Innervation of the developing kidney in vivo and in vitro.

Within the adult kidney, renal neurites can be observed alongside the arteries where they play a role in regulating blood flow. However, their role and localization during development has so far not been described in detail. In other tissues, such as the skin of developing limb buds, neurons play an important role during arterial differentiation. Here, we aim to investigate whether renal nerves could potentially carry out a similar role during arterial development in the mouse kidney. In order to do so, we used whole-mount immunofluorescence staining to identify whether the timing of neuronal innervation correlates with the recruitment of arterial smooth muscle cells. Our results show that neurites innervate the kidney between day 13.5 and 14.5 of development, arriving after the recruitment of smooth muscle actin-positive cells to the renal arteries.

Introducing blood flow in kidney explants by engraftment onto the chick chorioallantoic membrane is not sufficient to induce arterial smooth muscle cell development.

Kidney explant cultures are an important tool to gain insights into developmental processes, insights that can be used to develop strategies for engineering kidneys from stem cells. However, explants are not connected to a perfused vascular system. This limits their survival and limits physiological studies, for example of blood filtration, the main function of the kidney. Previous studies have shown that grafting kidneys onto avian chorioallantoic membrane (CAM) can establish perfusion and enable glomerular vascularization, but the realism and maturity of the resultant vasculature has not been examined. Here, we show that vasculature of kidney explants grafted onto CAM is very different from natural kidney vasculature, showing excessive growth of endothelial cells, absence of a hierarchical arterio-venous network and no vascular smooth muscle cell recruitment. The model therefore has serious limits.

Production of kidney organoids arranged around single ureteric bud trees, and containing endogenous blood vessels, solely from embryonic stem cells.

There is intense worldwide effort in generating kidney organoids from pluripotent stem cells, for research, for disease modelling and, perhaps, for making transplantable organs. Organoids generated from pluripotent stem cells (PSC) possess accurate micro-anatomy, but they lack higher-organization. This is a problem, especially for transplantation, as such organoids will not be able to perform their physiological functions. In this study, we develop a method for generating murine kidney organoids with improved higher-order structure, through stages using chimaeras of ex-fetu and PSC-derived cells to a system that works entirely from embryonic stem cells. These organoids have nephrons organised around a single ureteric bud tree and also make vessels, with the endothelial network approaching podocytes.

Long-term live imaging and multiscale analysis identify heterogeneity and core principles of epithelial organoid morphogenesis.

BACKGROUND: Organoids are morphologically heterogeneous three-dimensional cell culture systems and serve as an ideal model for understanding the principles of collective cell behaviour in mammalian organs during development, homeostasis, regeneration, and pathogenesis. To investigate the underlying cell organisation principles of organoids, we imaged hundreds of pancreas and cholangiocarcinoma organoids in parallel using light sheet and bright-field microscopy for up to 7 days. RESULTS: We quantified organoid behaviour at single-cell (microscale), individual-organoid (mesoscale), and entire-culture (macroscale) levels. At single-cell resolution, we monitored formation, monolayer polarisation, and degeneration and identified diverse behaviours, including lumen expansion and decline (size oscillation), migration, rotation, and multi-organoid fusion. Detailed individual organoid quantifications lead to a mechanical 3D agent-based model. A derived scaling law and simulations support the hypotheses that size oscillations depend on organoid properties and cell division dynamics, which is confirmed by bright-field microscopy analysis of entire cultures. CONCLUSION: Our multiscale analysis provides a systematic picture of the diversity of cell organisation in organoids by identifying and quantifying the core regulatory principles of organoid morphogenesis.

Differentiation of a Contractile, Ureter-Like Tissue, from Embryonic Stem Cell-Derived Ureteric Bud and Ex Fetu Mesenchyme.

BACKGROUND: There is intense interest in replacing kidneys from stem cells. It is now possible to produce, from embryonic or induced pluripotent stem cells, kidney organoids that represent immature kidneys and display some physiologic functions. However, current techniques have not yet resulted in renal tissue with a ureter, which would be needed for engineered kidneys to be clinically useful. METHODS: We used a published sequence of growth factors and drugs to induce mouse embryonic stem cells to differentiate into ureteric bud tissue. We characterized isolated engineered ureteric buds differentiated from embryonic stem cells in three-dimensional culture and grafted them into ex fetu mouse kidney rudiments. RESULTS: Engineered ureteric buds branched in three-dimensional culture and expressed Hoxb7, a transcription factor that is part of a developmental regulatory system and a ureteric bud marker. When grafted into the cortex of ex fetu kidney rudiments, engineered ureteric buds branched and induced nephron formation; when grafted into peri-Wolffian mesenchyme, still attached to a kidney rudiment or in isolation, they did not branch but instead differentiated into multilayer ureter-like epithelia displaying robust expression of the urothelial marker uroplakin. This engineered ureteric bud tissue also organized the mesenchyme into smooth muscle that spontaneously contracted, with a period a little slower than that of natural ureteric peristalsis. CONCLUSIONS: Mouse embryonic stem cells can be differentiated into ureteric bud cells. Grafting those UB-like structures into peri-Wolffian mesenchyme of cultured kidney rudiments can induce production of urothelium and organize the mesenchyme to produce rhythmically contracting smooth muscle layers. This development may represent a significant step toward the goal of renal regeneration.

Sfrp3 modulates stromal-epithelial crosstalk during mammary gland development by regulating Wnt levels.

Mammary stroma is essential for epithelial morphogenesis and development. Indeed, postnatal mammary gland (MG) development is controlled locally by the repetitive and bi-directional cross-talk between the epithelial and the stromal compartment. However, the signalling pathways involved in stromal-epithelial communication are not entirely understood. Here, we identify Sfrp3 as a mediator of the stromal-epithelial communication that is required for normal mouse MG development. Using Drosophila wing imaginal disc, we demonstrate that Sfrp3 functions as an extracellular transporter of Wnts that facilitates their diffusion, and thus, their levels in the boundaries of different compartments. Indeed, loss of Sfrp3 in mice leads to an increase of ductal invasion and branching mirroring an early pregnancy state. Finally, we observe that loss of Sfrp3 predisposes for invasive breast cancer. Altogether, our study shows that Sfrp3 controls MG morphogenesis by modulating the stromal-epithelial cross-talk during pubertal development.

Macrophages restrict the nephrogenic field and promote endothelial connections during kidney development.

The origins and functions of kidney macrophages in the adult have been explored, but their roles during development remain largely unknown. Here we characterise macrophage arrival, localisation, heterogeneity, and functions during kidney organogenesis. Using genetic approaches to ablate macrophages, we identify a role for macrophages in nephron progenitor cell clearance as mouse kidney development begins. Throughout renal organogenesis, most kidney macrophages are perivascular and express F4/80 and CD206. These macrophages are enriched for mRNAs linked to developmental processes, such as blood vessel morphogenesis. Using antibody-mediated macrophage-depletion, we show macrophages support vascular anastomoses in cultured kidney explants. We also characterise a subpopulation of galectin-3+ (Gal3+) myeloid cells within the developing kidney. Our findings may stimulate research into macrophage-based therapies for renal developmental abnormalities and have implications for the generation of bioengineered kidney tissues.