Extracellular vesicles (EVs) have been described as important mediators of cell communication, regulating several physiological processes, including tissue recovery and regeneration. In the kidneys, EVs derived from stem cells have been shown to support tissue recovery in diverse disease models and have been considered an interesting alternative to cell therapy. For this purpose, however, several challenges remain to be overcome, such as the requirement of a high number of EVs for human therapy and the need for optimization of techniques for their isolation and characterization. Moreover, the kidney’s complexity and the pathological process to be treated require that EVs present a heterogeneous group of molecules to be delivered. In this review, we discuss the recent advances in the use of EVs as a therapeutic tool for kidney diseases. Moreover, we give an overview of the new technologies applied to improve EVs’ efficacy, such as novel methods of EV production and isolation by means of bioreactors and microfluidics, bioengineering the EV content and the use of alternative cell sources, including kidney organoids, to support their transfer to clinical applications.

Clinical translation of stem cell therapies for heart disease requires electrical integration of transplanted cardiomyocytes. Generation of electrically matured human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) is critical for electrical integration. Here, we found that hiPSC-derived endothelial cells (hiPSC-ECs) promoted the expression of selected maturation markers in hiPSC-CMs. Using tissue-embedded stretchable mesh nanoelectronics, we achieved a long-term stable map of human three-dimensional (3D) cardiac microtissue electrical activity. The results revealed that hiPSC-ECs accelerated the electrical maturation of hiPSC-CMs in 3D cardiac microtissues. Machine learning–based pseudotime trajectory inference of cardiomyocyte electrical signals further revealed the electrical phenotypic transition path during development. Guided by the electrical recording data, single-cell RNA sequencing identified that hiPSC-ECs promoted cardiomyocyte subpopulations with a more mature phenotype, and multiple ligand-receptor interactions were up-regulated between hiPSC-ECs and hiPSC-CMs, revealing a coordinated multifactorial mechanism of hiPSC-CM electrical maturation. Collectively, these findings show that hiPSC-ECs drive hiPSC-CM electrical maturation via multiple intercellular pathways.

The prevalence of end-stage kidney disease (ESKD) is rapidly increasing with the need for regenerative therapies. Adult stem cell derived kidney tubuloids have the potential to functionally mimic the adult kidney tubule, but still lack the expression of important transport proteins needed for waste removal. Here, we investigated the potential of extracellular vesicles (EVs) obtained from matured kidney tubular epithelial cells to modulate in vitro tubuloids functional maturation. We focused on organic anion transporter 1 (OAT1), one of the most important proteins involved in endogenous waste excretion. First, we show that EVs from engineered proximal tubule cells increased the expression of several transcription factors and epithelial transporters, resulting in improved OAT1 transport capacity. Next, a more in-depth proteomic data analysis showed that EVs can trigger various biological pathways, including mesenchymal-to-epithelial transition, which is crucial in the tubular epithelial maturation. Moreover, we demonstrated that the combination of EVs and tubuloid-derived cells can be used as part of a bioartificial kidney to generate a tight polarized epithelial monolayer with formation of dense cilia structures. In conclusion, EVs from kidney tubular epithelial cells can phenotypically improve in vitro tubuloid maturation, thereby enhancing their potential as functional units in regenerative or renal replacement therapies. Graphical Abstract

Advanced kidney in vitro models such as organoids or tubuloids still lack the intrinsic expression of various transport proteins needed for active secretory function. Extracellular vesicles (EVs), cell-derived structures that constitute the organ's microenvironment, are known to regulate various cellular processes, including kidney development and regeneration across the nephron. In this study, we propose a new application of renal tubular epithelial cell EVs as modulators for tubuloid functional maturation by increasing the levels of various differentiation markers such as organic anion transport 1 (OAT1), a protein involved in endogenous waste excretion. First, we show that EVs from engineered proximal tubule cells increased the expression of several transcription factors and epithelial transporters in tubuloids that resulted in improved cellular transport capacity. Next, a more in-depth proteomic data analysis demonstrated that EVs can trigger various biological pathways, including mesenchymal-to-epithelial transition, which is crucial in the tubular epithelial maturation process. Moreover, we demonstrated that EV-treated tubuloid-derived cells in a 3D tubular conformation as part of a bioartificial kidney can generate a tight polarized epithelial monolayer with formation of dense cilia structures. In conclusion, EVs from renal tubular epithelial cells can phenotypically improve tubuloid maturation, thereby enhancing their potential as preclinical models and functional units in regenerative therapies.

Clinical translation of stem cell therapies for heart disease is limited by a risk of potentially life-threatening ventricular arrhythmias seen following cardiomyocyte delivery in large animal models. Enhancing cardiomyocyte maturation may reduce this arrhythmogenic risk by reducing automaticity of delivered cardiomyocytes. We tested whether human induced pluripotent stem cell (hiPSC)-derived endothelial cells can enhance maturation and suppress automaticity of iPSC-derived cardiomyocytes in vitro. We found that co-culture of hiPSC-derived endothelial cells with hiPSC-derived cardiomyocytes significantly increased protein expression of cardiac troponin T, cardiac troponin I, Kir2.1, connexin 43, and CD36. In addition, using a stretchable mesh nanoelectronics device, we found that hiPSC-derived endothelial cells accelerated electrical maturation of hiPSC-derived cardiomyocytes and progressively suppressed cardiomyocyte automaticity in vitro (Figure). Using single cell RNA-seq, we identified a subpopulation of hiPSC-derived cardiomyocytes that is eliminated upon co-culture with hiPSC-derived endothelial cells. Further work will investigate whether this subpopulation of cardiomyocytes is responsible for automaticity of cardiomyocyte cultures. Figure. Induced pluripotent stem cell (iPSC)-derived cardiomyocytes alone (CM only) or iPSC-derived cardiomyocytes co-cultured with iPSC-derived endothelial cells (CM+EC) were seeded onto a stretchable mesh nanoelectronics device. Unstimulated voltage tracings at day 30 of cardiomyocyte differentiation show cardiomyocytes with a slower beating rate and more narrow action potential when co-cultured with iPSC-derived endothelial cells compared to iPSC-derived cardiomyocytes alone.