The transition zone in Hirschsprung's bowel contains abnormal hybrid ganglia with characteristics of extrinsic nerves.

The aganglionic bowel in short-segment Hirschsprung's disease is characterized both by the absence of enteric ganglia and the presence of extrinsic thickened nerve bundles (TNBs). The relationship between the TNBs and the loss of enteric ganglia is unknown. Previous studies have described decreasing numbers of ganglia with increasing density of TNBs within the transition zone (TZ) between ganglionic and aganglionic gut, and there is some evidence of spatial contact between them in this region. To determine the cellular interactions involved, we have analysed the expression of perineurial markers of TNBs and enteric ganglionic markers for both neural cells and their ensheathing telocytes across four cranio-caudal segments consisting of most proximal ganglionic to most distal aganglionic from pull-through resected colon. We show that in the TZ, enteric ganglia are abnormal, being surrounded by perineurium cells characteristic of TNBs. Furthermore, short processes of ganglionic neurons extend caudally towards the aganglionic region, where telocytes in the TNB are located between the perineurium and nerve fibres into which they project telopodes. Thus, enteric ganglia within the TZ have abnormal structural characteristics, the cellular relationships of which are shared by the TNBs. These findings will help towards elucidation of the cellular mechanisms involved in the aetiology of Hirschsprung's disease.

Firefly luciferase offers superior performance to AkaLuc for tracking the fate of administered cell therapies.

INTRODUCTION: A novel, red-shifted bioluminescence imaging (BLI) system called AkaBLI has been recently developed for cell tracking in preclinical models and to date, limited data is available on how it performs in relation to existing systems. PURPOSE: To systematically compare the performance of AkaBLI and the standard Firefly luciferase (FLuc) systems to monitor the biodistribution and fate of cell therapies in rodents. METHODS: Umbilical cord mesenchymal stromal cells (MSCs) were transduced to produce two genetically engineered populations, expressing either AkaLuc or the engineered FLuc luc2. The bioluminescence of AkaLuc+ and FLuc+ cells was assessed both in vitro (emission spectra, saturation kinetics and light emission per cell) and in vivo (substrate kinetics following intraperitoneal and subcutaneous administration and biodistribution of the cells up to day 7). RESULTS: Introduction of the reporter genes has no effect on MSC phenotype. For BLI, the FLuc system is superior to AkaBLI in terms of (i) light output, producing a stronger signal after subcutaneous substrate delivery and more consistent signal kinetics when delivered intraperitoneally; (ii) absence of hepatic background; and (iii) safety, where the AkaLuc substrate was associated with a reaction in the skin of the mice in vivo. CONCLUSION: We conclude that there is no advantage in using the AkaBLI system to track the biodistribution of systemically administered cell-based regenerative medicine therapies in vivo.

Optical Tissue Clearing to Study the Intra-Pulmonary Biodistribution of Intravenously Delivered Mesenchymal Stromal Cells and Their Interactions with Host Lung Cells.

Mesenchymal stromal cells (MSCs) injected intravenously are trapped in the capillaries of the lungs and die within the first 24 h. Studying the biodistribution and fate of labelled therapeutic cells in the 3D pulmonary context is important to understand their function in this organ and gain insights into their mechanisms of action. Optical tissue clearing enables volumetric cell tracking at single-cell resolution. Thus, we compared three optical tissue-clearing protocols (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis (CUBIC), modified stabilised 3D imaging of solvent-cleared organs (s-DISCO) and ethyl cinnamate (ECi)) to evaluate their potential to track the biodistribution of human umbilical cord MSCs expressing the tdTomato fluorescence reporter and investigate how they interact with host cells in the mouse lung. The results showed that although CUBIC clearing is the only method that enables direct imaging of fluorescently labelled MSCs, combining s-DISCO or ECi with immunofluorescence or dye labelling allows the interaction of MSCs with endothelial and immune cells to be studied. Overall, this comparative study offers guidance on selecting an optical tissue-clearing method for cell tracking applications.

Functional molecules in mesothelial-to-mesenchymal transition revealed by transcriptome analyses.

Peritoneal fibrosis is a common complication of abdominal and pelvic surgery, and can also be triggered by peritoneal dialysis, resulting in treatment failure. In these settings, fibrosis is driven by activated myofibroblasts that are considered to be partly derived by mesothelial-to-mesenchymal transition (MMT). We hypothesized that, if the molecular signature of MMT could be better defined, these insights could be exploited to block this pathological cellular transition. Rat peritoneal mesothelial cells were purified by the use of an antibody against HBME1, a protein present on mesothelial cell microvilli, and streptavidin nanobead technology. After exposure of sorted cells to a well-known mediator of MMT, transforming growth factor (TGF)-ß1, RNA sequencing was undertaken to define the transcriptomes of mesothelial cells before and during early-phase MMT. MMT was associated with dysregulation of transcripts encoding molecules involved in insulin-like growth factor (IGF) and bone morphogenetic protein (BMP) signalling. The application of either recombinant BMP4 or IGF-binding protein 4 (IGFBP4) ameliorated TGF-ß1-induced MMT in culture, as judged from the retention of epithelial morphological and molecular phenotypes, and reduced migration. Furthermore, peritoneal tissue from peritoneal dialysis patients showed less prominent immunostaining than control tissue for IGFBP4 and BMP4 on the peritoneal surface. In a mouse model of TGF-ß1-induced peritoneal thickening, BMP4 immunostaining on the peritoneal surface was attenuated as compared with healthy controls. Finally, genetic lineage tracing of mesothelial cells was used in mice with peritoneal injury. In this model, administration of BMP4 ameliorated the injury-induced shape change and migration of mesothelial cells. Our findings demonstrate a distinctive MMT signature, and highlight the therapeutic potential for BMP4, and possibly IGFBP4, to reduce MMT. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The role of ERK5 in endothelial cell function.

Extracellular-signal-regulated kinase 5 (ERK5), also termed big MAPK1 (BMK1), is the most recently discovered member of the mitogen-activated protein kinase (MAPK) family. It is expressed in a variety of tissues and is activated by a range of growth factors, cytokines and cellular stresses. Targeted deletion of Erk5 in mice has revealed that the ERK5 signalling cascade is critical for normal cardiovascular development and vascular integrity. In vitro studies have revealed that, in endothelial cells, ERK5 is required for preventing apoptosis, mediating shear-stress signalling and regulating tumour angiogenesis. The present review focuses on our current understanding of the role of ERK5 in regulating endothelial cell function.

Multiparametric MRI based assessment of kidney injury in a mouse model of ischemia reperfusion injury.

Kidney diseases pose a global healthcare burden, with millions requiring renal replacement therapy. Ischemia/reperfusion injury (IRI) is a common pathology of acute kidney injury, causing hypoxia and subsequent inflammation-induced kidney damage. Accurate detection of acute kidney injury due to IRI is crucial for timely intervention. We used longitudinal, multi-parametric magnetic resonance imaging (MRI) employing arterial spin labelling (ASL), diffusion weighted imaging (DWI), and dynamic contrast enhanced (DCE)-MRI to assess IRI induced changes in both the injured and healthy contralateral kidney, in a unilateral IRI mouse model (n = 9). Multi-parametric MRI demonstrated significant differences in kidney volume (p = 0.001), blood flow (p = 0.002), filtration coefficient (p = 0.038), glomerular filtration rate (p = 0.005) and apparent diffusion coefficient (p = 0.048) between the injured kidney and contralateral kidney on day 1 post-IRI surgery. Identification of the injured kidney using principal component analysis including most of the imaging parameters demonstrated an area under the curve (AUC) of 0.97. These results point to the utility of multi-parametric MRI in early detection of IRI-induced kidney damage suggesting that the combination of various MRI parameters may be suitable for monitoring the extent of injury in this model.

Integration potential of mouse and human bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are a multipotent cell population which has been described to exert renoprotective and regenerative effects in experimental models of kidney injury. Several lines of evidence indicate that MSCs also have the ability to contribute to nephrogenesis, suggesting that the cells can be employed in stem cell-based applications aimed at de novo renal tissue generation. In this study we re-evaluate the capacity of mouse and human bone marrow-derived MSCs to contribute to the development of renal tissue using a novel method of embryonic kidney culture. Although MSCs show expression of some genes involved in renal development, their contribution to nephrogenesis is very limited in comparison to other stem cell types tested. Furthermore, we found that both mouse and human MSCs have a detrimental effect on the ex vivo development of mouse embryonic kidney, this effect being mediated through a paracrine action. Stimulation with conditioned medium from a mouse renal progenitor population increases the ability of mouse MSCs to integrate into developing renal tissue and prevents the negative effects on kidney development, but does not appear to enhance their ability to undergo nephrogenesis.

Intravenous Administration of Human Umbilical Cord Mesenchymal Stromal Cells Leads to an Inflammatory Response in the Lung.

Mesenchymal stromal cells (MSCs) administered intravenously (IV) have shown efficacy in preclinical models of various diseases. This is despite the cells not reaching the site of injury due to entrapment in the lungs. The immunomodulatory properties of MSCs are thought to underlie their therapeutic effects, irrespective of whether they are sourced from bone marrow, adipose tissue, or umbilical cord. To better understand how MSCs affect innate immune cell populations in the lung, we evaluated the distribution and phenotype of neutrophils, monocytes, and macrophages by flow cytometry and histological analyses after delivering human umbilical cord-derived MSCs (hUC-MSCs) IV into immunocompetent mice. After 2 hr, we observed a significant increase in neutrophils, and proinflammatory monocytes and macrophages. Moreover, these immune cells localized in close proximity to the MSCs, suggesting an active role in their clearance. By 24 hr, we detected an increase in anti-inflammatory monocytes and macrophages. These results suggest that the IV injection of hUC-MSCs leads to an initial inflammatory phase in the lung shortly after injection, followed by a resolution phase 24 hr later.

Multispectral optoacoustic tomography is more sensitive than micro-computed tomography for tracking gold nanorod labelled mesenchymal stromal cells.

Tracking the fate of therapeutic cell types is important for assessing their safety and efficacy. Bioluminescence imaging (BLI) is an effective cell tracking technique, but poor spatial resolution means it has limited ability to precisely map cells in vivo in 3D. This can be overcome by using a bimodal imaging approach that combines BLI with a technique capable of generating high-resolution images. Here we compared the effectiveness of combining either multispectral optoacoustic tomography (MSOT) or micro-computed tomography (micro-CT) with BLI for tracking the fate of luciferase+ human mesenchymal stromal cells (MSCs) labelled with gold nanorods. Following subcutaneous administration in mice, the MSCs could be readily detected with MSOT but not with micro-CT. We conclude that MSOT is more sensitive than micro-CT for tracking gold nanorod-labelled cells in vivo and depending on the route of administration, can be used effectively with BLI to track MSC fate in mice.

Fate of intravenously administered umbilical cord mesenchymal stromal cells and interactions with the host's immune system.

Mesenchymal stromal cells (MSCs) are multipotent cells showing promise in pre-clinical studies and currently used in many clinical trials. The regenerative potential of MSCs is mediated, at least in part, by direct and indirect immunomodulatory processes. However, the mechanism of action is not fully understood yet, and there are still concerns about possible undesired negative effects associated with the administration of living cells. In this study, we (i) compare the long-term fate and safety of umbilical cord (UC-)MSCs administered to immunocompetent and immunocompromised (severe combined immunodeficient (SCID) and non-obese diabetic (NOD)/SCID) animals, and (ii) investigate the immunological response of the host to the administered cells. Intravenous administration of firefly luciferase expressing UC-MSCs revealed that the cells get trapped in the lungs of both immunocompetent and immunocompromised animals, with > 95% of the cells disappearing within 72 h after administration. In 27% of the SCID and 45% of the NOD/SCID, a small fraction of the cells lived up to day 14 but in most cases they all disappeared earlier. One NOD/SCID mouse showed a weak signal up to day 31. Immunocompetent mice displayed elevated percentages of neutrophils in the lungs, the blood, and the spleen 2 h after the administration of the cells. The concentration of neutrophil chemoattractants (MCP1, CCL7, Gro-α and IP-10) were also increased in the plasma of the animals 2 h after the administration of the MSCs. Our results suggest that although the UC-MSCs are short-lived in mice, they still result in an immunological response that might contribute to a therapeutic effect.

Umbilical cord mesenchymal stromal cell-derived extracellular vesicles lack the potency to immunomodulate human monocyte-derived macrophages in vitro.

Mesenchymal stromal cells (MSCs) have been reported to display efficacy in a variety of preclinical models, but without long-term engraftment, suggesting a role for secreted factors, such as MSC-derived extracellular vesicles (EVs). MSCs are known to elicit immunomodulatory effects, an important aspect of which is their ability to affect macrophage phenotype. However, it is not clear if these effects are mediated by MSC-derived EVs, or other factors secreted by the MSCs. Here, we use flow cytometry to assess the effects of human umbilical cord (hUC) MSC-derived EVs on the expression of pro-inflammatory (CD80) and anti-inflammatory (CD163) surface markers in human monocyte-derived macrophages (hMDMs). hUC-MSC-derived EVs did not change the surface marker expression of the hMDMs. In contrast, when hMDMs were co-incubated with hUC-MSCs in indirect co-cultures, changes were observed in the expression of CD14, CD80 and CD163, particularly in M1 macrophages, suggesting that soluble factors are necessary to elicit a shift in phenotype. However, even though EVs did not alter the surface marker expression of macrophages, they promoted angiogenesis and phagocytic capacity increased proportionally to increases in EV concentration. Taken together, these results suggest that hUC-MSC-derived EVs are not sufficient to alter macrophage phenotype and that additional MSC-derived factors are needed.

Quantifying acute kidney injury in an Ischaemia-Reperfusion Injury mouse model using deep-learning-based semantic segmentation in histology.

This study focuses on ischaemia-reperfusion injury (IRI) in kidneys, a cause of acute kidney injury (AKI) and end-stage kidney disease (ESKD). Traditional kidney damage assessment methods are semi-quantitative and subjective. This study aims to use a convolutional neural network (CNN) to segment murine kidney structures after IRI, quantify damage via CNN-generated pathological measurements, and compare this to conventional scoring. The CNN was able to accurately segment the different pathological classes, such as Intratubular casts and Tubular necrosis, with an F1 score of over 0.75. Some classes, such as Glomeruli and Proximal tubules, had even higher statistical values with F1 scores over 0.90. The scoring generated based on the segmentation approach statistically correlated with the semiquantitative assessment (Spearman's rank correlation coefficient=0.94). The heatmap approach localised the intratubular necrosis mainly in the outer stripe of the outer medulla, while the tubular casts were also present in more superficial or deeper portions of the cortex and medullary areas. This study presents a CNN model capable of segmenting multiple classes of interest, including acute IRI-specific pathological changes, in a whole mouse kidney section and can provide insights into the distribution of pathological classes within the whole mouse kidney section.

Mesothelial Cells Exhibit Characteristics of Perivascular Cells in an In Vitro Angiogenesis Assay.

Mesothelial cells have been shown to have remarkable plasticity towards mesenchymal cell types during development and in disease situations. Here, we have characterized the potential of mesothelial cells to undergo changes toward perivascular cells using an in vitro angiogenesis assay. We demonstrate that GFP-labeled mesothelial cells (GFP-MCs) aligned closely and specifically with endothelial networks formed when human dermal microvascular endothelial cells (HDMECs) were cultured in the presence of VEGF-A165 on normal human dermal fibroblasts (NHDFs) for a 7-day period. The co-culture with GFP-MCs had a positive effect on branch point formation indicating that the cells supported endothelial tube formation. We interrogated the molecular response of the GFP-MCs to the angiogenic co-culture by qRT-PCR and found that the pericyte marker Ng2 was upregulated when the cells were co-cultured with HDMECs on NHDFs, indicating a change towards a perivascular phenotype. When GFP-MCs were cultured on the NHDF feeder layer, they upregulated the epithelial-mesenchymal transition marker Zeb1 and lost their circularity while increasing their size, indicating a change to a more migratory cell type. We analyzed the pericyte-like behavior of the GFP-MCs in a 3D cardiac microtissue (spheroid) with cardiomyocytes, cardiac fibroblasts and cardiac endothelial cells where the mesothelial cells showed alignment with the endothelial cells. These results indicate that mesothelial cells have the potential to adopt a perivascular phenotype and associate with endothelial cells to potentially support angiogenesis.

Harmonised culture procedures minimise but do not eliminate mesenchymal stromal cell donor and tissue variability in a decentralised multicentre manufacturing approach.

BACKGROUND: Mesenchymal stromal cells (MSCs), commonly sourced from adipose tissue, bone marrow and umbilical cord, have been widely used in many medical conditions due to their therapeutic potential. Yet, the still limited understanding of the underlying mechanisms of action hampers clinical translation. Clinical potency can vary considerably depending on tissue source, donor attributes, but importantly, also culture conditions. Lack of standard procedures hinders inter-study comparability and delays the progression of the field. The aim of this study was A- to assess the impact on MSC characteristics when different laboratories, performed analysis on the same MSC material using harmonised culture conditions and B- to understand source-specific differences. METHODS: Three independent institutions performed a head-to-head comparison of human-derived adipose (A-), bone marrow (BM-), and umbilical cord (UC-) MSCs using harmonised culture conditions. In each centre, cells from one specific tissue source were isolated and later distributed across the network to assess their biological properties, including cell expansion, immune phenotype, and tri-lineage differentiation (part A). To assess tissue-specific function, angiogenic and immunomodulatory properties and the in vivo biodistribution were compared in one expert lab (part B). RESULTS: By implementing a harmonised manufacturing workflow, we obtained largely reproducible results across three independent laboratories in part A of our study. Unique growth patterns and differentiation potential were observed for each tissue source, with similar trends observed between centres. Immune phenotyping verified expression of typical MSC surface markers and absence of contaminating surface markers. Depending on the established protocols in the different laboratories, quantitative data varied slightly. Functional experiments in part B concluded that conditioned media from BM-MSCs significantly enhanced tubulogenesis and endothelial migration in vitro. In contrast, immunomodulatory studies reported superior immunosuppressive abilities for A-MSCs. Biodistribution studies in healthy mice showed lung entrapment after administration of all three types of MSCs, with a significantly faster clearance of BM-MSCs. CONCLUSION: These results show the heterogeneous behaviour and regenerative properties of MSCs as a reflection of intrinsic tissue-origin properties while providing evidence that the use of harmonised culture procedures can reduce but do not eliminate inter-lab and operator differences.

VEGF Stimulates Activation of ERK5 in the Absence of C-Terminal Phosphorylation Preventing Nuclear Localization and Facilitating AKT Activation in Endothelial Cells.

Extracellular-signal-regulated kinase 5 (ERK5) is critical for normal cardiovascular development. Previous studies have defined a canonical pathway for ERK5 activation, showing that ligand stimulation leads to MEK5 activation resulting in dual phosphorylation of ERK5 on Thr218/Tyr220 residues within the activation loop. ERK5 then undergoes a conformational change, facilitating phosphorylation on residues in the C-terminal domain and translocation to the nucleus where it regulates MEF2 transcriptional activity. Our previous research into the importance of ERK5 in endothelial cells highlighted its role in VEGF-mediated tubular morphogenesis and cell survival, suggesting that ERK5 played a unique role in endothelial cells. Our current data show that in contrast to EGF-stimulated HeLa cells, VEGF-mediated ERK5 activation in human dermal microvascular endothelial cells (HDMECs) does not result in C-terminal phosphorylation of ERK5 and translocation to the nucleus, but instead to a more plasma membrane/cytoplasmic localisation. Furthermore, the use of small-molecule inhibitors to MEK5 and ERK5 shows that instead of regulating MEF2 activity, VEGF-mediated ERK5 is important for regulating AKT activity. Our data define a novel pathway for ERK5 activation in endothelial cells leading to cell survival.

Near infrared conjugated polymer nanoparticles (CPN™) for tracking cells using fluorescence and optoacoustic imaging.

Tracking the biodistribution of cell therapies is crucial for understanding their safety and efficacy. Optical imaging techniques are particularly useful for tracking cells due to their clinical translatability and potential for intra-operative use to validate cell delivery. However, there is a lack of appropriate optical probes for cell tracking. The only FDA-approved material for clinical use is indocyanine green (ICG). ICG can be used for both fluorescence and photoacoustic imaging, but is prone to photodegradation, and at higher concentrations, undergoes quenching and can adversely affect cell health. We have developed novel near-infrared imaging probes comprising conjugated polymer nanoparticles (CPNs™) that can be fine-tuned to absorb and emit light at specific wavelengths. To compare the performance of the CPNs™ with ICG for in vivo cell tracking, labelled mesenchymal stromal cells (MSCs) were injected subcutaneously in mice and detected using fluorescence imaging (FI) and a form of photoacoustic imaging called multispectral optoacoustic tomography (MSOT). MSCs labelled with either ICG or CPN™ 770 could be detected with FI, but only CPN™ 770-labelled MSCs could be detected with MSOT. These results show that CPNs™ show great promise for tracking cells in vivo using optical imaging techniques, and for some applications, out-perform ICG.

Extracellular Vesicles from Human Umbilical Cord-Derived MSCs Affect Vessel Formation In Vitro and Promote VEGFR2-Mediated Cell Survival.

Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have emerged as novel tools in regenerative medicine. Angiogenesis modulation is widely studied for the treatment of ischaemic diseases, wound healing, and tissue regeneration. Here, we have shown that EVs from human umbilical cord-derived MSCs can affect VEGFR2 signalling, a master regulator of angiogenesis homeostasis, via altering the phosphorylation of AKT. This translates into an inhibition of apoptosis, promoting exclusively cell survival, but not proliferation, in human microvascular endothelial cells. Interestingly, when comparing EVs from normoxic cells to those obtained from hypoxia (1% O2) preconditioned cells, hypoxia-derived EVs appear to have a slightly enhanced effect. Furthermore, when studied in a longer term endothelial-fibroblast co-culture angiogenesis model in vitro, both EV populations demonstrated a positive effect on vessel formation, evidenced by increased vessel networks with tubes of significantly larger diameters. Our data reveals that EVs selectively target components of the angiogenic pathway, promoting VEGFR2-mediated cell survival via enhancement of AKT activation. Our data show that EVs are able to enhance specific components of the VEGF signalling pathway and may have therapeutic potential to support endothelial cell survival.

Murine models of renal ischemia reperfusion injury: An opportunity for refinement using noninvasive monitoring methods.

BACKGROUND: Renal ischemia reperfusion injury (R-IRI) can cause acute kidney injury (AKI) and chronic kidney disease (CKD), resulting in significant morbidity and mortality. To understand the underlying mechanisms, reproducible small-animal models of AKI and CKD are needed. We describe how innovative technologies for measuring kidney function noninvasively in small rodents allow successful refinement of the R-IRI models, and offer the unique opportunity to monitor longitudinally in individual animals the transition from AKI to CKD. METHODS: Male BALB/c mice underwent bilateral renal pedicle clamping (AKI) or unilateral renal pedicle clamping with delayed contralateral nephrectomy (CKD) under isoflurane anesthetic. Transdermal GFR monitoring and multispectral optoacoustic tomography (MSOT) in combination with statistical analysis were used to identify and standardize variables within these models. RESULTS: Pre-clamping anesthetic time was one of the most important predictors of AKI severity after R-IRI. Standardizing pre-clamping time resulted in a more predictably severe AKI model. In the CKD model, MSOT demonstrated initial improvement in renal function, followed by significant progressive reduction in function between weeks 2 and 4. Performing contralateral nephrectomy on day 14 enabled the development of CKD with minimal mortality. CONCLUSIONS: Noninvasive monitoring of global and individual renal function after R-IRI is feasible and reproducible. These techniques can facilitate refinement of kidney injury models and enable the degree of injury seen in preclinical models to be translated to those seen in the clinical setting. Thus, future therapies can be tested in a clinically relevant, noninvasive manner.

CHMP5 is essential for late endosome function and down-regulation of receptor signaling during mouse embryogenesis.

Charged MVB protein 5 (CHMP5) is a coiled coil protein homologous to the yeast Vps60/Mos10 gene and other ESCRT-III complex members, although its precise function in either yeast or mammalian cells is unknown. We deleted the CHMP5 gene in mice, resulting in a phenotype of early embryonic lethality, reflecting defective late endosome function and dysregulation of signal transduction. Chmp5-/- cells exhibit enlarged late endosomal compartments that contain abundant internal vesicles expressing proteins that are characteristic of late endosomes and lysosomes. This is in contrast to ESCRT-III mutants in yeast, which are defective in multivesicular body (MVB) formation. The degradative capacity of Chmp5-/- cells was reduced, and undigested proteins from multiple pathways accumulated in enlarged MVBs that failed to traffic their cargo to lysosomes. Therefore, CHMP5 regulates late endosome function downstream of MVB formation, and the loss of CHMP5 enhances signal transduction by inhibiting lysosomal degradation of activated receptors.