Kidney Organoids: Protocols, Morphology, and Vascular Insights

Researchers are developing kidney organoids to better understand renal development, model diseases, and explore therapeutic applications. These lab-grown structures mimic key aspects of kidney architecture and function, providing a valuable tool for nephrology research. However, challenges remain in refining protocols, improving vascularization, and ensuring functional maturation.

Optimizing kidney organoid models requires precise control over cell sources, culture conditions, and molecular signaling.

Fundamental Cell Sources

Kidney organoid development relies on specific cell populations that can replicate nephrogenesis in vitro. Human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are the primary sources due to their ability to differentiate into nephron progenitor cells. These progenitors form key kidney structures, including podocytes, proximal tubules, and collecting ducts. iPSCs offer a patient-specific approach, enabling disease modeling and potential autologous transplantation without immune rejection. Studies have shown that iPSC-derived organoids can replicate genetic kidney disorders, such as polycystic kidney disease, by maintaining patient-specific mutations.

Direct differentiation of hPSCs into kidney organoids follows a developmental trajectory that mimics embryonic kidney formation. This process involves the induction of intermediate mesoderm, a transient embryonic tissue that gives rise to nephron progenitors and stromal components. Protocols typically use growth factors such as bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and retinoic acid to guide differentiation. Precise timing and concentration of these signals are critical, as deviations can lead to incomplete or aberrant organoid formation. Excessive WNT activation during early differentiation, for instance, can result in off-target mesodermal derivatives, reducing nephron formation efficiency.

Alternative cell sources have been explored to enhance organoid development. Adult renal progenitor cells from human kidney biopsies can generate nephron-like structures under appropriate conditions, though their limited proliferative capacity and donor variability pose challenges. Additionally, transdifferentiation approaches—reprogramming somatic cells such as fibroblasts into nephron progenitors—offer a way to bypass pluripotency, though further refinement is needed for functional integration within organoid models.

3D Culturing Protocols

A robust 3D culturing system is essential for kidney organoid self-organization and maturation. Transitioning from traditional two-dimensional cultures to three-dimensional structures allows cells to interact in a spatially relevant manner, closely resembling in vivo kidney development. Suspension culture methods, such as low-attachment plates or rotating bioreactors, prevent cell adhesion to flat surfaces, promoting spheroidal aggregate formation. Embedding these aggregates in extracellular matrix hydrogels, such as Matrigel or collagen, supports tissue organization by mimicking native renal basement membrane composition.

The differentiation medium plays a key role in guiding kidney-specific structures. Basal media formulations supplemented with growth factors like activin A, BMP7, and FGF9 drive lineage specification by mimicking embryonic signaling gradients. Temporal modulation of WNT and retinoic acid pathways ensures the sequential emergence of nephron progenitors, stromal components, and epithelialized tubules. Pulsatile exposure to CHIR99021, a WNT agonist, enhances nephron patterning, though prolonged activation can lead to aberrant mesodermal differentiation. Fine-tuning these signaling cues is necessary to balance nephrogenesis without inducing off-target cell populations.

Oxygen availability presents another challenge, as diffusion limitations can lead to hypoxic stress in the organoid core. Bioreactor systems, such as spinner flasks or microfluidic devices, improve oxygen and nutrient exchange by maintaining dynamic flow conditions. Perfusion culture platforms have been particularly effective in enhancing tubular lumen formation and epithelial polarity, as demonstrated in studies using microengineered chips that simulate fluid shear forces. These approaches mitigate central necrosis and promote more physiologically relevant tissue organization.

Morphological Features

Kidney organoids exhibit a complex architecture resembling the developing human kidney. Nephron-like units emerge, consisting of distinct compartments such as podocytes, proximal tubules, and distal segments. These components mirror the nephrogenic niche, where epithelialized tubules form alongside stromal elements. Bright-field and immunofluorescence imaging reveal well-defined tubular networks with apical-basal polarity, a hallmark of differentiated renal epithelium. The presence of a basement membrane, rich in laminin and collagen IV, reinforces epithelial integrity, enabling selective solute transport.

As differentiation progresses, podocyte-like cells develop with characteristic foot processes and slit diaphragm-like structures. Transmission electron microscopy has captured the ultrastructural features of these cells, including cytoplasmic projections resembling those in mature glomerular podocytes. Although podocytes express nephrin and podocalyxin, their functional maturation remains incomplete due to the absence of perfusable capillaries, limiting their ability to generate true filtration pressure.

Tubular compartments within kidney organoids display segment-specific markers confirming their differentiation into proximal and distal nephron structures. Proximal tubule-like cells express megalin and cubilin, proteins involved in endocytosis, while distal tubules show enrichment of Na+/K+-ATPase, reflecting their ion transport function. These segments exhibit lumen formation and microvilli development, enhancing absorptive capacity. However, their organization lacks the precise cortico-medullary patterning observed in native kidneys, likely due to the absence of external signaling gradients.

Key Signaling Pathways

Kidney organoid formation and maturation depend on multiple signaling pathways governing nephrogenesis. WNT signaling plays a central role in kidney lineage specification, particularly through WNT9B, which is critical for nephron progenitor self-renewal and mesenchymal-to-epithelial transition. Experimental modulation of WNT activity using small molecules like CHIR99021 has shown that transient activation enhances nephron differentiation, while prolonged exposure disrupts organoid organization.

Beyond WNT signaling, BMP and FGF pathways regulate nephron segmentation and stromal interactions. BMP7 promotes nephron progenitor survival, while FGF9 supports the expansion of intermediate mesoderm. Studies using recombinant BMP7 have shown improved nephron patterning. Concurrently, NOTCH signaling directs cell fate decisions within renal epithelium. Loss-of-function experiments targeting NOTCH1 and NOTCH2 have demonstrated impaired proximal tubule formation, highlighting the necessity of this pathway for proper nephron differentiation.

Methods of Molecular Characterization

Molecular characterization is essential for evaluating kidney organoid development and function. Transcriptomic analyses, including single-cell RNA sequencing (scRNA-seq), provide insights into cell population heterogeneity. By comparing organoid-derived cell profiles to human fetal kidney datasets, researchers have identified transcriptional signatures confirming nephron progenitors, podocytes, and tubular epithelial cells. These comparisons also reveal discrepancies, such as incomplete maturation of specific nephron segments and the presence of off-target cell populations, guiding protocol refinements. Epigenetic profiling, including ATAC-seq and ChIP-seq, further elucidates chromatin accessibility and histone modifications influencing nephrogenesis.

Proteomic approaches validate kidney-specific marker expression at the protein level. Mass spectrometry-based proteomics quantifies structural and functional proteins, confirming nephrin in podocytes, aquaporins in collecting duct-like structures, and transporters such as SGLT2 in proximal tubules. Immunostaining techniques, including confocal microscopy and flow cytometry, provide spatial resolution of these markers. Functional assays, such as albumin uptake in proximal tubule-like cells or dextran filtration in glomerular compartments, offer additional confirmation of organoid performance. These molecular characterization techniques refine kidney organoid models, ensuring they more accurately replicate native renal tissue complexity.

Vascular-Like Structures

A major challenge in kidney organoid research is developing functional vascular networks that support filtration and nutrient exchange. While organoids self-assemble into nephron-like structures, they lack a fully integrated vascular system. Endothelial cells expressing markers such as CD31 and VE-cadherin emerge but fail to form perfusable capillaries, limiting physiological relevance. This results in diffusion constraints, where cells in the organoid core experience hypoxic stress, impairing long-term viability and maturation. Efforts to enhance vascularization have included co-culturing kidney organoids with endothelial progenitors or embedding them in engineered hydrogels that promote angiogenesis.

Advances in microfluidic platforms and bioprinting technologies have introduced new strategies for integrating vascular structures. Organoids grown in perfusion bioreactors exhibit improved endothelial organization, with some studies demonstrating partial capillary ingrowth when transplanted into in vivo models. Additionally, vasculogenic growth factors such as VEGF and angiopoietin-1 show promise in stabilizing endothelial networks within organoid cultures. Despite these advancements, achieving a fully functional capillary network that supports glomerular filtration remains an ongoing challenge. Future work aims to refine these vascularization methods, potentially enabling kidney organoids to serve as more effective models for renal physiology and disease.