From patterning of the vasculature and neuronal networks to the development of diverse organs such as the lungs, mammary glands, and kidneys, branching morphogenesis is a common phenomenon observed during animal development. Perturbations in normal branching caused by genetic or exogenous factors can lead to disease, or abnormalities in surrounding tissues. In the kidney, correct patterning of the collecting duct is central to renal function, allowing appropriate filtration and drainage into the bladder. In addition, the ureteric bud and tip cells that will become the mature collecting duct are essential for the development of the kidney’s main processing unit, the nephron. Thus, perturbations in the process of branching morphogenesis in the developing kidney can lead to numerous pathologies and so studying the mechanisms behind this process are of great value. A platform to study branching of ureteric bud cells in vitro is a useful way of dissecting some of these mechanisms, and may also be a key part of advancing renal regenerative medicine.
Previous work has shown that it is possible to coax ureteric bud tips in vitro to propagate and branch in the absence of metanephric mesenchyme (MM) or MM-conditioned media. This was in itself an important step forward, since proliferation and branching of the ureteric bud normally requires signaling from the MM. However, serum was still required in addition to growth factors and signaling factors that would normally be secreted by the mesenchymal population. This is a problem for studying the process of branching in vitro, since serum contains numerous known and unknown factors that can act as confounding variables.
In their recent Stem Cell Reports publication, Yuri et al. have used a systematic approach to reveal a defined set of factors for the maintenance, proliferation and branching of dissected UB tips as well as dispersed ureteric bud (UB) cells. Crucially, their culture method does not require serum, making it ideal for studying pathways involved in branching. The authors began by culturing UBs from e11.5 mouse kidneys in factors known to be important for UB cell maintenance – specifically, GDNF and FGF1. These two factors together, or FGF1 alone, allowed the UB cells to survive and proliferate but did not allow the survival of tip cells or allow branching – this was a problem since the tip cells represent the stem cells of the collecting duct. It has previously been shown by Bridgewater et al and Marose et al that Wnt / β-catenin signaling is necessary for maintaining UB tip cell identity, so the authors hypothesized that addition of the GSK-B inhibitor CHIR99021 might encourage tip cell proliferation and maintenance. In fact, addition of all three factors (GDNF, FGF1 and CHIR) induced extensive branching and led to the enrichment of tip cells. Interestingly, direct inclusion of Wnts as a substitute for the WNT activator CHIR99021 did not recapitulate the effects, suggesting a more global pathway. To investigate this, the authors turned to a mouse knockout of an R-spondin protein receptor with abnormal UB branching. Rspondin (RSPO) proteins are known agonists of WNT-B-catenin signaling so they hypothesized that RSPO proteins may be able to activate WNT in their culture system similarly to CHIR99021. In fact they found that substituting either RSPO1 or RSPO3 for CHIR99021 in the culture system preserved the maintenance of tip cell identity and ability to branch extensively.
Continuing their systematic approach to identifying the defined factors required for in vitro propagation of branching UBs, the authors then investigated the role or retinoic acid (RA). RA is known to be crucial to kidney development as shown by the Mendelsohn lab (Batourina et al and Rosselot et al) and indeed the authors found that culturing UBs in RA alone allowed the cells to survive – but not branch. Adding GDNF to the RA encouraged proliferation and maintenance of tip markers; this highlights the essential role of RA in maintaining tip cell identity and the interplay of RA and GDNF/Ret signaling in proliferation and branching. This is of potentially crucial importance for renal regenerative medicine; RA is mainly secreted by the stromal cells and so inclusion of this population of cells in any approaches to renal tissue engineering may be required.
In addition to defining serum-free factors that allow the proliferation and branching of UBs in vitro, the authors proceeded to identify a defined set of factors that would allow single (dispersed) UB cells to propagate. This would be a useful addition to the toolkit of renal biologists, allowing dispersed UB cells to be manipulated (e.g. transfected with plasmids) and then their proliferation and branching studied either alone or in the context of other cell types. Stunningly, the authors were able to combine the factors above – FGF1, GDNF, RA – to not only allow proliferation but also branching of single UB cells, and inclusion of CHIR99021 into the cocktail induced further branching in an additive manner. Just as impressively, the authors mixed UB branched structures formed from single, dispersed UB cells with metanephric mesenchyme, and showed that they were still able to induce the mesenchyme to form nephrons, demonstrating the potential power of this approach.
This work is an impressive step forward in the propagation of UB cells, opening up avenues for studies of branching morphogenesis and kidney development in a serum-free manner. It will be interesting to see if this approach will hold true in human cells differentiated from pluripotent stem cells – if so it could be a valuable addition to the field of renal regenerative medicine.
Melanie Lawrence