Biology subjects

Little, M. H.

Publications and source records attributed to Little, M. H..

10 recordsLinked to original sources

bioRxiv2026

S3-enriched kidney proximal nephrons from stem cells facilitate tubular injury modelling

Chronic kidney disease (CKD) is a major global health challenge affecting >840 million people, with rising prevalence driven by common conditions including diabetes, hypertension, obesity, and drug toxicity. The proximal tubule (PT) is central to kidney function yet is highly injury-prone, particularly within the S3 segment which plays key roles in drug clearance and CKD pathology. Despite decades of in vitro PT model development, achieving accurate functional and structural segmentation of the PT with distinct PT sub-cell types has remained challenging. Here, we report an enrichment of S3-like cells within PT-enhanced kidney organoids (PT-EKO) that confers segment-specific functionality and injury susceptibility. This advanced platform facilitated physiologically relevant modelling of hyperglycaemia-induced damage, including rapid detection of injury biomarker Kidney Injury Molecule-1 (KIM-1). Integrating with both static and organ-on-chip culture systems, the translational potential of S3-enriched PT-EKO was underscored by its amenability to scale-up via cryopreservation of day 13 progenitors with retained differentiation capacity. PT-EKO applications were further broadened as an expandable high-yield source of isolatable PT cells, retaining PT characteristics across multiple passages and cryopreservation. Together, these findings present a high-fidelity platform for modelling tubular injury and advancing translational applications including CKD drug development, cell-specific nephrotoxicity testing and cellular therapies. SIGNIFICANCE STATEMENTAdvancing stem cell-based proximal nephron models, here we identify proximal tubule-enhanced kidney organoids (PT-EKO) as an enriched source of the nephrons most injury-prone cell type; S3 proximal tubule cells. This advanced platform provides physiologically-relevant modelling of tubular injury and a scalable source of high-quality PT cells, paving the way for more accurate modelling, screening, and cellular therapy applications.

bioengineering↗

bioRxiv2025

Nephron segmentation and patterning in kidney organoids can be modulated by distinct FGF subfamily members

Human pluripotent stem cell-derived kidney organoids replicate embryonic nephrogenesis in a 3D-culture system. Recent advances suggest that refining the culture environment to replicate spatiotemporal cues present during embryonic organogenesis improves patterning. Here, this paradigm was applied to FGF signalling, a key regulator of embryonic nephron progenitor maintenance, nephrogenesis and ureteric branching. Both FGF8b and FGF10 signalling is sufficient to support nephrogenesis, with each having distinct effects on nephron patterning. FGF10 enhanced the initial WT1+ mesenchymal population, leading to proximally biased nephrons, while FGF8b biased toward early distal patterning, leading to the formation of cells with connecting segment identity. The addition of both FGF8b and FGF10 together had an additive effect, leading to a balance of proximal and distal patterning. This differential patterning was retained in tissue transplanted under the murine renal capsule, with FGF8b-treated organoids displaying increased distal/connecting segments. These findings highlight plasticity during organoid nephrogenesis that can be modulated by FGF signalling and identify an approach to refine nephrogenesis toward key cell types.

developmental biology↗

bioRxiv2025

In vitro plasticity between ureteric epithelial and distal nephron identity and maturity is controlled by extracellular signals.

Several studies have described human pluripotent stem cell (hPSC)-derived ureteric epithelium, which in the embryo arises from the nephric duct and forms the collecting ducts of the kidney. However, hPSC-derived distal nephron epithelium can also adopt a ureteric phenotype, despite this not occurring during embryogenesis. In this study, RETtdTomato and GATA3mCherry reporter lines were used to further investigate this plasticity. Induction of anterior intermediate mesoderm resulted in the spontaneous formation of an epithelial plexus with a nephric duct-like identity. Subsequent addition of RSPO1 induced patterning of distalized nephrons, including distal convoluted tubule and thick ascending limb of loop of Henle but lacking proximal segments or glomeruli. This epithelium showed a capacity to adopt ureteric epithelial or nephric duct-like states in ex vivo co-culture in response to external cues. The same epithelium seeded as single cells in Matrigel formed epithelial spheroids and adopted a RET+ ureteric tip identity. This in vitro continuum between nephric duct, ureteric epithelium and distal nephron illustrates the role of the microenvironment in cellular identity. Highlights and eTOCO_ST_ABSHighlightsC_ST_ABSO_LIDistal renal epithelium shows substantial plasticity in vitro C_LIO_LIIdentity can be shifted towards distal nephron, including distal convoluted tubule and loop of Henle, in response to prolonged RSPO1 C_LIO_LIDistal epithelial identity can shift in ex vivo culture in response to surrounding signals C_LIO_LICollecting duct is supported by Matrigel culture C_LI eTOC blurbKairath and colleagues investigate the plasticity of induced renal epithelium in response to external signalling cues in vitro and ex vivo. Initial patterning generates a plastic epithelium able to pattern to specific distal nephron. The same epithelium, cultured in Matrigel patterns to ureteric epithelium.

developmental biology↗

bioRxiv2025

Classification of indeterminate and off-target cell types within human kidney organoid differentiation

Human pluripotent stem cell-derived organoids are multicellular models of developing tissues proposed to recapitulate developmental stages of lineage commitment across time. Assessing how accurately protocols recapitulate development is challenged by the lack of accurate reference data sets for early human development, the presence of off-target states and the potential to form cellular states not present in vivo. This study addresses these challenges with respect to differentiation of human pluripotent stem cells to kidney organoids. Based on a factorial single-cell transcriptomic analysis across a 27-day differentiation protocol, including 150,957 cells collected at five time points, we present a comprehensive classification of predicted identity, representing an in vitro temporal transcriptional atlas. For early stages, cellular identity was defined in relation to existing human developmental data across time and organ system. As renal structures arose, this was coupled with the kidney-specific classification methods. In this way we identify predicted cellular states, predictable off-target cell types based on embryology and transitional mesodermal populations with no clear in vivo equivalent. An analysis of all existing single cell data on this same protocol suggests the presence of the same off-target trajectories irrespective of cell line or laboratory. This study provides a valuable resource to better identify and understand how off-target populations arise in vitro, benefiting the advancement of kidney organoid technologies towards therapeutic outcomes.

cell biology↗

bioRxiv2025

Generation of multi-lineage kidney assembloids with integration between nephrons and a single exiting collecting duct

A functional kidney requires a patent connection between all nephrons and the collecting duct network to ensure an exit path for the urinary filtrate. While it is now possible to separately direct the differentiation of pluripotent stem cells to nephron-forming or nephric duct-forming populations, the controlled integration of these two systems has not been demonstrated in human. We report the formation of an integrated kidney assembloid via the co-culture of distinct nephric duct progenitor and nephron-forming cultures in the absence of any requirement for cell enrichment. Nephrons within these kidney assembloids connect to a single exiting nephric duct with this connection forming with the early distal nephron prior to nephron segmentation. Using constitutional reporter lines, we show that the nephrons arise from the nephric progenitors and fuse with the nephric duct while the nephric duct progenitors form a single common duct without any branching. The stromal component of the nephric duct progenitor population also contributes to the medullary stroma surrounding the nephrons. Such modular assembloids address the challenge of nephron unification in an engineered kidney tissue model.

bioengineering↗

bioRxiv2021

Enhanced metanephric specification to functional proximal tubule enables toxicity screening and infectious disease modelling in kidney organoids

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.

bioengineering↗

bioRxiv2021

DevKidCC allows for robust classification and direct comparisons of kidney organoid datasets

Kidney organoids provide a valuable resource to understand kidney development and disease. Clustering algorithms and marker genes fail to accurately and robustly classify cellular identity between human pluripotent stem cell (hPSC)-derived organoid datasets. Here we present a new method able to accurately classify kidney cell subtypes, a hierarchical machine learning model trained using comprehensive reference data from single cell RNA-sequencing of human fetal kidney (HFK). We demonstrate the tools (DevKidCC) performance by application to all published kidney organoid datasets and a novel dataset. DevKidCC is available on Github and can be used on any kidney single cell RNA-sequence data.

bioinformatics↗

bioRxiv2018

Bioprinted pluripotent stem cell-derived kidney organoids provide opportunities for high content screening.

Recent advances in the directed differentiation of human pluripotent stem cells to kidney brings with it the prospect of drug screening and disease modelling using patient-derived stem cell lines. Development of such an approach for high content screening will require substantial quality control and improvements in throughput. Here we demonstrate the use of the NovoGen MMX 3D bioprinter for the generation of highly reproducible kidney organoids from as few as 4,000 cells. Histological and immunohistochemical analyses confirmed the presence of renal epithelium, glomeruli, stroma and endothelium, while single cell RNAseq revealed equivalence to the cell clusters present within previously described organoids. The process is highly reproducible, rapid and transferable between cell lines, including genetically engineered reporter lines. We also demonstrate the capacity to bioprint organoids in a 96-well format and screen for response to doxorubicin toxicity as a proof of concept for high content compound screening.

cell biology↗

bioRxiv2018

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.

cell biology↗

bioRxiv2017

Bayesian inference of agent-based models: a tool for studying kidney branching morphogenesis

The adult mammalian kidney has a complex, highly-branched collecting duct epithelium that arises as a ureteric bud sidebranch from an epithelial tube known as the nephric duct. Subsequent branching of the ureteric bud to form the collecting duct tree is regulated by subcellular interactions between the epithelium and a population of mesenchymal cells that surround the tips of outgrowing branches. The mesenchymal cells produce glial cell-line derived neurotrophic factor (GDNF), that binds with RET receptors on the surface of the epithelial cells to stimulate several subcellular pathways in the epithelium. Such interactions are known to be a prerequisite for normal branching development, although competing theories exist for their role in morphogenesis. Here we introduce the first agent-based model of ex vivo kidney uretic branching. Through comparison with experimental data, we show that growth factor-regulated growth mechanisms can explain early epithelial cell branching, but only if epithelial cell division depends in a switch-like way on the local growth factor concentration; cell division occurring only if the driving growth factor level exceeds a threshold. We also show how a recently-developed method, \"Approximate Approximate Bayesian Computation\", can be used to infer key model parameters, and reveal the dependency between the parameters controlling a growth factor-dependent growth switch. These results are consistent with a requirement for signals controlling proliferation and chemotaxis, both of which are previously identified roles for GDNF.\n\nAuthor SummaryA number of important congenital disorders arise due to incomplete development of the mammalian kidney. Elucidating the cause of these conditions requires an understanding of the mechanisms that contribute to kidney morphogenesis. Whilst experimental work has suggested several candidate mechanisms, their importance is still not well understood. Here we develop a computational model of kidney morphogenesis at the individual cell level to compare these different hypotheses. Guided by existing experimental evidence we propose that a generic growth factor, that we term \"GDNF\", produced from the mesenchyme surrounding the epithelium, can drive a number of cellular responses. Simulations of our agent-based model reveal that diffusion of GDNF, coupled with GDNF-stimulated epithelial cell division, can generate the branching patterns seen in ex vivo kidney explant experiments. We also find that branching depends on the sensitivity of cell proliferation to changes in GDNF levels. In particular our model only generates realistic branching when there is significant variation in GDNF levels along the boundary of the epithelium, and most cells divide only if the local concentration of GDNF exceeds a threshold value. We conclude that feedback between mesenchymal cells that produce GDNF, and epithelial cells that consume it, is vital for normal kidney organogenesis.

developmental biology↗