.

The intermediate mesoderm is located between the somites and the lateral plate mesoderm and gives rise to renal progenitors that contribute to the three mammalian kidney types (pronephros, mesonephros and metanephros). In this review, focusing largely on murine kidney development, we examine how the intermediate mesoderm forms during gastrulation/axis elongation and how it progressively gives rise to distinct renal progenitors along the rostro-caudal axis. We highlight some of the potential signalling cues and core transcription factor circuits that direct these processes, up to the point of early metanephric kidney formation.

Turning mesoderm into kidney.

Mind the gap: renal tubule responses to injury and the role of Cxcl12 and Myc.

The hepatocyte nuclear factor-1β (Hnf1b) transcription factor is a key regulator of kidney tubule formation and is associated with a syndrome of renal cysts and early onset diabetes. To further our understanding of Hnf1b in the developing zebrafish kidney, we performed RNA sequencing analysis of proximal tubules from -deficient larvae. This analysis revealed an enrichment of gene transcripts encoding transporters of the solute carrier (SLC) superfamily, including multiple members of  and  glucose transporters. An investigation of expression of ,  and  as well as a poorly studied glucose/mannose transporter encoded by  revealed that these genes undergo dynamic spatiotemporal changes during tubule formation and maturation. A comparative analysis of zebrafish SLC genes with those expressed in mouse proximal tubules showed a substantial overlap at the level of gene families, indicating a high degree of functional conservation between zebrafish and mammalian proximal tubules. Taken together, our findings are consistent with a role for Hnf1b as a critical determinant of proximal tubule transport function by acting upstream of a large number of SLC genes and validate the zebrafish as a physiologically relevant model of the mammalian proximal tubule.

Transcriptional profiling of the zebrafish proximal tubule.

Acute kidney injury (AKI) remains a major global healthcare problem, and there is a need to develop human-based models to study AKI in vitro. Toward this goal, we have characterized induced pluripotent stem cell-derived human kidney organoids and their response to cisplatin, a chemotherapeutic drug that induces AKI and preferentially damages the proximal tubule. We found that a single treatment with 50 µM cisplatin induces hepatitis A virus cellular receptor 1 () and C-X-C motif chemokine ligand 8 () expression, DNA damage (γH2AX), and cell death in the organoids but greatly impairs organoid viability. DNA damage was not specific to the proximal tubule but also affected the distal tubule and interstitial cell populations. This lack of specificity correlated with low expression of proximal tubule-specific organic cation transporter 2 () for cisplatin. To improve viability, we developed a repeated low-dose regimen of 4 × 5 µM cisplatin over 7 days and found this caused less toxicity while still inducing a robust injury response that included secretion of known AKI biomarkers and inflammatory cytokines. This work validates the use of human kidney organoids to model aspects of cisplatin-induced injury, with the potential to identify new AKI biomarkers and develop better therapies.

Evaluation of cisplatin-induced injury in human kidney organoids.

Acute kidney injury (AKI) is a common clinical condition associated with diverse etiologies and abrupt loss of renal function. In patients with sepsis, rhabdomyolysis, cancer, and cardiovascular disorders, the underlying disease or associated therapeutic interventions can cause hypoxia, cytotoxicity, and inflammatory insults to renal tubular epithelial cells (RTECs), resulting in the onset of AKI. To uncover stress-responsive disease-modifying genes, here we have carried out renal transcriptome profiling in three distinct murine models of AKI. We find that  nerve growth factor inducible gene up-regulation is a common transcriptional stress response in RTECs to ischemia-, cisplatin-, and rhabdomyolysis-associated renal injury. The  gene encodes a secretory peptide precursor protein that has critical neuroendocrine functions; however, its role in the kidneys remains unknown. Our functional studies show that RTEC-specific  gene ablation exacerbates ischemia-, cisplatin-, and rhabdomyolysis-associated AKI  and cisplatin-induced RTEC cell death  Importantly, aggravation of cisplatin-induced renal injury caused by  gene ablation is partly reversed by TLQP-21, a Vgf-derived peptide. Finally,  and  mechanistic studies showed that injury-induced  up-regulation in RTECs is driven by the transcriptional regulator Sox9. These findings reveal a crucial downstream target of the Sox9-directed transcriptional program and identify  as a stress-responsive protective gene in kidney tubular epithelial cells.

SOX9 promotes stress-responsive transcription of VGF nerve growth factor inducible gene in renal tubular epithelial cells.

Kidney organoids represent a physiologically advanced model for studying the mechanisms of kidney development and disease. Here, we describe a simple two-step protocol for the differentiation of human pluripotent stem cells into kidney organoids. Our approach involves suspension culture that allows for rapid and cost-effective bulk production of organoids, which is well suited for large-scale assays such as drug screening. The organoids correspond to fetal human kidney tissue and may be of limited use for modeling adult kidney function. For complete details on the use and execution of this protocol, please refer to Przepiorski et al. (2018).

Article	Year
Kidney injury and regeneration in zebrafish. Seminars in nephrology\| PubMed ID: 25217272	2014
The small molecule probe PT-Yellow labels the renal proximal tubules in zebrafish. Chemical communications (Cambridge, England)\| PubMed ID: 25407666	2015
The Vital Dye CDr10b Labels the Zebrafish Mid-Intestine and Lumen. Molecules (Basel, Switzerland)\| PubMed ID: 28335401	2017
A Simple Bioreactor-Based Method to Generate Kidney Organoids from Pluripotent Stem Cells. Stem cell reports\| PubMed ID: 30033089	2018
Turning mesoderm into kidney. Seminars in cell & developmental biology\| PubMed ID: 30172050	2019
Mind the gap: renal tubule responses to injury and the role of Cxcl12 and Myc. Annals of translational medicine\| PubMed ID: 31032310	2019
Transcriptional profiling of the zebrafish proximal tubule. American journal of physiology. Renal physiology\| PubMed ID: 31188030	2019
Evaluation of cisplatin-induced injury in human kidney organoids. American journal of physiology. Renal physiology\| PubMed ID: 32150447	2020
SOX9 promotes stress-responsive transcription of VGF nerve growth factor inducible gene in renal tubular epithelial cells. The Journal of biological chemistry\| PubMed ID: 32887795	2020
Protocol for Large-Scale Production of Kidney Organoids from Human Pluripotent Stem Cells. STAR protocols\| PubMed ID: 33377044	2020

Veronika Sander

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