Significant strides have been made in the last few years towards the goal of engineering human renal tissue. Protocols to differentiate nephrons in vitro either on their own (and here) or in a mixed population with other renal cell types have being published. The ability to induce and maintain nephron progenitors in vitro has also recently been established, an important tool for both tissue engineering and developmental studies. While the formation of nephrons in vitro is a significant step forward and may be useful for some applications including basic research and toxicology, the architecture of the endogenous organ is key to function, and therefore any real strides towards physiologically relevant organoids will need to include appropriate gross morphology.
In the mixed population of renal progenitors and structures that results from the Takasato method, renal organoids can be formed that do appear to contain Gata3-containing epithelial tubules, however, these epithelial tubules do not branch or respond to the nephron progenitor signals in the same way as normal ureteric bud. In addition to the lack of higher-order structure comprising a central collecting duct, then, is the lack of branch-competent ureteric bud (UB) formation. There may be several reasons for this lack of branching. The stromal progenitors are important for maintenance of Ret expression in UB tips and so perhaps there is a problem with this population in the differentiated organoids. In addition, applying the same set of factors to all the tissue ignores the normal, localised antero-posterior differences that exist within the developing embryo.
In their recent paper, Taguchi and Nishinakamura have taken a “root and branch” approach, by paying close attention to the developmental cues and localised differences in normal development. They wanted to first develop a protocol for differentiating branch-competent ureteric bud from stem cells, and then try to add a single branching UB rudiment to metanephric mesenchyme (MM) in order to form a more anatomically correct organoid.
Using a reverse engineering method, the authors started by working backwards in development to identify signalling pathways essential for differentiation of the UB in vivo in mice. While it is true that this assumed the information gleaned from mice would be transferable to human, it was sensible in that mouse tissue is more readily available at precise developmental timings, has fewer ethical issues associated with it, and much is already known about the signalling pathways during mouse embryonic development.
The authors began by identifying the point at which branching capacity is achieved in mouse development. Using a transgenic Hoxb7-GFP mouse line and established reaggregation methods, they placed anterior or posterior GFP+ Wolffian Duct from E9.5, E10.5 and E11.5 with isolated metanephric mesenchyme, and also a GFP+ ureteric bud tip from E11.5 embryos with isolated MM. This showed them that branching capability was attained progressively as development went on, with robust branching from both WD and UB by E11.5, and less branching at earlier ages. The anterior or posterior WD branched equally in the presence of MM, driving home that the more anterior mesonephric mesenchyme is functionally different to the MM in its ability to induce branching.
They then used gene expression array analysis to identify useful markers for monitoring progression of differentiation. Looking at the gene expression analysis, they diligently worked their way back, identifying important factors for each stage of progression and then testing the ability of each factor to induce the next stage. For the progression from E9.5 WD to E11.5 UB, they isolated E9.5 GFP+ WD by cell sorting and then tried out combinations of factors identified in the gene analysis. Fgf9, retinoic acid (RA) and Wnt (in this case a Wnt agonist, CHIR) were identified as essential for progression of WD to UB, and addition of GDNF was needed to maintain Wnt11 expression and induce branching.
Having worked out the factors required for WD to branching UB, they then moved a step further back in developmental timings. The Hoxb7-GFP transgenic line shows clear expression of GFP at E8.75 in the anterior of the embryo suggesting that there are already WD-committed localised cells at this stage. They again identified factors from the array data and then tested their ability to maintain WD gene expression profile at E8.75, and to induce differentiation to UB. FGF9, CHIR and RA were again found to be required for maintenance of E8.75 WD gene profile, with lower levels of Wnt required and RA being essential. Interestingly, GDNF was dispensible at this stage to maintain Wnt11 expression, suggesting that different gene modules are active in the earlier embryo for WD maturation. Adding GDNF and higher Wnt concentration during a 3-day induction period from E8.75 led to branching UB-like cells.
Finally, the authors wanted to identify the differentiation steps between mouse ES cells and E8.75 committed WD cells. Using their microarray analysis, they identified two cell-surface proteins that could be reliably used to specifically sort E8.75 WD, giving them a tool to quantify successful differentiation from mESC to WD committed cells. Using a similar reverse engineering approach, and drawing on existing knowledge about early embryogenesis, they were able to define a protocol for differentiation of UB from mouse ESCs. The authors had previously discovered that shorter exposure to Wnt leads to anterior intermediate mesoderm (AIM) identity, whereas prolonged exposure to Wnt leads to posterior intermediate mesoderm (PIM) identity. This is because Wnt signalling maintains T+ nascent mesoderm, and the longer the cells remain in this state the more likely they are to take on PIM identity. In fact, they found that there is a very narrow window, at day 4.5 of differentiation, for Wnt exposure in this early stage of differentiation in order to induce AIM identity and subsequent WD/UB progression. Of course, Takasato et al. already showed that limiting Wnt exposure tips the balance to an anterior fate and produces more Gata3-positive epithelia in their organoids, in agreement with these data. In identifying the signals required for differentiation from ESC to WD, they also found that cell fate patterning of nephron progenitors and UB starts even before formation of immature mesoderm at the epiblast/primitive streak stages, in agreement with previous work (and here) showing early stage mesoderm patterning in the avian embryo.
The authors now had a method for differentiating WD from mouse ESCs. But, like most things, the proof is in the pudding. Could these induced WDs (iWDs) go on to differentiate into branching UBs? They established mESCs from Hoxb7-GFP mice, used their protocol to induce WD, and then sorted the differentiated WD cells. These were reaggregated and cultured with the factors identified by “reverse engineering”, namely, Fgf9, CHIR and RA, adding in GDNF for the final stage. These produced cells with UB identity that, crucially, were able to branch robustly. Using the Ganeva method, they isolated single UB nodules from these reaggregates, and added E11.5 metanephric mesenchyme dissected from mouse embryos. This produced branching organoids with a single collecting system, where the collecting duct tissue was differentiated from ESCs. Nephron progenitor gene expression was maintained and the distal tubules of differentiated nephrons were linked up to the collecting ducts. Strikingly, these induced UBs could also be induced to branch using a previously-established branching protocol without MM, showing that they truly are fully branch-competent. In a further step, the authors used their earlier published protocol for inducing nephron progenitors from mESCs, and added these to the induced UBs (instead of dissected MM), along with sorted E11.5 stromal progenitors. When both stromal progenitors (from the embryo) and induced nephron progenitors were included, the induced UBs could branch nicely, but removing either the stroma or NPs abolished or significantly reduced the branching, further underlining the importance of stromal progenitors in UB branching and renal differentiation.
Making branching UBs from ES cells was a major step forward, opening up the tantalising possibility of making renal organoids fully derived from human stem cells that had realistic macro-anatomy as well as functional nephrons. The authors applied their differentiation protocols to human iPS cells. With some amendments (necessary due to interspecies developmental and maturation differences), they were able to successfully induce branching tubules in 3D gel from human iPS cells, in an MM-free culture system, although it was much slower. This likely reflects the longer differentiation timeline of human renal maturation vs mouse.
In an attempt to make a fully engineered human organoid, they then used human iPSC-derived nephron progenitors combined with hiPSC-derived UB – this induced some weak evidence of initial branching, but did not progress past 7 days. This is likely consistent with the absence of human primary stromal progenitors although it is of course possible that the human protocol is not yet optimised in the same way as the mouse protocol. Interestingly, there appeared to be robust nephrogenesis in these cultures (seen in Supplementary Figure S7C) based on the brightfield images, suggesting that the human induced UB can at least induce MET in the nephron progenitors.
It is clear that Taguchi and Nishinakamura have contributed significantly to the field of renal tissue engineering with this work. It is now even more urgent to develop a protocol for inducing stromal progenitors from mouse and/or human stem cells, in order to test the hypothesis that the missing link for anatomically realistic organoids in human is stromal cells. There is no doubt that the field is progressing rapidly, and once the hurdle of human stem cell-derived organoids with adequate macro-anatomy has been broached, the field can move on to issues such as vascularisation and innervation, both of which may be crucial to the full maturation and growth of these engineered kidneys.