Disease Modeling With Kidney Organoids Ⅱ

4. Strategies to Improve Kidney Organoid Platforms 

Current kidney organoids exhibit kidney-relevant structures but do not resemble functional adult kidneys. Thus, the protocols described in Section 2.2 give rise to kidney organoids that lack an in vivo-like biophysical environment, including fluid flow through the tissues. Recent studies have improved organoid resemblance to the adult kidney, using techniques such as transplantation and combining different organoid types to achieve higher-order kidney structures. These techniques may be combined with bioengineering approaches to explore and improve organoid maturation. Below, we will highlight those strategies and discuss new considerations for generating next-generation kidney organoid systems. 

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4.1. Introducing Gradients 

We know that during normal embryogenesis the mesonephros, metanephros, and UB send signals to one another to pattern the developing kidney [6]. In addition, biochemical gradients established in the early embryo are essential to directing proper kidney patterning and development [47]. A signifificant issue in our current kidney organoid development protocols is their isolation from other bodily tissues during development, and thus lack of biochemical gradients established via interactions with other co-developing tissue systems of an embryo. Therefore, it is worthwhile to include those interactions in organoid development. Taguchi and Nishinakamura (2017) and Tsujimoto et al. (2020) have considered some of these interactions to facilitate organoid maturation [17,24]. In their strategies, they first used PIM and AIM-specific factors to develop independent organoids, then transplanted co-cultured MM and UB organoids into mice [17,24]. Kidney organoids developed in this manner exhibited higher-order structures but could not recapitulate extensive in vivo-like UB branching. While these studies underscore the importance of co-culturing neighboring tissues, they call for the improvement of the organoid via established gradients included from the onset of organoid development.

As it is nearly impossible to include every tissue from the developing embryo and optimize its conditions, we propose a simulation of an environment, where the cells experience the effects of neighboring tissue. This may be achieved by engineering a biochemical gradient to grow cells, wherein the gradient can mimic the presence of neighboring tissues. The gradient may then direct both AIM and PIM lineages within the same suspension of cells, and thus induce UB and MM formation simultaneously. For example, we could establish a GDNF gradient and advance UB branching within the MM to induce the formation of the intricate collecting duct system of the adult kidney.

Although these gradient-based approaches have not been explored to establish kidney organoids, they have been used to improve the maturation of other organoids. For example, Ben-Reuven and Reiner et al. (2020) embedded morphogen-releasing beads in a hydrogel to help pattern the anterior–posterior axis of brain organoids [48]. In another study, Kamperman et al. (2019) established cell substratum with biotinylated morphogens [49]. Besides these static approaches, researchers have also developed microfluidic chips with fellow-driven morphogen addition. For example, Cui et al. (2020) used a chamber with chemotaxis chambers for morphogen distribution and chambers into which stem cells may be cultured in hydrogels [50]. Thus, researchers may apply similar systems to program kidney organoid development using specifific factors that are spatially controlled to mediate their effects. This would establish a gradient and mimic the naturally occurring morphogen distributions relevant to embryonic kidney development. For example, beads with factors specifific to AIM and PIM differentiation may be seeded at opposite sides of a hydrogel to mimic natural AIM-PIM gradients. Alternatively, to control hiPSC differentiation, cells or organoids may be seeded into microfluidic devices, such that cells/organoids of different lineages are on opposite sides (see Figure 5A). HiPSCs embedded in hydrogels may be cultured in between these two structures, which may serve as sources of morphogenic factors. This approach may be utilized to mimic interactions between the mesonephros and metanephros, which the current kidney organoid protocols lack in their setup (see Figure 5A). Thus, future work should aim to mimic biochemical cues from multiple tissue types that are relevant to kidney development, including those between the UB and MM, as well as between the developing kidney and vasculature. The related system design can explore the options of establishing gradients to mimic those interactions.

4.2. Perfusing Organoids with Microfluidics 

Fluid flow is vital for kidney physiology since it initiates a cascade of intracellular events in response to mechanosensing and facilitates transport across tissues [51]. Therefore, if we wish to generate functional kidney organoids and sustain them for extended periods of time, we must integrate stable and perfusable vascular networks into our organoid model systems. Fluid flow has been introduced to kidney organoids in two main ways, microfluidics and transplantation. Homan et al. (2019) included biophysical cues in a 3D bio-printed chip, wherein organoids were cultured in a chamber with dynamic fluid flow [52]. This setup simulated microfluidic fellow and promoted vascular network formation, but it did not recapitulate perfusion through vascular and tubule structures to replicate kidney physiology. Nevertheless, this study highlighted the importance of the biophysical environment in organoid maturation.

Figure 5. Proposed improvements to kidney organoid platforms. (A) Approaches to generate gradients in which hiPSCs may be cultured to evolve into kidney organoids. From left to right: iPSC cluster in hydrogel flanked by morphogen-emitting beads, hiPSC cluster in hydrogel flanked by morphogen-emitting organoids, multi-chambered chip system with hiPSC cluster cultured in a hydrogel and bilaterally exposed to morphogens of NP and UB lineage contained in a liquid. (B) Proposed chip system to culture a kidney organoid beside a branching endothelial channel, with follow through in a hydrogel (e.g., collagen matrix). (C) Proposed scaffolding setups, in which kidney organoids can be grown and subsequently transplanted. From left to right: porous scaffold with branching endothelial channels (red), seeded kidney organoids, and seeded morphogen beads (red and blue spheres); porous scaffold with NP organoids surrounding UB organoids

We believe that one can develop organoids in a channeled microfluidic organ-on-chip platform to model the human kidney, as has been performed in other spheroidal systems. For example, unlike Homan et al. (2019), who placed an organoid in a fluid-fellow chamber, Nashimoto et al. (2017) cultured human lung fibroblast spheroids in an extracellular matrix (ECM) between two endothelial channels [52,53]. The channels gave rise to a perfusible vascular network formed via angiogenesis. Using a somewhat similar approach to Nashimoto et al. (2017), we propose placing a hiPSC cluster next to a perfusable endothelial vessel that can be built into a chip, as shown in Figure 5B [53]. With the subsequent addition of sprouting kidney-organoid-specific signals, the vascular channel can be directed to branch and invade the organoid as it starts to differentiate. This configuration would replicate the early stages of kidney development [54]. With sustained perfusion of the necessary morphogenetic signals for kidney development, kidney organoids should be able to better replicate actual human kidney formation processes. It is also important that system parameters, such as fluid fellow stress, ECM composition and configuration, and organoid stage are carefully considered and optimized. Furthermore, since Schumacher et al. (2021) have shown that hypoxia leads to increased angiogenesis in kidney organoids, such an ex vivo system may be cultured in the presence of decreased oxygen to more efficiently induce vascularization [55]. Ultimately, these strategies should facilitate organoid maturation, recapitulate vascular and tubular perfusion, and perform physiological functions as they occur in vivo. 

4.3. Advancing Organoid Maturation via Transplantation 

Studies have indicated that transplanting kidney organoids into a host, such as a mouse, chicken egg, or rat, may provide a viable route for vascularization [16,33,56]. However, transplanted organoids exhibit limited success as functional structures. A major hurdle in this strategy is recapitulating the extensive UB branching within the MM of transplanted organoids. Additionally, nephron segments within organoids lack proper orientation towards a collecting duct. These features are essential for the transplanted organoids to perform an adult human kidney’s function. Moreover, post-transplantation organoids integrate with the host, making it difficult to distinguish the organoid vs. host tissue boundaries, and thus limiting the organoid-specific analysis. 

Tissue engineering strategies provide a wide range of tools to improve kidney organoid development in terms of maturation and functional integration. Typically, tissue engineering approaches employ scaffolds (e.g., silk fibroin scaffolds) in various configurations, such as films, mats, hydrogels, and sponges, to support the arrangement of cells and to recapitulate the different biophysical cues of the desired issues. For example, in one study, Gupta et al. (2019) used porous silk scaffolds to grow organoids, followed by transplantation [31]. In this approach, the scaffolding biomaterial and configuration provided a framework to support organoid development and confer stability upon transplantation. However, this system lacked functional cues to support organoid maturation, such as the co-development of vasculature with the fellow in proximity to other developing structures.

Future studies can develop similar strategies and include functional cues to support organoid maturation. For example, as outlined in Figure 5C, a porous scaffold can be configured with perfusable channels and loaded with morphogenic factors to develop a relevant organoid development system via transplantation. Pre-seeding with endothelial cells can support directed vessel development, whereas the open porous structure can be seeded with hiPSCs and programmed via morphogenetic factors for organoid development. Alternatively, the extracellular matrix (ECM) secreted from kidney tissues can, by itself, serve as a scaffolding material for tissue engineering a kidney organoid. For example, decellularized kidneys have been used to support the organoid’s maturation. Those matrices could be used to culture and transplant organoids or to derive tissue-engineering scaffolds from those biomaterials.

Furthermore, scaffolds present an untapped opportunity to embed multiple organoid lineages together in chosen positions. For example, Taguchi and Nishinakamura (2017) and Tsujimoto et al. (2020) transplanted both UB and NP organoids, allowing them to vascularize [17,24]; however, they lacked an extracellular scaffold to orient their multi-lineage organoids. By utilizing scaffolds, we could mimic nephron generation around a single collecting structure by orienting UB organoids in the center and bottom of a scaffold. At the same time, we can seed NP organoids along the exterior (see Figure 5C). Similar tissue engineering approaches can be developed to advance organoid maturation. Nevertheless, when designing a tissue engineering-based approach for transplanting the organoids, parameters such as biomaterials’ configuration and compatibility should all be carefully selected, as per the needs of the kidney

5. Conclusions 

The development of the kidney organoid is a major breakthrough in vitro kidney disease modeling. Kidney organoids allow for the individualized study of genetic kidney diseases and drug screening in a human-derived platform. While they hold tremendous promise, kidney organoids are immature and isolated in their current form. To truly model the human kidney and related diseases, we need to mature them by recapitulating developing kidney interactions. In particular, we need to advance the interactions between the UB and MM in vitro and introduce human-derived vasculature to kidney organoids. We must also consider gradient-based morphogen approaches to parallel in vivo human embryonic development. Kidney organoids hold great potential for genetic studies of adult and fetal kidney disease and drug screening. Their advancement may lead to the development of life-saving kidney disease treatments in a way that is faster and more accurate than ever before.

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