Ala Little Melissa Model 19
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The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration. Regenerating the kidney requires the induction of the more than 20 distinct cell types required for excretion and the regulation of pH, and electrolyte and fluid balance. We have previously described the simultaneous induction of progenitors for both collecting duct and nephrons via the directed differentiation of human pluripotent stem cells. Paradoxically, although both are of intermediate mesoderm in origin, collecting duct and nephrons have distinct temporospatial origins. Here we identify the developmental mechanism regulating the preferential induction of collecting duct versus kidney mesenchyme progenitors. Using this knowledge, we have generated kidney organoids that contain nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells. Within these organoids, individual nephrons segment into distal and proximal tubules, early loops of Henle, and glomeruli containing podocytes elaborating foot processes and undergoing vascularization. When transcription profiles of kidney organoids were compared to human fetal tissues, they showed highest congruence with first trimester human kidney. Furthermore, the proximal tubules endocytose dextran and differentially apoptose in response to cisplatin, a nephrotoxicant. Such kidney organoids represent powerful models of the human organ for future applications, including nephrotoxicity screening, disease modelling and as a source of cells for therapy.
While pluripotent stem cell-derived kidney organoids are now being used to model renal disease, the proximal nephron remains immature with limited evidence for key functional solute channels. This may reflect early mispatterning of the nephrogenic mesenchyme and/or insufficient maturation. Here we show that enhanced specification to metanephric nephron progenitors results in elongated and radially aligned proximalised nephrons with distinct S1 - S3 proximal tubule cell types. Such PT-enhanced organoids possess improved albumin and organic cation uptake, appropriate KIM-1 upregulation in response to cisplatin, and improved expression of SARS-CoV-2 entry factors resulting in increased viral replication. The striking proximo-distal orientation of nephrons resulted from localized WNT antagonism originating from the organoid stromal core. PT-enhanced organoids represent an improved model to study inherited and acquired proximal tubular disease as well as drug and viral responses.
Using fluorescent reporter lines and lineage tracing in human kidney organoids, we have confirmed both the presence of a SIX2+ nephron progenitor population and the contribution of these cells to nephrogenesis via MET in kidney organoids19,20. However, the possibility exists that we are modelling mesonephric rather than metanephric nephrogenesis, potentially contributing to poor PT patterning and maturation (reviewed in ref. 21). It is also possible that suboptimal maintenance of progenitor identity during iPSC differentiation in vitro limits nephron maturation. Several media have been described that are able to support the maintenance of isolated nephron progenitors in vitro22,23,24,25. While each media contains low levels of canonical WNT activity and FGF2/9, distinct differences in nephron patterning result from the inclusion of a variety of TGFβ superfamily agonists (BMP4, BMP7, Activin A) and antagonists (A83-01, LDN193189), NOTCH inhibition (DAPT), and other growth factors (TGFα, IGF1/2, LIF). The inclusion of LDN193189 (inhibitor of BMP receptor-mediated SMAD1/5/8) supported tubular patterning but not formation of glomeruli22. In contrast, the addition of LIF and either dual-SMAD inhibition (LDN193189 and A83-01) or NOTCH inhibition (DAPT) resulted in the formation of nephrons with podocytes but different nephron morphologies23,25. Finally, while proximodistal nephron patterning in mouse has previously been shown to be influenced by relative Wnt, Bmp, and Notch signalling in mouse26, these data suggest that distinct nephron progenitor states may show varying competence for different nephron segments, or that distinct SIX2 populations give rise to different regions of the nephron.
Here we show that patterning to a posterior metanephric SIX2+ nephron progenitor population by extending the duration of mesodermal patterning, while simultaneously enhancing nephron progenitor expansion, specifies progenitors with improved metanephric identity without influencing anteroposterior/mediolateral patterning. These progenitors form strongly proximalised, elongated, and spatially aligned nephrons. The PTs within these nephrons display distinct segmentation into S1, S2 and S3 cell types, upregulation of key solute channels and transporters, and functional uptake of albumin and organic cations. Treatment with cisplatin upregulates Kidney Injury Marker-1 (KIM-1), while increased expression of key viral entry factors enables improved SARS-CoV-2 infection and replication compared to standard protocols. Notably, striking nephron alignment results from localised WNT antagonism, supporting a role for WNT gradients in human nephron proximodistal patterning. Taken together, this study suggests a requirement for optimal nephron progenitor commitment for appropriate PT identity. Such PT-enhanced kidney organoids represent a model of the human proximal nephrons with likely applications for infectious and genetic disease research, drug development, and nephrotoxicity evaluation.
Kidney organoids have previously proven useful to model inherited, early-onset kidney disease3,5,40,79,80,81,82,83,84,85. More recently, organoids have been successfully applied to understanding the pathogenesis of the infectious respiratory disease COVID-19, with SARS-CoV-2 viral infection and replication being achieved in a range of stem cell-derived tissues86,87,88,89,90. Driven by the occurrence of AKI in COVID-19 patients91,92,93,94, a handful of studies have explored kidney organoids as a potential model of COVID-1995,96. While it is still debated whether kidney damage results from direct viral infection or a combination of inflammatory responses and drug nephrotoxicity (reviewed in ref. 97, human PTs show high expression of the key SARS-CoV-2 receptor ACE255,98 and evidence of viral infection99,100,101,102,103,104,105.
The utility of human PSC-derived kidney organoids as accurate models for disease research applications will rely upon their nephron maturation and functionality. To date, proximal tubules characterised within kidney organoids have lacked significant evidence of functional solute transport. In this study, we have shown that prolonged maintenance and delayed epithelialisation of the nephron progenitor population improved PT maturation and functionality compared to standard organoid protocols. Critically, this approach promoted development of distinct S1, S2 and S3 cell populations within the PT, a feature not previously identified in a kidney organoid. The application of DevKidCC in the current study enabled an unbiased and quantitative transcriptional comparison to previous published kidney organoid and human fetal kidney datasets, providing a reliable readout of cell identity and maturation and minimising the caveats associated with comparing restricted marker panels13.
Treatment strategies for coronavirus infections, including SARS-CoV and MERS-CoV, are still in their infancy with progress reliant upon an improved understanding of virus biology and interaction with host factors114. Despite the rapid accumulation of information on SARS-CoV-2, findings have often been conflicting or challenging to interpret, including reported heterogeneity in the expression of viral entry factors and the correlation between expression levels and disease outcome115,116,117. The utility of kidney organoids to study such aspects of infection has been illustrated by recent studies, including the demonstration of reduced infectivity following blocking of the ACE2 receptor95,96. PT-enhanced organoids show clear apical ACE2 protein staining of the proximal tubules via immunofluorescence, together with ACE2 expression within the PT cell clusters. Previous reports have shown ACE2 protein in organoids derived from separate protocols96,113 and ACE2/SARS-CoV-2 interaction was previously detected in the cell membrane protein fraction extracted from kidney organoids96,113. Compared to line- and age-matched standard organoids, PT-enhanced organoids exhibited improved expression of a range of previously identified viral entry factors compared to standard organoids, translating to superior infectivity. Along with their robust response to the nephrotoxic chemotherapeutic cisplatin, these findings underscore the advantage of organoids with enhanced PT patterning and functionality for disease modelling and drug screening.
Despite enhancing PT development, this protocol faces some limitations with respect to nephron patterning and off-target populations. While providing a powerful model of PT function, reduced patterning to distal tubular segments highlights the challenge of simultaneously generating all kidney cell types in a single protocol, as previously described in mouse (refs. 3, 4, 7, 131). In addition, the formation of pre-cartilage cells is problematic for any potential clinical application, albeit not unique to this approach. Cartilage development has been observed in organoids from several protocols following transplantation132,133,134. In PT-enhanced organoids, this may represent a side-effect of prolonged BMP signalling that could potentially be suppressed through timed SMAD1/5/8 inhibition. The presence of central pre-cartilage within the cortical stroma population of the organoid core resulted in strong central WNT antagonism (SFRP2) that contributed to the striking nephron alignment observed. The establishment of a sink and source of WNT activity along the length of the tubule, driving nephron directionality, is in agreement with our current understanding of proximodistal patterning during mouse development26, while the cortical stroma population likely supports and promotes the proximal nephron development62. Interestingly, while standard organoids develop regions of cartilage post transplantation, they do not display this characteristic nephron spatial arrangement either before or after transplant. It is possible that this core is the result of altered biophysical parameters. We have previously shown that higher density standard organoids favour the development of a central unpatterned core, whereas a bioprinted sheet does not69. Such observations indicate that an interplay between cell deposition density and the patterning of the mesodermal population in the enhanced protocol facilitated the strong centralised source of WNT antagonism. Together this suggests an approach to further control the spatial organisation of bioengineered tissue through manipulation of signalling gradients. 153554b96e
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