Direct reprogramming into tubule cells

Many regenerative medicine approaches are based on the reprogramming of somatic cells to iPS cells, followed by directed differentiation to the desired cell type. This is a powerful possibility, in the decade since the first publication on iPS cells the reprogramming has become pretty much routine, and the directed differentiation protocols are rapidly becoming more efficient, more specific and more diverse. Differentiation towards renal fates is no exception to this. However, this method of generating cells of a specific type is, of course, indirect, so will take longer and every step needed that does not have 100% efficiency (and what in stem cell biology is 100% efficient?) will lead to reduced overall efficiency. Moreover, going via a fully pluripotent state followed by directed differentiation will always run the risk of incomplete differentiation and teratoma or other types of cancer development. In this respect, it is also important to remember that incomplete in vivo reprogramming with the Yamanaka factors leads to tumours resembling Wilms’ tumours.

The alternative is to transdifferentiate cells directly from a somatic cell type to the desired type without a pluripotent intermediate.Several examples of this are now known. Previously, the lab of Melissa Little presented data on the transdifferentiation of HK2 cells, an adult human proximal tubule cell line, into CITED1-positive nephron progenitors. However, so far there have been no follow-up publications, and especially the demonstration of this on primary cells will be essential for therapeutic use. And obviously, for this additional routes to use nephron progenitors in a therapeutic setting are required.

Now, in the December issue of Nature Cell Biology, Kaminski et al identify a method to transdifferentiate mouse and human fibroblasts into kidney tubule cells, which they refer to as induced tubular renal epithelial cells, or iRECs. In a reprogramming factor discovery approach reminiscent of the original iPS publication they first use a bioinformatics approach to identify 55 candidate reprogramming factors, which using expression analysis in Xenopus and involvement in human congenital renal disease and mouse phenotypes was further reduced to 13 candidates. These 13 were tested in their capacity to active GFP expression from a Ksp/Cdh16-Cre driven reporter MEFs. Use of all 13 factors gave a low (0.1%) efficiency of reporter activation. Subsequent removal of factors identified four factors, Emx2Hnf1b, Hnf4a and Pax8 which combined were sufficient to increase efficiency to 0.6% after transduction and 11.2% after 31 days of culture. Further optimisation led to efficiencies of 23.8% after 5 weeks or  5.4% after one week, although the latter required co-expression of SV-largeT which might not be desirable for therapeutic purposes.  The iRECs showed different epithelial characteristics and their expression profile resembled that of different segments of primary tubules. Interestingly,  further removal of some of the factors resulted in more cells resembling more specific markers. Functionally, iRECs closely resemble tubule cells, they are polarised with correct expression of different polarity markers, they take up fluorescent albumin and they are sensitive to nephrotoxins. the cells can integrate into tubules of disaggregated embryonic kidneys, and they could form tubules in decellularized kidneys. Finally, as icing on the cake, the authors showed it is possible to make iRECs from postnatal (tail) mouse fibroblasts and human fibroblasts.

Ther data presented in this paper convincingly show that it is possible to directly reprogram fibroblasts into renal tubular cells. This gives important new opportunities for regenerative medicine and further illustrates the plasticity of fully differentiated cells when transfected with the right reprogramming factors.

Peter Hohenstein

 

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More NPC culture methods

Two new developments recently in the culture methods for NPCs. First of all, Tanigawa and colleagues continued on their previous work on the culture of rat NPCs with a new study in Cell Reports on the culture of mouse and human NPCs. Slightly different conditions from the rat protocol and different from the protocol from the Oxburgh lab.  Although the new Tanigawa method can only maintain the the cells in culture for 19 days with full developmental potential (but still 1800-fold expansion which could be enough for many applications), importantly this includes the potential to form the potential to form proximal structures and glomeruli. Very elegantly, the authors also show that the NPCs can be cultured using in vitro differentiated ES/iPS cells as starting material.

This was followed today by a paper in Cell Stem Cell by Li and colleagues. It is tempting to consider this an ‘another one’ paper, but there are a lot of clearly impressive feats reported in this paper. For instance, the self-renewal capacity is incredible, p110 after 17 months and counting. In contrast to other protocols it seems to be equally  efficient on NPCs from different stage embryonic kidneys, and they show full developmental potential in vitro and in vivo, they demonstrate methods for genome editing of these cultures, they show that paracrine factors from these cultures can improve  survival in an acute kidney injury model, and slightly different but apparently just as effective conditions are described for human NPCs. One could reasonably argue that there is too much in this paper and by necessity many things are just being touched on. But the great thing is there is so much in this paper, even if it only touches on many things :-). This should get many people think about new experiments, and luckily the protocols included in the supplementary data look extremely detailed. .

Having now several different protocols for the culture of NPCs it gets interesting to pay more attention to the the differences. Species differences might point to biological differences between these species with respect to the NPC niche, but even differences between the protocols for the same species could, coupled to the differences in developmental capacity and stage of origin of the cells that can be used, give new leads to further define the niche of these cells at different time-points, and by extension differences between these cells and developmental stages. In this respect table S1 and S2 in the Li et al Cell Stem Cell paper is a treat. For instance, as already pointed out by Tanigawa et al and confirmed by Li et al, differences in proximal developmental capacity might be linked to the exact activity of the β-catenin pathway and be explained by our work on the post-MET nephron patterning. Likewise, close comparison between growth factors, concentration and the exact capabilities of the different protocols might give interesting new insights.

 

Peter Hohenstein

A Wnt gradient visualized

Ever since the idea of Wnt molecules as a morphogen gradient emerged, the visualization of that gradient has remained elusive. Antibodies against Wnt molecules are notoriously bad, and epitope tags had a tendency to to disrupt the normal function of the proteins. This lack of visualization has led to much uncertainty about the nature of Wnt gradients and the distance over which they can function. Now, for the first time Farin et al have succeeded in generating an HA-tagged version of the endogenous Wnt3 gene in mice giving homozygous viable and healthy animals by placing the epitope not at either end of the protein but in a lowly conserved region opposite from the interaction site with the Frizzled receptors.

The detection of this epitope-tagged Wnt3 in the gut is still problematic, but with a multistep detection protocol (rat anti-HA / rabbit anti-rat / goat anti-rabbit poly HRP / tyramide signal amplification) they succeed in visualizing the protein. Thus, Farin et al found that Wnt3 produced in and secreted by a Paneth cell binds to Frz receptors on the neighboring cell, the Lgr5+ intestinal stem cell where Frz keeps it tethered to the membrane. Next, cell division of these cells dilutes the surface-bound Wnt3 over daughter cells, thus creating a short-range (mainly one, occasionally two cell) gradient.

Of course, it is always dangerous to translate findings from one biological system to the next. This paper only looks ant Wnt3 and only looks at the Paneth cell / Lgr5+ stem cell system. This system is characterized by direct contact between the niche (Paneth cell) and stem cell (Lgr5+ cell). If we assume the same short-range mechanism holds true for Wnt9b secreted from the ureteric bud and its role in controlling the nephron progenitor cells, what does this mean for the control of the nephron progenitors / cap mesenchyme? If the majority of Wnt9b secreted from the ureteric bud would only reach the neighboring cells in the cap as suggested for Wnt3, what is the exact role of the stromal Fat4 signal that controls the Yap/Taz  and directs the β-catenin signal towards differentiation targets, if there would hardly be any Wnt9b that reaches these cells in the first place? Is there a role for proliferation within the cap in further distributing the Wnt9b signal? Or is the Wnt3/Paneth/Lgr5 cell system not a good model for the mechanism via which Wnt9b controls the NPCs, and does this require longer-range activity (as discussed by Farin et al)? And how about the other Wnt family members having a role in the developing kidney? It is clear there is still much to be described about the role of Wnt signalling and this paper provides an elegant way to do this.

 

Peter Hohenstein

Kidney Development Hopes and Dreams for 2016

Happy New Year!

So what can we expect or hope for in 2016?

Since there were already several NPC culture methods discussed at the IWDN in Utah, it is likely we will see more of this published in the coming year (preferably with protocols as detailed as in the Oxburgh lab paper). Important things I would be looking for are cells that can give rise to all nephron cell types, including podocytes and other proximal fates. As we showed that β-catenin signalling is incompatible with proximal patterning, maybe further titration of the CHIR concentration might help here. Also, something a bit cheaper would be a treat for my lab budgets… Finally, I have no doubt CRISPR/Cas9 will work in these cells as it does in every other cell type, but would be great to see this published sooner rather than later.

No doubt there will also be a lot more on directed differentiation of ES/iPS cells. I would love to see published data on protocols for differentiation of mouse ES cells, this would allow use of all those mutants and, maybe even more important, reporter lines that have been made over the years. It will also allow the use of many of the standard kidney failure models to see if these differentiated cells can repair kidneys. Obviously, human-compatible systems are the final goal but I don’t think we can do without mice yet… What else with these cells? More toxicological screening data? Developmental phenotypes?

I would also be very interested to see data on the role of specific miRNAs in the developing kidney. I think by now Dicer and/or Drosha have been knocked out in every developing kidney compartment, always with severe phenotypes, and the identification last year of mutations in about every miRNA processor gene in Wilms’ tumours also confirms the important of this in the developing kidney. Would be really interesting to see if specific miRNAs can be coupled to specific functions.

And finally some day dreaming… definitively influenced by all the media attention of Tim Peak, the first UK astronaut in 25 years currently in the International Space Station… How would an embryonic kidney develop in a drop of medium in micro-gravity? Will they do anything or simply drop (well… more like float) dead for lack or air-liquid interface? Or would they even do better and form a kidney shape? Would patterning be normal? The cold storage method developed by Jamie Davies should easily get samples to the ISS fast enough. In my lab the record for getting half embryos send to us this way was 6 days in transit (Fedex doesn’t know where Edinburgh is), and the trip to the ISS is more like 6 hours. And the ISS has, or soon will have, a pretty fancy new culture system. Who will fund this?

 

Peter Hohenstein

 

 

 

 

 

Kidney Development in 2015

There’s no doubt 2015 was a very good year for kidney development research. Many important developments,interesting papers and important new ideas. For me, three things clearly stand out…

First, the differentiation of human pluripotent cells to kidney organoids is now really coming of age (Takasato et al). Great for the development of better kidney replacement therapies, and already useful for many developmental studies.

Second, and my personal favorite, the nephron progenitor culture protocol published by the Oxburgh lab (Brown et al). I’ve had one project get stuck myself because of the impossibility to expand these cells in a proper manner/ This will male many new things possible. Especially the inclusion of a details and complete protocol is a treat.

And third on my list, the paper by Chen et al showing the first details on young and old nephrons. This provides an extra layer of complexity that cannot be ignored, and might be very relevant for our own work on Wilms’ tumours.

Exciting times, and promising a lot for 2016 :-).

Peter Hohenstein

A Kidney Development blog

Did you ever google ‘kidney development’? Lots of information on kidney development, but not a recent overview of the latest things in kidney development… And let’s face, there is enough to talk about and discuss… So here it is…

Feel free to comment on posts, feel free to discuss papers, feel free to discuss new or unpublished stuff, feel free to discuss ideas, feel free to follow the blog on twitter @kiddevonline. If you would like to blog from here yourself, just drop me a line…

Peter Hohenstein