Jessica M. Vanslambrouck

Direct Transcriptional Reprogramming of Adult Cells to Embryonic Nephron Progenitors

Direct reprogramming involves the enforced re-expression of key transcription factors to redefine a cellular state. The nephron progenitor population of the embryonic kidney gives rise to all cells within the nephron other than the collecting duct through a mesenchyme-to-epithelial transition, but this population is exhausted around the time of birth. Here, we sought to identify the conditions under which adult proximal tubule cells could be directly transcriptionally reprogrammed to nephron progenitors. Using a combinatorial screen for lineage-instructive transcription factors, we identified a pool of six genes (SIX1, SIX2, OSR1, EYA1, HOXA11, and SNAI2) that activated a network of genes consistent with a cap mesenchyme/nephron progenitor phenotype in the adult proximal tubule (HK2) cell line. Consistent with these reprogrammed cells being nephron progenitors, we observed differential contribution of the reprogrammed population into the Six2(+) nephron progenitor fields of an embryonic kidney explant. Dereplication of the pool suggested that SNAI2 can suppress E-CADHERIN, presumably assisting in the epithelial-to-mesenchymal transition (EMT) required to form nephron progenitors. However, neither TGFβ-induced EMT nor SNAI2 overexpression alone was sufficient to create this phenotype, suggesting that additional factors are required. In conclusion, these results suggest that reinitiation of kidney development from a population of adult cells by generating embryonic progenitors may be feasible, opening the way for additional cellular and bioengineering approaches to renal repair and regeneration.

Renal Subcapsular Transplantation of PSC-Derived Kidney Organoids Induces Neo-vasculogenesis and Significant Glomerular and Tubular Maturation In Vivo

Summary Human pluripotent stem cell (hPSC)-derived kidney organoids may facilitate disease modeling and the generation of tissue for renal replacement. Long-term application, however, will require transferability between hPSC lines and significant improvements in organ maturation. A key question is whether time or a patent vasculature is required for ongoing morphogenesis. Here, we show that hPSC-derived kidney organoids, derived in fully defined medium conditions and in the absence of any exogenous vascular endothelial growth factor, develop host-derived vascularization. In vivo imaging of organoids under the kidney capsule confirms functional glomerular perfusion as well as connection to pre-existing vascular networks in the organoids. Wide-field electron microscopy demonstrates that transplantation results in formation of a glomerular basement membrane, fenestrated endothelial cells, and podocyte foot processes. Furthermore, compared with non-transplanted organoids, polarization and segmental specialization of tubular epithelium are observed. These data demonstrate that functional vascularization is required for progressive morphogenesis of human kidney organoids.

Direct transcriptional reprogramming to nephron progenitors

The direct reprogramming of one cell fate to another represents an attractive option for the generation of specific endpoints for cellular therapy. This appears to require both the reactivation of critical transcription factor regulatory networks and chromatin remodelling. The direct reprogramming of mature renal epithelial cell lines to a nephron progenitor state has been reported. However, our limited knowledge of the optimal growth conditions to maintain this state remains a challenge for their therapeutic application. Here we examine whether nephron progenitors as an endpoint of direct reprogramming have been suitably defined and whether alternative options for reprogramming to kidney exist.

The Renal Papilla: An Enigma in Damage and Repair

It is now well established that organ-specific adult stem cells exist in a variety of tissues throughout the body where their survival, proliferation, and multipotency are regulated by the niche in which they reside. The controversy over whether such populations also exists in a relatively

Reprogramming Somatic Cells to a Kidney Fate

Recent years have challenged the view that adult somatic cells reach a state of terminal differentiation. Although the ultimate example of this, somatic cell nuclear transfer, has not proven feasible in human beings, dedifferentiation of mature cell types to a more primitive state, direct reprogramming from one mature state to another, and the reprogramming of any adult cell type to a pluripotent state via enforced expression of key transcription factors now all have been shown. The implications of these findings for kidney disease include the re-creation of key renal cell types from more readily available and expandable somatic cell sources. The feasibility of such an approach recently was shown with the dedifferentiation of proximal tubule cells to nephrogenic mesenchyme. In this review, we examine the technical and clinical challenges that remain to such an approach and how new reprogramming approaches also may be useful for kidney disease.

Self-organisation after embryonic kidney dissociation is driven via selective adhesion of ureteric epithelial cells

ABSTRACT Human pluripotent stem cells, after directed differentiation in vitro, can spontaneously generate complex tissues via self-organisation of the component cells. Self-organisation can also reform embryonic organ structure after tissue disruption. It has previously been demonstrated that dissociated embryonic kidneys can recreate component epithelial and mesenchymal relationships sufficient to allow continued kidney morphogenesis. Here, we investigate the timing and underlying mechanisms driving self-organisation after dissociation of the embryonic kidney using time-lapse imaging, high-resolution confocal analyses and mathematical modelling. Organotypic self-organisation sufficient for nephron initiation was observed within a 24 h period. This involved cell movement, with structure emerging after the clustering of ureteric epithelial cells, a process consistent with models of random cell movement with preferential cell adhesion. Ureteric epithelialisation rapidly followed the formation of ureteric cell clusters with the reformation of nephron-forming niches representing a later event. Disruption of P-cadherin interactions was seen to impair this ureteric epithelial cell clustering without affecting epithelial maturation. This understanding could facilitate improved regulation of patterning within organoids and facilitate kidney engineering approaches guided by cell-cell self-organisation. Summary: Time-lapse imaging, high-resolution confocal analyses and mathematical modelling demonstrate how component cells of a developing tissue can reform complex multicellular structures, even after dissociation.

Fate-mapping within human kidney organoids reveals conserved mammalian nephron progenitor lineage relationships.

While mammalian kidney morphogenesis has been well documented, human kidney development is poorly understood. Here we combine reprogramming, CRISPR/Cas9 gene-editing and organoid technologies to study human nephron lineage relationships in vitro. Early kidney organoids contained a SIX2+ population with a transcriptional profile akin to human nephron progenitors. Lineage-tracing using gene-edited induced pluripotent stem cell (iPSC) lines revealed that SIX2-expressing cells contribute to nephron formation but not to the putative collecting duct epithelium. However, Cre-mediated temporal induction of the SIX2+ lineage revealed a declining capacity for these cells to contribute to nephron formation over time. This suggests human kidney organoids, unlike the developing kidney in vivo, lack a nephron progenitor niche capable of both self-renewal and ongoing nephrogenesis. Nonetheless, human iPSC-derived kidney tissue maintains previously identified lineage relationships supporting the utility of pluripotent stem cell-derived kidney organoids for interrogating the molecular and cellular basis of early human development.

Directing the Differentiation of Human Es Cells Towards a Renal Fate

Aim: Patency after percutaneous balloon angioplasty (PTA) for haemodialysis fistula stenosis is highly variable. This study aimed to assess factors associated with patency following first episode of treatment with PTA. Background: Restenosis recurs commonly after PTA. Previous studies have shown that some intrinsic fistula and biochemical factors may influence patency after PTA. Methods: We retrospectively reviewed all endovascular procedures performed by nephrologists between 2007 and 2012 at a single centre. Anatomical, clinical, biochemical and medication information was subjected to cox regression analysis to identify factors influencing post-intervention patency. Results: 120 patients were identified as having first episode treatment with PTA. During a median follow-up period of 22.66 months (5.24–53 months), 171 follow-up procedures were performed. Post-intervention primary patency rates at 6, 12 and 18 months were 46%, 25% and 15% respectively. Cumulative (functional) patency rates at 6, 12 and 18 months were 97%, 94 and 92% respectively with 1.4 additional procedures per patient. In univariate cox regression analysis, the presence of multiple lesions (p = 0.037) was associated with early restenosis at 6 months, while upper arm fistulae were associated with early restenosis (p = 0.004) and shorter primary patency (p = 0.001). Other anatomical characteristics (fistula age, lesion length, pre-procedure stenosis), clinical history (diabetes, coronary and peripheral artery disease), medications, and biochemical parameters (HbA1c, CRP, albumin and lipids) did not influence patency. Conclusion: Multiple stenoses and upper arm fistulae may be associated with shorter patency after PTA. More large volume prospective studies are required to further assess factors associated with patency after PTA in haemodialysis fistulae, particularly the role of metabolic and inflammatory markers.