Nephron progenitors rhythmically alternate between renewal and differentiation phases that synchronize with kidney branching morphogenesis.

The mammalian kidney achieves massive parallelization of function by exponentially duplicating nephron-forming niches during development. Each niche caps a tip of the ureteric bud epithelium (the future urinary collecting duct tree) as it undergoes branching morphogenesis, while nephron progenitors within niches balance self-renewal and differentiation to early nephron cells. Nephron formation rate approximately matches branching rate over a large fraction of mouse gestation, yet the nature of this apparent pace-maker is unknown. Here we correlate spatial transcriptomics data with branching 'life-cycle' to discover rhythmically alternating signatures of nephron progenitor differentiation and renewal across Wnt, Hippo-Yap, retinoic acid (RA), and other pathways. We then find in human stem-cell derived nephron progenitor organoids that Wnt/ß-catenin-induced differentiation is converted to a renewal signal when it temporally overlaps with YAP activation. Similar experiments using RA activation indicate a role in setting nephron progenitor exit from the naive state, the spatial extent of differentiation, and nephron segment bias. Together the data suggest that nephron progenitor interpretation of consistent Wnt/ß-catenin differentiation signaling in the niche may be modified by rhythmic activity in ancillary pathways to set the pace of nephron formation. This would synchronize nephron formation with ureteric bud branching, which creates new sites for nephron condensation. Our data bring temporal resolution to the renewal vs. differentiation balance in the nephrogenic niche and inform new strategies to achieve self-sustaining nephron formation in synthetic human kidney tissues.

Highly-parallel production of designer organoids by mosaic patterning of progenitors.

Human organoids are a promising approach for disease modeling and regenerative medicine. However, organoid variability and limited control over morphological outcomes remain significant challenges. Here we extend a DNA 'velcro' cell patterning approach, precisely controlling the number and ratio of human stem cell-derived progenitors contributing to nephron and mosaic nephron/ureteric bud organoids within arrays of microwells. We demonstrate long-term control over organoid size and morphology, decoupled from geometric constraints.

Rho/ROCK activity tunes cell compartment segregation and differentiation in nephron-forming niches.

Controlling the time and place of nephron formation in vitro would improve nephron density and connectivity in next-generation kidney replacement tissues. Recent developments in kidney organoid technology have paved the way to achieving self-sustaining nephrogenic niches in vitro. The physical and geometric structure of the niche are key control parameters in tissue engineering approaches. However, their relationship to nephron differentiation is unclear. Here we investigate the relationship between niche geometry, cell compartment mixing, and nephron differentiation by targeting the Rho/ROCK pathway, a master regulator of the actin cytoskeleton. We find that the ROCK inhibitor Y-27632 increases mixing between nephron progenitor and stromal compartments in native mouse embryonic kidney niches, and also increases nephrogenesis. Similar increases are also seen in reductionist mouse primary cell and human induced pluripotent stem cell (iPSC)-derived organoids perturbed by Y-27632, dependent on the presence of stromal cells. Our data indicate that niche organization is a determinant of nephron formation rate, bringing renewed focus to the spatial context of cell-cell interactions in kidney tissue engineering efforts.

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

Transmembrane protein 135 regulates lipid homeostasis through its role in peroxisomal DHA metabolism.

Transmembrane protein 135 (TMEM135) is thought to participate in the cellular response to increased intracellular lipids yet no defined molecular function for TMEM135 in lipid metabolism has been identified. In this study, we performed a lipid analysis of tissues from Tmem135 mutant mice and found striking reductions of docosahexaenoic acid (DHA) across all Tmem135 mutant tissues, indicating a role of TMEM135 in the production of DHA. Since all enzymes required for DHA synthesis remain intact in Tmem135 mutant mice, we hypothesized that TMEM135 is involved in the export of DHA from peroxisomes. The Tmem135 mutation likely leads to the retention of DHA in peroxisomes, causing DHA to be degraded within peroxisomes by their beta-oxidation machinery. This may lead to generation or alteration of ligands required for the activation of peroxisome proliferator-activated receptor a (PPARa) signaling, which in turn could result in increased peroxisomal number and beta-oxidation enzymes observed in Tmem135 mutant mice. We confirmed this effect of PPARa signaling by detecting decreased peroxisomes and their proteins upon genetic ablation of Ppara in Tmem135 mutant mice. Using Tmem135 mutant mice, we also validated the protective effect of increased peroxisomes and peroxisomal beta-oxidation on the metabolic disease phenotypes of leptin mutant mice which has been observed in previous studies. Thus, we conclude that TMEM135 has a role in lipid homeostasis through its function in peroxisomes.

A mutation in transmembrane protein 135 impairs lipid metabolism in mouse eyecups.

Aging is a significant factor in the development of age-related diseases but how aging disrupts cellular homeostasis to cause age-related retinal disease is unknown. Here, we further our studies on transmembrane protein 135 (Tmem135), a gene involved in retinal aging, by examining the transcriptomic profiles of wild-type, heterozygous and homozygous Tmem135 mutant posterior eyecup samples through RNA sequencing (RNA-Seq). We found significant gene expression changes in both heterozygous and homozygous Tmem135 mutant mouse eyecups that correlate with visual function deficits. Further analysis revealed that expression of many genes involved in lipid metabolism are changed due to the Tmem135 mutation. Consistent with these changes, we found increased lipid accumulation in mutant Tmem135 eyecup samples. Since mutant Tmem135 mice have similar ocular pathologies as human age-related macular degeneration (AMD) eyes, we compared our homozygous Tmem135 mutant eyecup RNA-Seq dataset with transcriptomic datasets of human AMD donor eyes. We found similar changes in genes involved in lipid metabolism between the homozygous Tmem135 mutant eyecups and AMD donor eyes. Our study suggests that the Tmem135 mutation affects lipid metabolism as similarly observed in human AMD eyes, thus Tmem135 mutant mice can serve as a good model for the role of dysregulated lipid metabolism in AMD.

Different Human Immune Lineage Compositions Are Generated in Non-Conditioned NBSGW Mice Depending on HSPC Source.

NBSGW mice are highly immunodeficient and carry a hypomorphic mutation in the c-kit gene, providing a host environment that supports robust human hematopoietic expansion without pre-conditioning. These mice thus provide a model to investigate human hematopoietic engraftment in the absence of conditioning-associated damage. We compared transplantation of human CD34+ HSPCs purified from three different sources: umbilical cord blood, adult bone marrow, and adult G-CSF mobilized peripheral blood. HSPCs from mobilized peripheral blood were significantly more efficient (as a function of starting HSPC dose) than either cord blood or bone marrow HSPCs at generating high levels of human chimerism in the murine blood and bone marrow by 12 weeks post-transplantation. While T cells do not develop in this model due to thymic atrophy, all three HSPC sources generated a human compartment that included B lymphocytic, myeloid, and granulocytic lineages. However, the proportions of these lineages varied significantly according to HSPC source. Mobilized blood HSPCs produced a strikingly higher proportion of granulocyte lineage cells (~35% as compared to ~5%), whereas bone marrow HSPC output was dominated by B lymphocytic cells, and cord blood HSPC output was enriched for myeloid lineages. Following transplantation, all three HSPC sources showed a shift in the CD34+ subset towards CD45RA+ progenitors along with a complete loss of the CD45RA-CD49f+ long-term HSC subpopulation, suggesting this model promotes mainly short-term HSC activity. Mice transplanted with cord blood HSPCs maintained a diversified human immune compartment for at least 36 weeks after the primary transplant, although mice given adult bone marrow HSPCs had lost diversity and contained only myeloid cells by this time point. Finally, to assess the impact of non-HSPCs on transplantation outcome, we also tested mice transplanted with total or T cell-depleted adult bone marrow mononuclear cells. Total bone marrow mononuclear cell transplants produced significantly lower human chimerism compared to purified HSPCs, and T-depletion rescued B cell levels but not other lineages. Together these results reveal marked differences in engraftment efficiency and lineage commitment according to HSPC source and suggest that T cells and other non-HSPC populations affect lineage output even in the absence of conditioning-associated inflammation.

Modulation of Tmem135 Leads to Retinal Pigmented Epithelium Pathologies in Mice.

Purpose: Aging is a critical risk factor for the development of retinal diseases, but how aging perturbs ocular homeostasis and contributes to disease is unknown. We identified transmembrane protein 135 (Tmem135) as a gene important for regulating retinal aging and mitochondrial dynamics in mice. Overexpression of Tmem135 causes mitochondrial fragmentation and pathologies in the hearts of mice. In this study, we examine the eyes of mice overexpressing wild-type Tmem135 (Tmem135 TG) and compare their phenotype to Tmem135 mutant mice. Methods: Eyes were collected for histology, immunohistochemistry, electron microscopy, quantitative PCR, and Western blot analysis. Before tissue collection, electroretinography (ERG) was performed to assess visual function. Mouse retinal pigmented epithelium (RPE) cultures were established to visualize mitochondria. Results: Pathologies were observed only in the RPE of Tmem135 TG mice, including degeneration, migratory cells, vacuolization, dysmorphogenesis, cell enlargement, and basal laminar deposit formation despite similar augmented levels of Tmem135 in the eyecup (RPE/choroid/sclera) and neural retina. We observed reduced mitochondria number and size in the Tmem135 TG RPE. ERG amplitudes were decreased in 365-day-old mice overexpressing Tmem135 that correlated with reduced expression of RPE cell markers. In Tmem135 mutant mice, RPE cells are thicker, smaller, and denser than their littermate controls without any signs of degeneration. Conclusions: Overexpression and mutation of Tmem135 cause contrasting RPE abnormalities in mice that correlate with changes in mitochondrial shape and size (overfragmented in TG vs. overfused in mutant). We conclude proper regulation of mitochondrial homeostasis by TMEM135 is critical for RPE health.