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Cellular processes including lysosomal and mitochondrial dysfunction are implicated in the development of many diseases. Quantitative visualization of mitochondria and lysosoesl is crucial to understand how these organelles are dysregulated during disease. To address a gap in live-imaging tools, we developed GEM-SCOPe (Genetically Encoded and Modular SubCellular Organelle Probes), a modular toolbox of fluorescent markers designed to inform on localization, distribution, turnover, and oxidative stress of specific organelles. We expressed GEM-SCOPe in differentiated astrocytes and neurons from a human pluripotent stem cell PRKN-knockout model of Parkinson's disease and identified disease-associated changes in proliferation, lysosomal distribution, mitochondrial transport and turnover, and reactive oxygen species. We demonstrate GEM-SCOPe is a powerful panel that provide critical insight into the subcellular mechanisms underlying Parkinson's disease in human cells. GEM-SCOPe can be expanded upon and applied to a diversity of cellular models to glean an understanding of the mechanisms that promote disease onset and progression.
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Inducible pluripotent stem cells (iPSCs) derived from patient samples have significantly enhanced our ability to model neurological diseases. Comparative studies of dopaminergic (DA) neurons differentiated from iPSCs derived from siblings with Gaucher disease discordant for parkinsonism provides a valuable avenue to explore genetic modifiers contributing to GBA1-associated parkinsonism in disease-relevant cells. However, such studies are often complicated by the inherent heterogeneity in differentiation efficiency among iPSC lines derived from different individuals. To address this technical challenge, we devised a selection strategy to enrich dopaminergic (DA) neurons expressing tyrosine hydroxylase (TH). A neomycin resistance gene (neo) was inserted at the C-terminus of the TH gene following a T2A self-cleavage peptide, placing its expression under the control of the TH promoter. This allows for TH+ DA neuron enrichment through geneticin selection. This method enabled us to generate comparable, high-purity DA neuron cultures from iPSC lines derived from three sisters that we followed for over a decade: one sibling is a healthy individual, and the other two have Gaucher disease (GD) with GBA1 genotype N370S/c.203delC+R257X (p.N409S/c.203delC+p.R296X). Notably, the younger sister with GD later developed Parkinson disease (PD). A comprehensive analysis of these high-purity DA neurons revealed that although GD DA neurons exhibited decreased levels of glucocerebrosidase (GCase), there was no substantial difference in GCase protein levels or lipid substrate accumulation between DA neurons from the GD and GD/PD sisters, suggesting that the PD discordance is related to of other genetic modifiers.
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Parkinson's disease (PD) is characterized pathologically by the loss of dopaminergic (DA) neurons in the substantia nigra (SN). Whether cell types beyond DA neurons in the SN show vulnerability in PD remains unclear. Through transcriptomic profiling of 315,867 high-quality single nuclei in the SN from individuals with and without PD, we identified cell clusters representing various neuron types, glia, endothelial cells, pericytes, fibroblasts, and T cells and investigated cell type-dependent alterations in gene expression in PD. Notably, a unique neuron cluster marked by the expression of RIT2, a PD risk gene, also displayed vulnerability in PD. We validated RIT2-enriched neurons in midbrain organoids and the mouse SN. Our results demonstrated distinct transcriptomic signatures of the RIT2-enriched neurons in the human SN and implicated reduced RIT2 expression in the pathogenesis of PD. Our study sheds light on the diversity of cell types, including DA neurons, in the SN and the complexity of molecular and cellular changes associated with PD pathogenesis.
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Células Endoteliales , Enfermedad de Parkinson , Humanos , Animales , Ratones , Enfermedad de Parkinson/genética , Sustancia Negra , Neuronas Dopaminérgicas , NeuroglíaRESUMEN
Accumulation of advanced glycation end products (AGEs) on biopolymers accompanies cellular aging and drives poorly understood disease processes. Here, we studied how AGEs contribute to development of early onset Parkinson's Disease (PD) caused by loss-of-function of DJ1, a protein deglycase. In induced pluripotent stem cell (iPSC)-derived midbrain organoid models deficient for DJ1 activity, we find that lysosomal proteolysis is impaired, causing AGEs to accumulate, α-synuclein (α-syn) phosphorylation to increase, and proteins to aggregate. We demonstrated these processes are at least partly driven by astrocytes, as DJ1 loss reduces their capacity to provide metabolic support and triggers acquisition of a pro-inflammatory phenotype. Consistently, in co-cultures, we find that DJ1-expressing astrocytes are able to reverse the proteolysis deficits of DJ1 knockout midbrain neurons. In conclusion, astrocytes' capacity to clear toxic damaged proteins is critical to preserve neuronal function and their dysfunction contributes to the neurodegeneration observed in a DJ1 loss-of-function PD model.
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Enfermedad de Parkinson , Humanos , Enfermedad de Parkinson/genética , Proteostasis , Astrocitos , Proteolisis , Mesencéfalo , Organoides , LisosomasRESUMEN
The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer's disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.
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Enfermedad de Alzheimer , Angiopatía Amiloide Cerebral , Humanos , Barrera Hematoencefálica/metabolismo , Células Endoteliales/metabolismo , Encéfalo/metabolismo , Sistema Nervioso Central/metabolismo , Enfermedad de Alzheimer/metabolismoRESUMEN
Patient-specific, human-based cellular models that integrate biomimetic BBB, immune, and myelinated neuron components are critically needed to enable translationally relevant and accelerated discovery of neurological disease mechanisms and interventions. By engineering a brain-mimicking 3D hydrogel and co-culturing all six major brain cell types derived from patient iPSCs, we have constructed, characterized, and utilized a multicellular integrated brain (miBrain) immuno-glial-neurovascular model with in vivo- like hallmarks. As proof of principle, here we utilized the miBrain to model Alzheimer's Disease pathologies associated with APOE4 genetic risk. APOE4 miBrains differentially exhibit amyloid aggregation, tau phosphorylation, and astrocytic GFAP. Unlike the co-emergent fate specification of glia and neurons in organoids, miBrains integrate independently differentiated cell types in a modular system with unique utility for elucidating cell-type specific contributions to pathogenesis. We here harness this feature to identify that risk factor APOE4 in astrocytes promotes tau pathogenesis and neuronal dysregulation through crosstalk with microglia. One-Sentence Summary: A novel patient-specific brain model with BBB, neuronal, immune, and glial components was developed, characterized, and harnessed to model Alzheimer's Disease-associated pathologies and APOE4 genetic risk.
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The hypothalamus is a region of the brain that plays an important role in regulating body functions and behaviors. There is a growing interest in human pluripotent stem cells (hPSCs) for modeling diseases that affect the hypothalamus. Here, we established an hPSC-derived hypothalamus organoid differentiation protocol to model the cellular diversity of this brain region. Using an hPSC line with a tyrosine hydroxylase (TH)-TdTomato reporter for dopaminergic neurons (DNs) and other TH-expressing cells, we interrogated DN-specific pathways and functions in electrophysiologically active hypothalamus organoids. Single-cell RNA sequencing (scRNA-seq) revealed diverse neuronal and non-neuronal cell types in mature hypothalamus organoids. We identified several molecularly distinct hypothalamic DN subtypes that demonstrated different developmental maturities. Our in vitro 3D hypothalamus differentiation protocol can be used to study the development of this critical brain structure and can be applied to disease modeling to generate novel therapeutic approaches for disorders centered around the hypothalamus.
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The blood-brain barrier (BBB) is a key physiological component of the brain, protecting the brain from peripheral processes and pathogens. The BBB is a dynamic structure that is heavily involved in cerebral blood flow, angiogenesis, and other neural functions. However, the BBB also creates a challenging barrier for the entry of therapeutics into the brain, blocking more than 98% of drugs from contact with the brain. Neurovascular comorbidities are common in several neurological diseases including Alzheimer's and Parkinson's Disease, suggesting that BBB dysfunction or break down likely has a causal role in neurodegeneration. However, the mechanisms by which the human BBB is formed, maintained, and degenerated in diseases remain largely unknown due to limited access to human BBB tissue. To address these limitations, we have developed an in vitro induced human BBB (iBBB) derived from pluripotent stem cells. The iBBB model can be used for discovery of disease mechanisms, drug targets, drug screening, and medicinal chemistry studies to optimize brain penetration of central nervous system therapeutics. In this chapter, we will explain the steps to differentiate the three cellular components (endothelial cells, pericytes, and astrocytes) from induced pluripotent stem cells, and how to assemble them into the iBBB.
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Barrera Hematoencefálica , Células Madre Pluripotentes Inducidas , Humanos , Células Endoteliales , Astrocitos , EncéfaloRESUMEN
Non-invasive Gamma ENtrainment Using Sensory stimulation (GENUS) at 40Hz reduces Alzheimer's disease (AD) pathology such as amyloid and tau levels, prevents cerebral atrophy, and improves behavioral testing performance in mouse models of AD. Here, we report data from (1) a Phase 1 feasibility study (NCT04042922, ClinicalTrials.gov) in cognitively normal volunteers (n = 25), patients with mild AD dementia (n = 16), and patients with epilepsy who underwent intracranial electrode monitoring (n = 2) to assess safety and feasibility of a single brief GENUS session to induce entrainment and (2) a single-blinded, randomized, placebo-controlled Phase 2A pilot study (NCT04055376) in patients with mild probable AD dementia (n = 15) to assess safety, compliance, entrainment, and exploratory clinical outcomes after chronic daily 40Hz sensory stimulation for 3 months. Our Phase 1 study showed that 40Hz GENUS was safe and effectively induced entrainment in both cortical regions and other cortical and subcortical structures such as the hippocampus, amygdala, insula, and gyrus rectus. Our Phase 2A study demonstrated that chronic daily 40Hz light and sound GENUS was well-tolerated and that compliance was equally high in both the control and active groups, with participants equally inaccurate in guessing their group assignments prior to unblinding. Electroencephalography recordings show that our 40Hz GENUS device safely and effectively induced 40Hz entrainment in participants with mild AD dementia. After 3 months of daily stimulation, the group receiving 40Hz stimulation showed (i) lesser ventricular dilation and hippocampal atrophy, (ii) increased functional connectivity in the default mode network as well as with the medial visual network, (iii) better performance on the face-name association delayed recall test, and (iv) improved measures of daily activity rhythmicity compared to the control group. These results support further evaluation of GENUS in a pivotal clinical trial to evaluate its potential as a novel disease-modifying therapeutic for patients with AD.
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Enfermedad de Alzheimer , Demencia , Animales , Ratones , Enfermedad de Alzheimer/terapia , Proyectos Piloto , Estudios de Factibilidad , AtrofiaRESUMEN
APOE4 is the strongest genetic risk factor for Alzheimer's disease1-3. However, the effects of APOE4 on the human brain are not fully understood, limiting opportunities to develop targeted therapeutics for individuals carrying APOE4 and other risk factors for Alzheimer's disease4-8. Here, to gain more comprehensive insights into the impact of APOE4 on the human brain, we performed single-cell transcriptomics profiling of post-mortem human brains from APOE4 carriers compared with non-carriers. This revealed that APOE4 is associated with widespread gene expression changes across all cell types of the human brain. Consistent with the biological function of APOE2-6, APOE4 significantly altered signalling pathways associated with cholesterol homeostasis and transport. Confirming these findings with histological and lipidomic analysis of the post-mortem human brain, induced pluripotent stem-cell-derived cells and targeted-replacement mice, we show that cholesterol is aberrantly deposited in oligodendrocytes-myelinating cells that are responsible for insulating and promoting the electrical activity of neurons. We show that altered cholesterol localization in the APOE4 brain coincides with reduced myelination. Pharmacologically facilitating cholesterol transport increases axonal myelination and improves learning and memory in APOE4 mice. We provide a single-cell atlas describing the transcriptional effects of APOE4 on the aging human brain and establish a functional link between APOE4, cholesterol, myelination and memory, offering therapeutic opportunities for Alzheimer's disease.
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Apolipoproteína E4 , Encéfalo , Colesterol , Fibras Nerviosas Mielínicas , Oligodendroglía , Animales , Humanos , Ratones , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Colesterol/metabolismo , Oligodendroglía/metabolismo , Oligodendroglía/patología , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Mielínicas/patología , Autopsia , Células Madre Pluripotentes Inducidas , Neuronas/metabolismo , Neuronas/patología , Heterocigoto , Transporte Biológico , Homeostasis , Análisis de la Célula Individual , Memoria , Envejecimiento/genética , Perfilación de la Expresión Génica , Vaina de Mielina/metabolismo , Vaina de Mielina/patologíaRESUMEN
Children with congenital heart disease (CHD) are often restricted from some types of physical activity (PA) despite the lack of evidence regarding the need to restrict recreational PA, apart from those with rhythm disorders. This study retrospectively investigated the associations between parent-reported activity restrictions (on-going need to restrict exertion, body contact or competition) and measures of health-related fitness among 236 children (8.2 ± 2.1 years, range 4−12 years) treated for single ventricle (n = 104), tetralogy of Fallot (n = 48), transposition of the great arteries (n = 47) or atrial septal defect (n = 37). Body mass index (BMI), moderate-to-vigorous physical activity (MVPA; 7 day accelerometry), strength, flexibility, and movement skill assessment results were collected from the baseline assessment research records for two studies completed in Ontario, Canada. A subset of 62 children also had physician-reported activity restrictions. Regression models empirically tested the goodness of fit between the dependent and independent variables. Participants with body contact restrictions from both parents and physicians had significantly higher BMI z-scores (0.23 ± 1.19 vs. −0.32 ± 0.85; t = 2.55; p = 0.04 and 0.66 ± 1.33 vs. −0.02 ± 0.98; t = 2.25; p = 0.02 for CDC and WHO scores, respectively). Otherwise, BMI z-score was not associated with patient variables (p > 0.36; sex, cardiac diagnosis, age, or activity restriction). Children with any type of parent-reported restriction (0.98 ± 2.06 vs. −0.08 ± 1.99; t = 3.77; p = 0.0002) were less flexible. Movement skill (TGMD-2) scores were 50% lower (25.1 ± 31.2 vs. 52.6 ± 28.6; F = 6.93; p = 0.009) among children with parent-reported competitive sport restrictions. Weekly MVPA (p > 0.18) and strength (p > 0.05) were not associated with activity restriction. Children whose parents reported PA restrictions were less flexible, and had decreased movement skill and increased BMI z-scores if the restrictions impacted competitive sport or body contact, respectively. Future research is recommended to confirm these results among larger samples of children who have both parent- and physician-specified PA restrictions.
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Cardiopatías Congénitas , Transposición de los Grandes Vasos , Acelerometría , Índice de Masa Corporal , Niño , Ejercicio Físico , Cardiopatías Congénitas/cirugía , Humanos , Ontario , Aptitud Física , Estudios Retrospectivos , Transposición de los Grandes Vasos/cirugíaRESUMEN
Alzheimer disease (AD) is the most prevalent type of dementia. It is marked by severe memory loss and cognitive decline, and currently has limited effective treatment options. Although individuals with AD have common neuropathological hallmarks, emerging data suggest that the disease has a complex polygenic aetiology, and more than 25 genetic loci have been linked to an elevated risk of AD and dementia. Nevertheless, our ability to decipher the cellular and molecular mechanisms that underlie genetic susceptibility to AD, and its progression and severity, remains limited. Here, we discuss ongoing efforts to leverage genomic data from patients using cellular reprogramming technologies to recapitulate complex brain systems and build in vitro discovery platforms. Much attention has already been given to methodologies to derive major brain cell types from pluripotent stem cells. We therefore focus on technologies that combine multiple cell types to recreate anatomical and physiological properties of human brain tissue in vitro. We discuss the advances in the field for modelling four domains that have come into view as key contributors to the pathogenesis of AD: the blood-brain barrier, myelination, neuroinflammation and neuronal circuits. We also highlight opportunities for the field to further interrogate the complex genetic and environmental factors of AD using in vitro models.
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Enfermedad de Alzheimer/patología , Encéfalo/patología , Modelos Anatómicos , Modelos Neurológicos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/fisiopatología , Animales , Encéfalo/fisiología , Encéfalo/fisiopatología , Predisposición Genética a la Enfermedad , HumanosRESUMEN
While gene expression profiling has traditionally been the method of choice for large-scale perturbational profiling studies, proteomics has emerged as an effective tool in this context for directly monitoring cellular responses to perturbations. We previously reported a pilot library containing 3400 profiles of multiple perturbations across diverse cellular backgrounds in the reduced-representation phosphoproteome (P100) and chromatin space (Global Chromatin Profiling, GCP). Here, we expand our original dataset to include profiles from a new set of cardiotoxic compounds and from astrocytes, an additional neural cell model, totaling 5300 proteomic signatures. We describe filtering criteria and quality control metrics used to assess and validate the technical quality and reproducibility of our data. To demonstrate the power of the library, we present two case studies where data is queried using the concept of "connectivity" to obtain biological insight. All data presented in this study have been deposited to the ProteomeXchange Consortium with identifiers PXD017458 (P100) and PXD017459 (GCP) and can be queried at https://clue.io/proteomics .
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Antineoplásicos/toxicidad , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Cardiotoxinas/toxicidad , Inhibidores de Proteínas Quinasas/toxicidad , Proteómica , Línea Celular Tumoral , Humanos , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ProteomaRESUMEN
COVID-19 has unfortunately halted lab work, conferences, and in-person networking, which is especially detrimental to researchers just starting their labs. Through social media and our reviewer networks, we met some early-career stem cell investigators impacted by the closures. Here, they introduce themselves and their research to our readers.
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In Alzheimer's disease, amyloid deposits along the brain vasculature lead to a condition known as cerebral amyloid angiopathy (CAA), which impairs blood-brain barrier (BBB) function and accelerates cognitive degeneration. Apolipoprotein (APOE4) is the strongest risk factor for CAA, yet the mechanisms underlying this genetic susceptibility are unknown. Here we developed an induced pluripotent stem cell-based three-dimensional model that recapitulates anatomical and physiological properties of the human BBB in vitro. Similarly to CAA, our in vitro BBB displayed significantly more amyloid accumulation in APOE4 compared to APOE3. Combinatorial experiments revealed that dysregulation of calcineurin-nuclear factor of activated T cells (NFAT) signaling and APOE in pericyte-like mural cells induces APOE4-associated CAA pathology. In the human brain, APOE and NFAT are selectively dysregulated in pericytes of APOE4 carriers, and inhibition of calcineurin-NFAT signaling reduces APOE4-associated CAA pathology in vitro and in vivo. Our study reveals the role of pericytes in APOE4-mediated CAA and highlights calcineurin-NFAT signaling as a therapeutic target in CAA and Alzheimer's disease.
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Apolipoproteína E4/genética , Barrera Hematoencefálica/metabolismo , Calcineurina/metabolismo , Angiopatía Amiloide Cerebral/genética , Factores de Transcripción NFATC/genética , Pericitos/metabolismo , Péptidos beta-Amiloides/metabolismo , Apolipoproteína E3/genética , Apolipoproteína E3/metabolismo , Apolipoproteína E4/metabolismo , Barrera Hematoencefálica/citología , Humanos , Técnicas In Vitro , Células Madre Pluripotentes Inducidas , Factores de Transcripción NFATC/metabolismo , Permeabilidad , RNA-Seq , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Background: The Canadian Assessment of Physical Literacy (CAPL) accurately and reliably assesses the physical literacy level of young children. However, in preliminary analyses ceiling effects were noted among 183 older children. The purposes of this study were to assess the validity of a modified CAPL designed for older children (CAPL 789) and to offer descriptive results.Methods: CAPL 789 assessed the physical literacy of Canadian children in grades 7-9. Data were collected from schools and recreation facilities in the provinces of Alberta and Ontario (Canada), yielding a new sample of 245 participants (129 girls, 13.7 ± 0.9 years). Descriptive statistics were calculated for all CAPL domains. Age and gender effects were examined to support the validity.Results: Physical competence score (/32) increased with age (F = 4.90, p < 0.05), the means in grades 7-9 being 18.9 ± 3.2, 20.9 ± 4.4 and 21.2 ± 3.4, respectively. Girls scored significantly higher (t = -2.29, p < 0.05) than boys (6.6 ± 1.2 vs 6.3 ± 1.3) in the knowledge assessment (/10).Conclusion: The CAPL 789 was feasible among children from grades 7-9. Additional research is required to establish the psychometric properties in children 12 to 16 years.
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Conducta del Adolescente , Conducta Infantil , Ejercicio Físico/psicología , Conductas Relacionadas con la Salud , Educación y Entrenamiento Físico/métodos , Encuestas y Cuestionarios , Adolescente , Factores de Edad , Composición Corporal , Niño , Femenino , Humanos , Masculino , Motivación , Actividad Motora , Educación y Entrenamiento Físico/estadística & datos numéricos , Psicometría , Reproducibilidad de los Resultados , Factores SexualesRESUMEN
Cardiorespiratory fitness (CRF) is a good summative measure of the body's ability to perform continuous, rhythmic, dynamic, large-muscle group physical activity, and exercise. In children, CRF is meaningfully associated with health, independent of physical activity levels, and it is an important determinant of sports and athletic performance. Although gas-analyzed peak oxygen uptake is the criterion physiological measure of children's CRF, it is not practical for population-based testing. Field testing offers a simple, cheap, practical alternative to gas analysis. The 20-m shuttle run test (20mSRT)-a progressive aerobic exercise test involving continuous running between 2 lines 20 m apart in time to audio signals-is probably the most widely used field test of CRF. This review aims to clarify the international utility of the 20mSRT by synthesizing the evidence describing measurement variability, validity, reliability, feasibility, and the interpretation of results, as well as to provide future directions for international surveillance. The authors show that the 20mSRT is an acceptable, feasible, and scalable measure of CRF and functional/exercise capacity, and that it has moderate criterion validity and high to very high reliability. The assessment is pragmatic, easily interpreted, and results are transferable to meaningful and understandable situations. The authors recommend that CRF, assessed by the 20mSRT, be considered as an international population health surveillance measure to provide additional insight into pediatric population health.