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

Differentiation of a contractile, ureter-like tissue, from Embryonic Stem cell-derived ureteric bud and <i>Ex-Fetu</i> 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

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

Funder: Giersch FoundationBACKGROUND: 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

Design and testing of ex vivo vascularization methods for kidney explants in order to overcome the diffusion limit of oxygen and nutrients in 3D-culture-systems

The best way of treating end-stage renal disease is the transplantation of a donor organ. However, the number of patients requiring a transplant exceeds the number of available kidneys. Growing kidneys from induced pluripotent stem cells could help to close the gap between organ demand and supply and could omit the need to take immunosuppressants if the engineered kidneys were to be generated from the patient’s own cells. Kidney organoids from murine kidney cells are similar to embryonic kidneys but, with traditional culture methods, the organoids, as well as the kidneys themselves, grow rather flat and spread out, which does not represent the overall shape of a kidney grown in vivo. Embedding kidney explants in type 1 collagen resulted in a more spherical shape, but also in central necrosis, most likely caused by hypoxia. Staining of the explants for endothelial markers revealed the presence of capillaries, which remained immature. In some tissues, arterial differentiation is induced by neurons. To analyse whether the renal arterial maturation is controlled by neurons, developing kidneys were isolated and co-stained for vascular and neuronal markers. The co-staining revealed that innervation occurs after formation of the smooth muscle cell lining and during upregulation of calponin 1, which is involved in smooth muscle cell contraction. Several studies link vascular differentiation to mechanical stimuli caused by the onset of blood flow. To detect whether connecting the kidneys to the vasculature of a host would enhance vascular maturity, the explants were grafted onto the chick chorioallantoic membrane. Injection of the chick vessels following isolation and staining of the explant confirmed blood flow through the graft vessels. However, vascular maturity was not improved as indicated by the lack of vascular smooth muscle cells. The use of fertilized eggs makes it difficult to influence the growth environment of the explants. Isolating blood vessels and co-culturing them under perfusion with embryonic kidneys in vitro would allow more control over available growth factors and mechanical cues by adjusting the stiffness of the surrounding matrix as well the flow rate through the blood vessels. For this purpose, I have designed a culture device that allows the long-term perfusion of arteries and veins. Using this device, I have demonstrated that isolated blood vessels respond to proangiogenic treatment by forming sprouts. In co-cultures of the blood vessels with embryonic kidneys, endothelial sprouts were seen between both tissues and appeared to form a connection between the vessel and the explant, which sets the basis for the in vitro vascularisation of kidney explants. Vascularizing kidney explants can aid identifying methods to vascularize kidney organoidsin vitro. This is a critical step in renal tissue engineering as it would improve organoid growth and enable testing their functionality in terms of blood filtration. A functional vasculature will also be essential in generation transplantable renal tissue, which could on day help to treat kidney disease

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

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.MINECO (BFU2015-71244-ERC; BFU2014-52125-REDT; BFU2014-57831), and Fundacion Ramón Areces to F.M-B. M.D.B. was supported by a MINECOFPI 2015 PhD fellowship. M.H. was supported by a MINECO-FPI 2012 PhD fellowship. M.B.F. was supported by a La-Caix

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. It can therefore be concluded that neurons are not required to initiate arterial smooth muscle cell recruitment within the kidney. The dataset is related to the upcoming publication Tarnick et al. (in submission), "Innervation of the developing kidney in vivo and in vitro"

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

Abstract 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