Amyotrophic lateral sclerosis
ICD-10 Code G12.21
What is the clinical spectrum of Amyotrophic Lateral Sclerosis?
Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a progressive and lethal disorder that causes motor neuron degeneration, resulting in muscle weakness, paralysis, weight loss, and respiratory failure Brotman et al., StatPearls, Feb 2024.
While ALS can be both of sporadic and of familial origin, over 90% of ALS cases are sporadic.
The clinical presentation of ALS is heterogeneous, which is believed to be the result of interaction of multiple genetic, lifestyle, and environmental factors Feldman et al., Lancet, Oct 2022.
The hallmark clinical feature of ALS is the coexistence of symptoms triggered by degeneration of upper motor neurons (UMN), in the brain and the spinal cord, and lower motor neurons (LMN), in the anterior horn of the spinal cord and the brainstem Meyer et al., Neurol. Res. Pract., Apr 2025.
In limb-onset ALS, LMN lesions are associated with muscle atrophy and flaccid paralysis (loss of muscle tone, muscle weakness, reduced reflexes), while UMN lesions present with hyperreflexia, poor dexterity, incoordination, and spastic paralysis (muscle spasms, involuntary jerky movements).
In the less frequently occurring bulbar onset ALS, the most common symptoms are dysarthria (slurred speech) and dysphagia (difficulty swallowing).
Respiratory-onset ALS, which occurs in around 5% of ALS cases, begins with progressive weakness of the diaphragm controlled by LMN, leading to shortness of breath during exertion, and orthopnea (breathing difficulty when lying down).
In progressive muscular atrophy, most patients initially present with asymmetric LMN lesion symptoms that progress to UMN lesion symptoms. Patients with primary lateral sclerosis initially present with UMN lesion symptoms and the disease usually progresses to LMN lesion symptoms. Symptoms and their rates of progression vary according to the disease type. Patients with primary lateral sclerosis typically have a slower progression and lack weight loss.
Cell types within affected regions include Betz cells in the primary motor cortex in UMN, anterior horn cells of the spinal cord in LMN, and lower cranial motor nuclei of the brainstem in LMN.
Small eosinophilic intraneuronal inclusions, termed Bunina bodies, are common in degenerating neurons. The abnormal accumulation of TDP-43 is present in most cases of sporadic ALS, ALS-Fronto-temporal dementia, and some cases of familial ALS Dong & Chen, Neurosci., Feb 2018.
Symptoms typically appear in adulthood, with an onset at 55 to 65 years of age in average.
Up to half of ALS patients develop varying degrees of cognitive impairment and of change in behaviour. Notably, up to 75% of ALS patients that carry C9ORF72 mutation experience cognitive impairment, with up to 45% meeting criteria for frontotemporal dementia (FTD) Jellinger, Int. J. Mol. Sci., Sep 2023.
Individuals with ALS usually die of respiratory failure within 3 to 5 years following the onset of symptoms.
What do we know about the etiology of Amyotrophic Lateral Sclerosis?
ALS is a multifactorial disease with a complex interplay of genetic, epigenetic, environmental and lifestyle triggering factors Pasinelli & Brown, Nature, Sep 2006, Ryan et al., JAMA Neur., Jul 2019.
The precise etiology of ALS is not fully understood, and is believed to involve multiple mechanisms, including genetic mutations, oxidative stress, excitotoxicity, defective RNA processing, intraneuronal protein aggregates, mitochondrial dysfunction, and neuroinflammation.
Mechanistic pathways may vary across affected individuals, with inter-individual variations in genetic background being a contributing factors.
Approximately 10–15% of ALS cases are considered familial ALS (FALS) and inherited in either an autosomal dominant, autosomal recessive, or X-linked mode, while the remaining ALS cases are sporadic (SALS).
Over 120 genes have so far been implicated in ALS and this number keeps growing as new data from ALS patient-population Genome-Wide Association Study (GWAS) and Whole Exome Sequencing (WES) keeps coming in.
About 70% of FALS and 15% of SALS have mutations in known ALS genes, including SOD1, FUS, TARDBP, C9ORF72, ATXN2, and others. However, the distinction between familial and sporadic ALS is not clear-cut Alsultan et al., Degener. Neurol. Neurom. Dis., May 2016, Al-Chalabi, Nature Rev. Neurol., Dec 2016, Wang et al., Front. Neurosci., May 2023.
Approx. 10%-20% of FALS and 1% of SALS cases are associated with mutations in superoxide dismutase 1 (SOD1), that regulates oxidative stress, lipid metabolism, and inflammation.
TARDBP gene, that encodes the transactive response DNA-binding protein 43 (TDP-43), that regulates various steps of RNA metabolism, including mRNA splicing, RNA transportation, translation, and miRNA biogenesis, is mutated in approx. 5% of FALS.
To date, more than 50 different mutations in fused in sarcoma (FUS) gene that encodes a multifunctional RNA binding protein involved in transcription, alternative splicing, mRNA transport, mRNA stability, and miRNA biogenesis, have been identified in patients with ALS, which together account for approx. 4% of FALS and fewer than 2% of SALS.
Chromosome 9 open Reading frame 72 hexanucleotide repeat expansions in the noncoding region of C9ORF72 are found in about 39% of FALS and 7% of SALS of European ancestry.
Screening of a large ALS cohort demonstrated that 14% of FALS and 2.6% of SALS cases had more than one potential pathogenic mutation in a known ALS gene, and these cases had a significantly earlier onset of disease.
ALS has a genetic architecture in which a few rare variants contribute to risk in each patient, rather than a polygenic architecture whereby the cumulative effects of many common variants increase risk.
ALS-associated genetic mutations trigger protein misfolding and aggregations that impair mitochondrial electron transport chain function, increasing reactive oxygen species (ROS) that exacerbates protein misfolding-induced endoplasmic reticulum (ER) stress. Persistent ER stress activates the unfolded protein response (UPR) machinery that, if overwhelmed, can result in cell apoptosis.
This pathological process functions in feedback loops that can form a vicious cycle. For example, high ROS levels, amplified by mitochondria damage, can cause DNA damage, lipid peroxidation, and protein oxidation, accelerating neuronal death.
Environmental stressors, such as heavy metals, pesticides, and physical trauma, are believed to act through overlapping molecular pathways that involve damage to mitochondrial electron transport, excessive ROS, ER damage, UPR and apoptosis.
Age-related decline in protein autophagy and in DNA repair, that would have allowed to mitigate the ALS pathophysiology, are a factor of vulnerability, explaining the onset of ALS at an advanced age.
Misfolded proteins act as damage-associated molecular patterns (DAMP) that activate microglia, the resident immune cells unique to the central nervous system, that clear misfolded proteins, apoptotic neurons and other cell debris Di Sabato et al., J. Neurochem., Mar 2016.
DAMP bind to microglia’s pattern recognition receptors (toll-like receptors, NOD-like receptors), triggering the NF-κB pathway and microglial release of cytokines IL-1β, TNF-α, and IL-6 that lock microglia into a M1 pro-inflammatory phenotype that sustains neuroinflammation. Cytokines signals that help microglia shift to a M2 reparative state are downregulated in ALS.
The complement cascade becomes dysregulated in ALS, leading to C3 and C1q marking of motor neurons and synapses for phagocytosis by microglia, contributing to neuronal loss Sta et al., Neurobio. Dis., Jun 2011.
In ALS, tight junction proteins (ZO-1, occludin, claudin-5) are downregulated, leading to vascular leakage Garbuzova-Davis et al., Brain Res., Aug 2012.
Blood-spinal cord barrier disruption allows infiltration of CD4/CD8 T cells that produce IFN-γ and IL-4, further exacerbating the shifting of microglia to proinflammatory M1 and astrocytes to A1 neurotoxic states that accelerate neuronal death Engelhardt et al., Arch. Neurol., Jan 1993, Rolfes et al., Brain Comm., Jul 2021.
Neuroinflammation triggers gliosis as a protective mechanism in which proliferating astrocytes and microglia attempt to prevent further injury by forming a fibrotic barrier composed of extracellular matrix (ECM) proteins. However, as ALS progresses, chronic neuroinflammation entertains a chronic gliosis, in which over-reactive astrocytes form a dense glial scar that ultimately prevents tissue regeneration Lawrence et al., Acta Neuropath. Comm., Mar 2023, Li et al., Tox. Res., Dec 2023.
Often linked to environmental or genetic factors, glutamate excitotoxicity is believed to be another major driver of ALS.
Transcranial magnetic stimulation assessment of the functional integrity of the motor cortex and its corticomotoneuronal projections had shown cortical hyperexcitability both in patients with SALS and FALS Vucic & Kiernan, H. Clin. Neurol., Jan 2013.
Possible causes of excitotoxicity include dysregulation of glutamate clearance by EAAT2 transporters, leading to accumulation of excitatory glutamate in the synaptic cleft, or change in AMPA and NMDA receptors, leading to sustained Ca2+ influx into post-synaptic neurons and subsequent mitochondrial dysfunction, oxidative stress, and apoptosis Van den Bosch et al., BBA Mol. Bas. Dis., Nov 2006.
How similar are human and animal neuromuscular systems?
This is not an exhaustive list of species-specific differences, nor can one be made given their unknown full extent, but rather an example of how these differences impact the face, construct, predictive, and intrinsic validity of animal models.
Not all species-specific differences can be accounted for in animal models, as there are hundreds of them, their relevance for amyotrophic lateral sclerosis (ALS) is unclear, and their interaction with other organ systems in the animal model makes it difficult to predict how they would have behaved within the human system.
Species-specific differences in corticomotoneuronal system anatomy and physiology
The corticomotoneuronal system has multiple human-specific features that play a key role in susceptibility to ALS in humans and that cannot be recapitulated in animal models of ALS.
The corticomotoneuronal (CM) system in humans comprises direct monosynaptic connections from cortical UMN, in the primary motor cortex, to LMN, in the spinal cord and brainstem, enabling fractionated and precise movements and other fine motor behaviours unique to primates, that are particularly developed in humans, and that therefore cannot be recapitulated in rodent models Eisen et al., J. Neurol. Neurosurg. Psych., Jul 2017.
Unlike primates, rodents lack direct CM connections between UMN and LMN. Instead, their corticospinal neurons primarily influence movement indirectly through interneurons.
The fact that humans have longer axons, and higher axonal transport of molecules, organelles, and proteins, also makes them uniquely vulnerable to mitochondrial dysfunction-reduced energy supply.
The increased dependency on direct UMN-LMN connectivity in humans, makes dual UMN-LMN degeneration even more devastating, likely accounting for a more severe distal motor deficits in ALS patients than in ALS animal models Lemon, Ann. Rev. Neurosci., Jul 2008.
Betz cells, a specialized subtype of L5 pyramidal excitatory neurons found in the UMN’s primary motor cortex, play a critical role in corticospinal motor control.
Human Betz cells are larger in diameter, more numerous per hemisphere and have a more extensive dendritic arborization than NHP or mouse Betz cells Nolan et al., J. Comp. Neurol., Jan 2024.
Larger Betz cells generate stronger action potentials and innervate multiple LMN, which both amplifies motor output and vulnerability to ALS-related excitotoxicity.
In Betz cells of ALS patients, TDP-43 accumulate more compared to mice, accelerating UMN loss Wu et al., Ag. Health Res., Mar 2025.
Similarly to Betz cell, a specific subclass of excitatory neurons called Von Economo neurons, primarily located in the frontoinsular and anterior cingulate cortex, also show transcriptional dysregulation in ALS state, including in stress response, synaptic vesicle cycling, and autophagy Pineda et al., Cell, Apr 2024.
Von Economo neurons, believed to be linked to higher order cognitive and emotional functions, are present in humans but absent in many of commonly used model organisms, including mice, rats, rabbits, cats, and dogs Hodge et al., Nature, Aug 2019.
Species-specific differences in gene orthologs and gene expression
In humans, ALS risk is driven by rare highly penetrant genetic variants rather than common polygenic factors.
Not all ALS-associated genes have orthologs in model organisms that have the same ALS-related function, and even when they do, their expression consistently differs across species, impacting the validity of the disease phenotype, relevance of insights on disease mechanisms, and applicability of therapeutic targets in model organisms.
For example, while C9ORF72 gene has a murine ortholog, its repeat expansion mutation does not naturally occur in mice Chew et al., Science, May 2015.
Comparative single-cell transcriptomics analysis of human and mouse cerebral cortex shows that despite basic transcriptomic similarities of cell types, there are numerous inter-species differences at the single-gene and gross-structural level. The most-divergent gene families include neurotransmitter receptors, ion channels, extracellular matrix (ECM) elements, and cell-adhesion molecules Hodge et al., Nature, Sep 2019, all of which intervene in ALS.
Alternatively spliced exons contribute to the molecular diversity, differentiation, and function of brain cell types Zhao et al., BMC Gen., Apr 2023. And yet, only about a quarter of alternatively spliced exons for a given transcript is conserved between humans and rodents Modrek & Lee, Nature Gen., May 2003, indicating possible inter-species differences is spliceosome machinery (small nuclear ribonucleoproteins, splicing factors, RNA-binding proteins).
Long noncoding RNA (lncRNA) can regulate gene expression in several manners, including by direct binding to pre-mRNA and interaction with transcription factors. Analysis of ALS patient-derived samples showed that expression of numerous lncRNA is dysregulated, sparking interest in lncRNA as disease progression biomarkers and therapeutic targets Yu et al., Sci. Rep., Sep 2024. However, genome, transcriptome, and evolutionary analysis suggest that the same lncRNA mechanisms that have rewired gene expression in a human-specific manner, including in susceptibility to neurodegenerative diseases like ALS, are not likely to be found in model organisms Zimmer-Bensch, Cells, Nov 2019, Lin et al., eLife, Dec 2023.
In agreement with these findings, differential expression analysis between human and mouse UMN and LMN motoneurons, which degenerate in ALS, has confirmed human-specific enrichment in statistically significant ALS-associated genes Yadav et al., Cell, Neuron, Feb 2023, Pineda et al., Cell, Apr 2024.
Species-specific differences in alternative splicing
There are ALS-associated genes that show variable mRNA splicing patterns across species, leading to species-specific protein diversity and function Barbosa-Morais et al., Science, Dec 2012.
One such example is inter-species variation in RNA-binding specificity of the TDP-43 protein, due to differences in TDP43 protein sequence, TDP43-binding RNA motifs, and cellular context Carmen-Orozco et al., Mol. Neurodeg., Jun 2024.
Inter-species differences in RNA binding motifs can arise as a result of variations across species in RNA sequence, in RNA secondary structure, and in RNA subcellular localisations.
Implications for study of ALS are inter-specific differences in phenotype of experimental animal models of ALS, in disease mechanisms and in relevance of therapeutic targets Gumina et al., Int. J. Mol. Sci., Nov 2019.
Species-specific differences in neuromuscular vascular physiology
ALS patients often present with pathological alterations in their small cerebral blood vessels, which has detrimental effects on the integrity of the blood brain barrier (BBB), blood–spinal cord barrier (BSCB), and blood–cerebrospinal fluid barrier (BCSFB) Leonardi et al., J. Neurol., Apr 1984.
Endothelial cells, astrocytes, and pericytes, are essential for production of tight junction proteins, regulation of tight junction integrity, and stabilization of tight junctions. Decreased transcription of tight junction proteins that are essential for the integrity of BSCB was observed in spinal cords from ALS patients Henkel et al., Neurol., May 2009.
Brain vascular and perivascular cell types (endothelial cells, pericytes, smooth muscle cells) exhibit divergent transcriptional profiles between humans and mice, conferring human-specific vulnerabilities and perturbations across the brain vasculature that do not overlap with mouse models of human neurodegenerative diseases Yang et al., Nature, Feb 2022.
The majority of genes that have been linked to risk of Alzheimer’s disease by GWAS are expressed in the human brain vasculature and are associated with endothelial protein transport, adaptive immunity, and ECM pathways. The current knowledge of disease mechanisms suggests that these human-specific pathways share commonalities with ALS pathophysiology, notably in neuroinflammation.
Brain pericytes (also known as Rouget cells), wrap around and communicate with the endothelial cells of capillaries and small blood vessels in the CNS, thus supporting the integrity of the blood brain/spinal cord barrier. Their loss or dysfunction is believed to contribute to ALS.
Comparison of mouse and human pericytes transcriptomes showed that 206 orthologous genes were consistently differentially expressed between human and mouse pericytes, of which 91 genes were specific/up-regulated in human and 115 in mouse pericytes Miao et al., Vasc. Pharmac., Dec 2024. Genes that were expressed in human but not in mouse brain pericytes, such as MAGI2, DLG2, GPC5, and HTR1F, may indirectly contribute to ALS pathology through BSCB dysfunction, excitotoxicity, and neuroinflammation.
Vascular cells in the adult human brain exhibit well-established zonation - specialized gene expression and functional roles - of vascular cells along the arterial-capillary-venous axis. In contrast, adult mouse brain vascular zonation differs significantly in gene expression, reflecting species-specific vascular physiology Crouch et al., Tr. Neurosci., Jul 2023. Owing to species-specific protective zonation genes, animal models of ALS may underrepresent the impact of BSCB dysfunction, neuroinflammation, and gliosis.
Species-specific differences in brain cells morphology, composition and function
Microglia and astrocyte cell types are directly involved in pathological neuroinflammation and gliosis in ALS.
Astrocytes play many important roles in the brain, including supply in nutrients, removal of waste, mechanical support to neurons, signaling to endothelial cells, regulation of neurogenesis, and repair of injury.
In human neocortex astrocytes are 2.6-fold larger in diameter and extend 10-fold more glial fibrillary acidic protein-positive primary processes than in rodents. Several anatomically defined subclasses of astrocytes are not represented in rodents Oberheim et al., J. of Neurosci., Mar 2009.
Inter-species differences in morphology also extend to non-human primates, since in humans, the subclass of interlaminar astrocytes is more abundant in comparison to chimpanzee. Human-specific astrocytic complexity is likely to have contributed to both the increased functional competence of the human brain and increased vulnerability to neurodegenerative disorders such as ALS.
Microglia, the resident immune cells of the CNS, are particularly under scrutiny for their role in human neurodegenerative Masuda et al., Nature, Feb 2019 and psychiatric disorders Rahimian et al., Neurosci. & Biobeh. Rev., Dec 2021.
Comparison of single-cell transcriptomics across ten species, revealed a larger heterogeneity in human microglia transcripts Geirsdottir et al., Cell, Dec 2019. Species-specific gene expression pathways in microglia are associated with complement system, phagocytosis, and metabolic pathways, all of which play a crucial role in ALS progression.
Species-specific differences in neuromuscular junction molecular and cellular architecture
The human neuromuscular junction (NMJ) is composed of presynaptic motor neurons, postsynaptic muscle fibers, and glial cells, that engage in crosstalk to facilitate signal transmission from motor neurons to muscle fibers.
Super-resolution imaging, and proteomic profiling showed that human NMJ were significantly smaller, less complex, more fragmented, and more stable across the entire adult lifespan than mouse NMJ Boehm et al., J. Anat., Jun 2020.
In comparison to other mammal species, human NMJ possess a distinctive distribution of active zone proteins and differential expression of core synaptic proteins and molecular pathways Jones et al., Cell Rep., Nov 2017.
This likely contributes to susceptibility to motoneurons degeneration in humans and suggests that mechanisms that underly motoneuron dysfunction in animal models of ALS may be different in ALS patients.
Species-specific differences in neurotransmission physiology
The neurotransmission physiology varies across species, likely due to both baseline inter-species differences and ALS-state inter-species differences in glutamate release, clearance, and uptake.
Since research on nervous system physiology is typically reliant on studies in animal model organisms and since findings in animal studies are not systematically compared to findings in human studies, the exact extent of inter-species differences in neurotransmitter physiology remains unknown, and therefore cannot be accounted for.
Therapies that focus on modulating neurotransmission physiology have massively failed to translate into improved patient care, showing that reliance on animal research has led to a lack of understanding of physiology of the human brain Egunlusi & Joubert, Pharmaceuticals, May 2024.
The following highlights some of the known human-specific features of post-synaptic glutamate dynamics in normal and ALS state, as well as plausible inter-species differences that are likely to have led to failure of EAAAT2 modulators, AMPA receptor antagonists, and NMDA receptor antagonists, in clinical trials.
In humans, glutamate is the primary excitatory neurotransmitter in the CNS, playing a crucial role in synaptic connections between UMN and LMN.
According to the "dying forward" hypothesis in ALS, hyperexcitable UMN drive excitotoxic damage to LMN Odierna et al., Brain, May 2024.
Due to previously-mentioned human-specific direct CM connections and greater connectivity, the function spread of glutamate released from presynaptic neurons is amplified and the baseline excitability in human motoneurons is higher compared to model organisms Arnold et al., Int. J. Mol. Sci., May 2024.
In ALS state, a variety of possible mechanisms (C9ORF72 mutations, SOD1 mutations, reduced GABAergic inhibition) are suspected to drive an increased glutamate release into synaptic cleft. Excessive glutamate release in combination with human-specific corticomotoneuronal features, makes humans more susceptible to downstream excitotoxicity than excessive glutamate release alone in SOD1 G93A mice.
In normal state in both humans and mice, the EAAT2 glutamate transporter is highly expressed in astrocytes, clearing 90% of synaptic glutamate.
In human ALS, EAAT2 expression is significantly reduced, through a variety of possible mechanisms (misfolded proteins, oxidative stress, neuroinflammation) Lin et al., Neuron, Mar 1998.
While ceftriaxone administered to SOD1-G93A mice slowed the disease course, preserved strength, and prolonged survival in mice Rothstein & Kuncl, J. Neurochem., Aug 1995, it did not significantly increase the survival time or significantly decrease the rate of decline in function in ALS patients Cudkowicz et al., Neurol., Feb 2013.
Based on this discrepancy in outcome between preclinical and clinical trials for Ceftriaxone, the reduction in EEAT2 expression could possibly be milder in ALS animal models than in ALS patients, which combined with other inter-species-differences, could result in less severe excitotoxicity, and subsequent overestimation of the effect of Ceftriaxone.
Postsynaptic AMPA receptors are tetramers that assemble in various combinations of subunits. In the absence of their GluR2 subunit, AMPA receptors are permeable to Ca2+, alongside Na+/K+ ions. In normal state, a low-level Ca2+ influx via AMPA receptors supports mitochondrial function and sufficient ATP production, however in disease state the excessive influx of Ca2+ leads to mitochondrial dysfunction, ROS production, ER stress, and apoptosis.
In ALS state, upregulation of GluR2-lacking AMPA receptors and downregulation of GluR2 editing, enhances Ca2+ influx and increases the risk of downstream excitotoxicity Wright & Vissel, Front. Mol. Neurosci., Apr 2012.
The AMPA receptor antagonist Talampanel has failed to reduce ALS-related functional deterioration in clinical trials Caroll, Fiercebiotech, May 2010, despite improvement of motor function in SOD1-G93A mice Tortarolo et al., J. Neurosci. Res., Dec 2005.
This mismatch in therapeutic outcomes could possibly be explained by a higher baseline level of GluR2-containing AMPA receptors in mice, and/or fewer Ca2+-permeable AMPA receptors in ALS mouse models, which combined with other inter-species-differences, may reduce Ca2+ influx and thus result in a less severe and more therapeutically manageable excitotoxicity in ALS mice than in ALS patients.
Postsynaptic NMDA receptors require both glutamate and depolarization to activate. GluN2B-rich NMDA receptors, that have higher Ca2+ permeability, add to the risk of excitotoxicity in humans Neagoe et al., Stem Cell Res., Apr 2018.
While treatment with NMDA receptor antagonist Memantine significantly delayed the disease progression and increased the life span of SOD1-G93A mice Wang & Zhang, Europ. J. Neurosci., Nov 2005, it failed to slow the progression of ALS in patients Bhai et al., Muscl. & Nerve, Nov 2024.
This disappointing outcome could possibly be explained by a higher baseline prevalence of GluN2B-rich NMDA receptors in humans compare to mice, which combined with other inter-species-differences, may produce a more severe and more rapidly progressive motoneuron degeneration in ALS patients than in ALS mouse models.
Species-specific differences in immune system
Immune-mediated motoneuron degeneration in ALS involves both innate and adaptive immune responses.
In ALS, innate immunity includes microglia activation to M1 pro-inflammatory state and complement system dysregulation. Adaptive immunity in ALS includes CD4/CD8 T cell Infiltration and production of autoantibodies.
Both types of immune responses differ across species, affecting how ALS progresses and leading to discrepancies in drug efficacy between ALS animal models and ALS patients.
Extensive differences between humans and mice were demonstrated in the structure of innate and adaptive immunity, including in balance of leukocyte subsets, defensins, Toll receptors, inducible NO synthase, the NK inhibitory receptor families Ly49 and KIR, FcR, Ig subsets, the B cell (BLNK, Btk, and λ5) and T cell (ZAP70 and common γ-chain) signaling pathway components, Thy-1, γδ T cells, cytokines and cytokine receptors, Th1/Th2 differentiation, costimulatory molecule expression and function, Ag-presenting function of endothelial cells, and chemokine and chemokine receptor expression Mestas & Hughes, J Immunol, Mar 2004.
In addition, it was shown that transcriptional responses to acute inflammatory stresses of different etiologies in mouse models do not correlate with human acute inflammatory diseases Seok et al., PNAS, Jan 2013
Major species-specific differences were also found in transcriptional regulation, chromatin state and higher order chromatin organization. Notably, cis-regulatory sequences next to immune-system-related genes have shown the most divergence, as they have apparently undergone more rapid evolution Yue et al., Nature, Nov 2014.
It was suggested that using animal models with a humanized immune system might improve translatability to humans, however, such an approach would face persistent, insurmountable challenges: the role of the human immune system in ALS is complex and not fully understood, the equivalence of humanized animals to the human immune system was never demonstrated by objective measures Davis, Cell, Dec 2008, and the cross-talk between the human immune system and the rest of human organ systems cannot be recapitulated in animals.
Species-specific differences in functional assessment
The ALS Functional Rating Scale-Revised (ALSFRS-R) is a validated rating instrument designed to assess the functional status and monitor the progression of disability in patients with ALS. It typically assesses human-relevant functions such as speech, salivation, cutting food, handwriting, hygiene, turning in bed and respiratory function.
However, there is no equivalent of ALSFRS-R for animal models.
Instead, researchers often use various behavioural and functional tests such as rotarod test, grip strength test, hindlimb extension reflex, and gait analysis. Such tests in animal models may not translate to function in ALS patients and can be difficult to analyse due to species-specific behaviours.
For instance, the rotarod test is one of the most commonly used tests to measure motor coordination in mice. Studies of inbred strains, selected lines, and transgenic animals have shown that rotarod performance in mice is highly influenced by the genetic background. In addition, there is little consensus on ideal parameters and test schedules to produce optimal results, despite wide use of rotarod tests in biomedical research.
The variability in size of rod diameters, rotation rate, training regimens, experimenter effects etc. contribute to poor reproducibility and reliability of the rotarod test Rustay et al., PNAS, Feb 2003.
There are no standard methods in animal neurobehavioral genetics, that would have allowed to understand how genetic variations influence behaviour, emotion, cognition and motor function in animals. Instead, most tests are done in a manner that is unique to each laboratory, making it difficult to compare findings and arrive to conclusions about the effects of ALS treatments in animals Wahlsten, Physiol. & Beh., Aug 2001.
Even if such standardized methods did exist, they would still fall short of translating to human-specific behaviour, emotion, cognition and motor function.
Face validity - How well does an animal model replicate the human disease phenotype?
Owing to human-specific genomic and physiological features, animal models of amyotrophic lateral sclerosis (ALS) have severe drawbacks, including absence of several key pathophysiological and clinical features of ALS, and presence of features that are not found in ALS patients.
Identification in patient populations of genes associated with ALS had prompted development of rodent, zebrafish, pig, non-human primate and other animal species models that carry mutations found in ALS patients Zhu et al., Transl. Neurodeg., Sep 2023, Morrice et al., Neural. Reg. Res., Dec 2018, Philips & Rothstein, Curr. Protocol. Pharmacol., Jun 2015, Zhou et al., Front. Neurosci., Feb 2024.
The most frequent animal models of ALS are transgenic animals that carry genes associated with the familial form of ALS (FALS), with an exception of a few genes that are also associated with sporadic ALS (SALS).
Over a dozen SOD1 transgenic rodent models have been developed by overexpressing missense, mutated, or truncated human ALS-associated gene variants, such as G93A, D83G, D85G, D86G, D90A, and G37R.
The most widely used mouse model of ALS, the SOD1 G93A mouse, carries a point mutation in the SOD1 gene at position 93 that leads to toxic gain-of-function. The SOD1 G93A mouse reproduces neuroinflammation, motor neuron degeneration in the spinal cord, muscle weakness, and limb paralysis.
A major limitation of the overwhelming majority of transgenic mouse models, including the SOD1 G93A mouse, is the absence of motor neuron degeneration in the cerebral cortex characteristic of ALS patients Philips & Rothstein, Curr. Protocol. Pharmacol., Jun 2015, Filipi et al., Sci. Rep., Apr 2023. This is particularly problematic, since cortical motor neuron degeneration is the fundamental feature of ALS and the basis for ALS diagnosis.
Although the SOD1 D83G transgenic mouse shows some motor neuron degeneration in the cerebral cortex, it does not recapitulate the clinical paralytic phenotype Zhu et al., Transl. Neurodeg., Sep 2023.
Another consequence of limited cortical involvement in SOD1 mice is that cognitive impairments, observed in some SOD1-related human ALS cases, are not recapitulated in these mouse models.
While in ALS patients microglia, astrocytes, and oligodendrocytes play a key role in neuroinflammation and gliosis, comprehensive study of glia in the cortex of the SOD1 G93A mouse, using single-cell RNA sequencing at four stages of the disease, shows minimal changes throughout the disease progression Filipi et al., Sci. Rep., Apr 2023, indicating that the complexities of human neuroinflammation and gliosis are not recapitulated in SOD1 G93A mice.
In SOD1 G93A mice, gait abnormalities, hindlimb tremors, and hindlimb weakness mimic human limb-onset, but the progression is much faster than in humans, which means that certain intermediary features and pathological mechanisms of human ALS may not be recapitulated in mice.
Although numerous features of BSCB disruption (endothelial cell degeneration, capillary leakage, perivascular edema, downregulation of tight junction proteins, and microhemorrhages) were represented, several other key features such pericyte degeneration, perivascular collagen IV expansion, and white matter capillary abnormalities were absent in SOD1 animal models of ALS Garbuzova-Davis & Sanberg, Front. Cell. Neurosci., Feb 2014.
This means that vascular remodeling, cortical and subcortical vascular dysfunction, and fibrotic processes cannot be studied in these animal models.
Based on known genetic variants of TDP-43 in patients with FALS, over 20 corresponding TDP-43 mouse models have been generated Zhu et al., Transl. Neurodeg., Sep 2023.
TDP-43 mice have shown inconsistent phenotypes depending on the genetic induction methods. In many cases it is not clear whether the genetically-induced phenotype in mice is related to the mutation itself or to overexpression of the mutated gene.
For instance, transgenic mouse models that overexpress exogenous human TDP-43 around twice the level of endogenous tdp-43, using Thy1.2 promoters that drive gene expression specifically in neuronal tissues, displayed accumulation of pathological aggregates of ubiquitinated proteins in specific neuronal populations, gliosis, varying degrees of spinal cord pathology, and progressive paralysis and death.
In contrast, the TDP-43 Q331K transgenic mouse, that overexpresses human mutant Q331K only up to 1.5 fold of endogenous TDP-43 mouse levels, displayed a limited period of progressive motor dysfunction and its ALS-like features did not result in death Arnold et al., PNAS, Feb 2013, Morrice et al., Neural. Reg. Res., Dec 2018.
Importantly, most TDP-43 mutant mice did not reproduce the critical cytoplasmic mis-localization of TDP-43 protein, raising concerns about the validity of these mouse models in studying ALS pathogenesis Philips & Rothstein, Curr. Protocol. Pharmacol., Jun 2015, Zhu et al., Transl. Neurodeg., Sep 2023.
Another Thy1.2-TDP-43 line, with 2 to 5 fold overexpression of endogenous tdp-43, developed early onset tremor and abnormal hindlimb reflexes, with TDP-43 negative ubiquitinated cytoplasmic inclusions. Although a moderate loss of large caliber motor axons and abnormal neuromuscular junction morphology was reported, there was no loss of motor neurons in cortex or spinal cord nor subsequent death in this mouse model of ALS.
The A315T TDP-43 overexpressing mouse model under mouse prion promoter control did not recapitulate neuromuscular deficits but instead showed clinical features that were not observed in ALS patients, such as gastrointestinal dysfunction. This outcome is possibly due to site-specific overexpression of human TDP-43, underscoring the impact of experimental induction methods and genetic backgrounds of mouse models on disease phenotype Hatzipetros et al., Brain Res., Oct 2014.
The effect of experimental induction method on susceptibility to ALS phenotype was also found to vary across rodent species.
For instance, in contrast to the TDP-43 M337V mouse model, rats overexpressing comparable levels of TDP-43 M337V showed a more severe immobility, paralysis, and early death phenotype than those overexpressing wild-type TDP-43.
While the TDP-43 M337V mouse exhibited splicing deregulation in the spinal cord, it did not develop detectable neurodegeneration or motor dysfunction Watanabe et al., Mol. Brain, Jan 2020.
ALS patients who experience cognitive impairment also exhibit motor degeneration. However, the motor degeneration, which is the defining feature of human ALS, is not recapitulated in TDP-43 Q331Klow and G348C mutant mice.
Instead, the TDP-43 G348C mice developed learning/memory deficits but without overt motor neuron degeneration and without paralysis Swarup et al., Brain, Sep 2011.
In C9ORF72-associated ALS, patients typically carry 30 to 1,600 repeats of the G4C2 hexanucleotide motif. In contrast to ALS patients, transgenic mice that carry approx. 500 and 1000 repeats of the G4C2 motif in the C9ORF72 gene did not show neurodegeneration, raising questions about the usefulness of this mouse model for studying human ALS Zhu et al., Transl. Neurodeg., Sep 2023.
The use of zebrafish as a model for ALS has a number of limitations, not least of which is the absence of upper motor neurons. The bisphenol A-induced motoneuron degeneration in zebrafish does not cause motoneuron-specific degeneration since other types of neurons also undergo cell death Morrice et al., Neural. Reg. Res., Dec 2018.
Large animal models, such as transgenic SOD1G93A and M337V TDP-43 pigs and TDP-43-overexpressing cynomolgus monkeys, are less frequently used as experimental models of ALS because of inefficiency of gene targeting in large animals, long reproductive cycles, complex maintenance, and high associated cost Zhu et al., Transl. Neurodeg., Sep 2023.
Construct validity - How well do the mechanisms of disease induction in animals reflect the currently understood etiology of the human disease?
Incomplete recapitulation of amyotrophic lateral sclerosis (ALS) symptoms in ALS animal models, and repeated clinical failures of drug candidates for ALS, challenge our understanding of disease mechanisms.
Familial ALS (FALS) is a multifactorial disease, where known ALS-associated genetic mutations act as causative factors alongside other disease-related genes and modifying factors that are absent in non-human species. Developing such a transgenic animal model would require multiple ALS-associated transgenes to be expressed on a patient-specific genetic background, which remains unachievable.
The complexity of sporadic ALS (SALS), driven by a combination of polygenic, epigenetic, environmental, and lifestyle factors, makes achieving strong construct validity in animal models virtually impossible.
In transgenic animal models of ALS, mutant genes usually need to be highly overexpressed in order for the animal to develop an ALS-like phenotype within its lifespan, which raises doubts as to relevance for FALS and SALS patients of disease-triggering mechanisms employed in animals.
The inadequacy of artificially induced animal models limits treatment options for ALS patients.
For instance, the SOD1G93A mouse, considered as the cornerstone of ALS animal research, typically carries between 8 and 25 copies of the human SOD1 transgene, while humans carry only one copy of the mutant gene.
Mice carrying such a disproportionally high number of human SOD1 transgenes may suffer from a phenotype that is so overdriven that no therapy outside of the direct inhibition of SOD1 will ever affect ALS-related survival.
Treatments by antibiotics minocycline and ceftriaxone, validated in SOD1G93A mice, did not provide a survival benefit for ALS patients, suggesting that the SOD1G93A mouse may be more susceptible to infections and other non-ALS related illnesses, and that it is this illness, rather than ALS that is alleviated by tested treatments Wobst et al., Med. Res. Rev., Feb 2020, Worp et al., Plos Medicine, Mar 2010.
In addition to transgene overexpression, other confounding factors that can directly impact the disease phenotype in mouse models include the propensity of transgenic mice carrying mutant human SOD1 to spontaneously delete the copy number, mouse-specific gene expression regulation, promoter-dependent effect, and dynamic interactions between numerous mouse-specific features from molecular to organism level.
Predictive validity - How well do animal models predict safety and efficiency of therapies in human patients?
In the same manner that it is easier to repair something when you know exactly how you have broken it, many therapy candidates for amyotrophic lateral sclerosis (ALS) have successfully reversed disease symptoms in engineered ALS animal models, only to fail in ALS patients.
Since the '90s, over 70 molecules investigated as a possible treatment for ALS have failed to demonstrate effectiveness in clinical trials Petrov et al., Front. Aging Neurosci., Mar 2017, Philips & Rothstein, Curr. Protocol. Pharmacol., Jun 2015.
Existing treatment options for ALS can slow down the progression of the disease but unfortunately do not cure it.
Riluzole, Edaravone, and sodium phenylbutyrate provide mild therapeutic and limited survival benefits.
Sanofi's glutamate inhibitor riluzol, approved by the FDA in 1995, reduces hyperexcitability by blocking glutamatergic neurotransmission.
Mitsubishi Tanabe Pharma’s edaravone, marketed as Radicava, was first developed in the 80's to treat stroke, and is believed to reduce oxidative stress and slow down motoneuron degeneration Morrice et al., Neural. Reg. Res., Dec 2018.
The histone deacetylase inhibitor, sodium phenylbutyrate, was approved by the FDA in 1996 to treat urea cycle disorders. In 2022, the FDA has approved the combination of sodium phenylbutyrate and taurursodiol, known under the name Relyvrio, for treatment of ALS. Relyvrio appeared to slow down neurodegeneration by reducing mitochondrial dysfunction and ER stress Brotman et al., StatPearls, Feb 2024. However, Relyvrio was pulled off the market in 2024, after failing to meet both primary and secondary endpoints in Phase 3 PHOENIX trial Amylyx Pharmaceuticals, Relyvrio, Apr 2024.
In 2023, the FDA granted accelerated approval for Biogen’s and Ionis’ antisense oligonucleotide (ASO) Qalsody (tofersen) that targets SOD1 mRNA FDA News Release, tofersen, Apr 2023 FDA News releas, Tofersen, Apr 2023, EMA authorisation, Tofersen, Jun 2024. Accelerated approval was based on the surrogate biomarker plasma neurofilament light chain (NfL), estimated to be reasonably predictive of a clinical benefit. Nevertheless, since NfL measures neuronal degeneration and not functional improvement, the long-term efficacy of tofersen remains to demonstrated in further patient studies.
Biogen’s and Ionis’ ASO BIIB105, designed to reduce expression of the ATXN2 gene, did not demonstrate an improvement in motor function and respiratory function, and was therefore discontinued in 2024 Biogen News Release, BIIB105, May 2024. Two years earlier, it was Biogen’s and Ionis’ ASO BIIB078, targeting the C9orf72 mRNA, that was discontinued for the same reason.
Earlier in 2024, Sanofi's receptor-interacting protein kinase 1 (RIPK1) inhibitor SAR443820 for ALS was discontinued after failing to show significant improvement in ALSFRS-R scores compared to placebo Denali Report, SAR443820/DNL788, Feb 2024.
Ethical validity - How well do animal experiments align with human ethical principles?
Preclinical
Ethics is a human-specific philosophical concept. Animal experimentation is unethical in essence by human standards, since it involves physical constraint, psychological suffering and deprivation of freedom, social interactions, natural environment and life purpose.
In addition to this baseline, animal experiments inflict severe clinical harm in animals De Vleeschauwer et al., Animals, Aug 2023:
Table S13: Severity classification of genetically altered (GA) lines
Neurology and sensory organs - Paresis and paralysis, GA lines resulting in severe impairment of animal’s motility and weight loss due to inability to reach food/water: up to severe clinical signs
Loss of coordination and gait - Marked abnormal gait impairing ability to move and function normally causing difficulties in eating and drinking: up to severe clinical signs
Behavior/emotional state - GA lines resulting in long-term moderate anxiety or short term severe anxiety: up to severe clinical signs
Cardiomyopathy - Up to severe clinical signs: Global heart failure with permanent respiratory distress and impairment of the general condition
Mortality, rodents older than 2 weeks - GA lines resulting in lethality (significantly earlier than WT strain) from 2 weeks post-partum on: severe
Table S4: Severity classification of clinical signs
Mobility - Abnormal gait, lameness: up to severe clinical signs
Respiration - Reduced rate at rest and when active. Irregular rhythm. Appears to require effort.: up to severe clinical signs
Clinical
While there is no consensus on whether an unethical act can be justified by a pursuit of a hypothetically ethical outcome, it was suggested that animal research was necessary to advance efficient and safe treatments for human diseases.
However, statistics consistently show that efficacy and safety of drugs developed and tested in animals for amyotrophic lateral sclerosis (ALS) are very poor, raising the question of whether it is ethical to put ALS patients’ health and life at risk.
For instance, although minocycline and topiramate had shown promising results in ALS mouse models, these drugs have led to degradation of motor function and severe adverse effects in ALS patients Gordon et al., Lancet, Dec 2007, Cudkowicz et al., Neur., Aug 2003.
Intrinsic validity - How well do animal models capture the clinical heterogeneity of the human disease?
The heterogeneity in genotype, symptoms, onset and progression patterns of amyotrophic lateral sclerosis (ALS) is not recapitulated in animal models of ALS.
Extrinsic validity - How well does animal experimentation generate reliable and reproducible outcomes?
It is often argued that although animal models have severe limitations, animal research enables to gather insights that may be valuable. However, the basic precondition for a hypothetical benefit is not met since the majority of animal experiments is irreproducible Freedman et al., PLOS Biol., Jun 2015.
Contributing factors include flawed experimental design, variation in animal strains and experimental conditions, and lack of transparency on methodology and results of animal studies.
In a bid to improve the quality of reporting of animal experiments, the ARRIVE - Animal Research: Reporting of In Vivo Experiments - guidelines, were published in 2010 and updated to ARRIVE 2.0 in 2020 Arrive guidelines website.
Nonetheless, and in spite of significant investment in dissemination, various incentives and training of animal researchers, the Arrive guidelines remain poorly implemented Percie du Sert et al., BMC Vet. Res., Jul 2020, Bazoit, BioRxiv, Feb 2025.
As a result of weak relevance, rigor and reliability of animal studies, erroneous and misleading hypothesis are generated, animal and human lives are needlessly sacrificed and dozens of billions of dollars are annually wasted Yarborough et al., PLOS Biol., Jun 2018.
Beyond the problem of experimental design and reporting of animal studies, there are deep-seeded cultural reasons that are not likely to be addressed any time soon, such as the "publish or perish" culture that discourages from unbiased analysis and reporting of negative results and rewards exaggerated and overhyped claims Smaldino & McElreath, Roy. Soc. Op. Sci., Sep 2016.
In Summary
Amyotrophic lateral sclerosis (ALS) is a progressive and lethal neurodegenerative disorder, leading to muscle weakness and respiratory failure.
As a result of a complex interplay of genetic, epigenetic, environmental and lifestyle factors, the clinical presentation of ALS is heterogeneous.
Owing to species-specific differences in corticomotoneuronal system anatomy and physiology, in gene expression, in neuromuscular vascular physiology, in brain cells morphology, composition and function, in neuromuscular junction molecular and cellular architecture, in neurotransmission physiology, and in immune system, animal models do not recapitulate some of the fundamental pathophysiological and clinical features of ALS.
The most commonly used animal models of ALS are genetically-engineered mice, which do not take account of human causative and modifier factors.
Modeling ALS in vivo causes severe suffering in animals, but also in ALS patients, since animal research has not translated to significant therapeutic benefits.
Human-based methods are needed to better understand the complex and heterogenous mechanisms of ALS, to identify new therapeutic targets, to developer safer and more effective treatments, and to bring to market personalized solutions for ALS patients.
How is Human-Based In Vitro the Answer to advance biomedical research into Amyotrophic Lateral Sclerosis
*To model heterogenous FALS and SALS phenotypes, using ALS patient-derived iPSC/primary 3D motoneuron tissues/motoneuron organoids/motoneuron-on-chip Pereira et al., Nature Comm., Aug 2021, Fujimori et al., Nature Med., Aug 2018.
*To determine the impact of rare genetic variants on ALS pathogenesis, by gene editing (base editing, overexpression, knock-out, etc.) and gene perturbation (CRISP interference, siRNA) in iPSC/primary 3D motoneuron tissues/organoids/motoneuron-on-chip Kim et al., Front Cell Neurosci., Nov 2020, Deneault et al., Methods, Jul 2022.
*To identify epigenetic drivers of motor neuron degeneration and to test potential epigenetic therapies for ALS, using epigenetic editing (CRISPR activation/repression, lncRNA) in healthy human/ALS patient-derived motoneuron tissues/organoids/motoneuron-on-chip.
*To study the genotype-phenotype relationship in ALS patient-derived iPSC 3D tissues/organoids/neuromuscular junction-on-chip with patient-specific genetic background Dittlau et al., Stem Cell Rep., Apr 2021.
*To identify mechanisms of SALS - By studying the role of microglia-to-astrocyte signaling in neurofilament accumulation, using cytokine array assay, immunocytochemistry, transcriptomics, and gene ontology analysis, in iPSC from a set of identical twins discordant for ALS Allison et al., Genes, Jan 2022.
-By studying mechanisms and effects of cell-to-cell propagation of pathogenic TDP-43 protein, using immunocytochemistry, multi-omics, apoptosis assay, and DNA damage assay, in SALS patient-derived iPSC/cerebral organoids/motoneuron-on-chip Tamaki et al., Plos Gen., Feb 2023.
-By studying mechanisms and effects of pathogenic alterations in autophagy pathways and cytokine secretion, using SALS patient-derived iPSC/cerebral organoids/motoneuron-on-chip Feng et al., Oxid. Med. Cell. Long., Aug 2022.
*To explore the cell-autonomous and non-cell autonomous mechanisms of FALS/SALS C9orf72 mutation, by coculturing mutant astrocytes with wild-type motoneurons and measuring neuronal firing patterns, ion channel activity, neuronal excitability, in ALS patient iPSC-derived astrocytes and motoneurons Zhao et al., Glia, Dec 2019.
*To study the effect of environmental and lifestyle contributors to ALS (heavy metals, pesticides, smoke) and to model inter-individual differences in ALS pathophysiology, by exposing the culture medium of healthy human 3D tissues, genetically edited for GWAS-identified variants, to nutrients and to toxicants, and by measuring resulting inflammation (cytokine assays), oxidative stress (DCFH-DA staining), gene expression change (RNA-seq), mitochondrial membrane potential (TMRM staining), and other endpoints.
*To study human-specific corticospinal connections and ALS-related degeneration by using calcium imaging, optogenetics, and glutamate caging, in healthy human/ALS patient-derived brain-spinal assembloids/(multi)organs-on-chip (cortical organoids fused with spinal motor neuron organoids).
*To investigate excitotoxicity, by combining glutamate caging (glutamate overload) with real-time calcium-sensitive fluorescent staining (excessive Ca influx), cell viability assays (neurodegeneration), in healthy human/ALS patient-derived NMJ organoids/brain-spinal assembloids/(multi)organs-on-chip.
*To study ALS-related alterations in human-specific alternative splicing by TDP-43, and to develop drugs that modulate the TDP-43 pathology, by using ALS patients-derived iPSC/postmortem tissues, that retain human RNA-binding motifs, human TDP43 protein sequence and human cellular machinery Miwa et al., Neurol. Clin. Neurosci., Jan 2025.
*To determine the contribution of each cell type to ALS, by using chimeric bioprinted tissues containing one or more diseased cell types in the context of a healthy background of other cell types - astrocytes, microglia, oligodendrocytes, endothelial cells, motor neurons -, in healthy/ALS patient-derived 3D tissues.
*To study the human-specific perturbations of the brain vasculature (BBB, BSCB, BCSFB), by measuring multi-omics, electrophysiology function, Ca imaging, barrier permeability, cytokines and other endpoints, in healthy/ALS patient-derived iPSC co-culture vascular-motoneurons assembloids/(multi)organs-on-chip. To study dysregulation of neurovascular interactions in ALS, by comparing the transcriptomes of vascular cells (endothelial cells, pericytes, smooth muscle cells) to transcriptomes of astrocytes, oligodendrocytes, immune, muscle, and neuron cells, in healthy and in ALS state.
*To identify new biological markers of ALS for an improved accuracy of diagnosis, monitoring of disease progression, and response to treatment, using patient-derived 3D tissues/organoids/organs-on-chip. To link these biomarkers to patients’ distinct phenotypes.
*To conduct reverse translational research by comparing safety and efficacy of candidate drugs for ALS both in clinical trials and in vitro Morimoto et al., Cell St. Cell, Jun 2023.
*To identify broadly effective therapeutic targets to treat multiple forms of ALS, using FALS and SALS patient-derived 3D tissues/organoids/organs-on-chip Okano et al., Trends Pharmacol. Sci., Jan 2020, Dafinca et al., Stem Cell Rep., Apr 2020.
*To test safety and efficacy of single and combination therapies, by high-throughput screening in ALS patient-derived organoids/organs-on-chip Osaki et al., Science Adv., Oct 2018, Iuliano et al., Adv. Mat. Tech., Oct 2023, Setsu et al., Stem Cell Rep., Dec 2024.
Although in vitro methods have inherent limitations, their relevance to human biology far exceeds that of animal research.
Animal model organisms were never comprehensively compared to humans and scientifically validated. Complementing in vitro methods with animal experiments is not effective for human patients, because species-specific differences prevent reliable integration and translation of results to humans.
While animal research benefits from experimenting on a complete organism, model organisms fail to replicate the interplay of thousands of human-specific features, from molecular level to organism level, and are therefore not representative of the complete human organism.
To address the challenges of individual human-based in vitro models, they can be integrated with other human-based in vitro methods, AI-driven analysis, clinical data, and real-world patient data.
Have you leveraged in vitro methods in unique ways? We would love to hear how! Join the conversation to exchange ideas, collaborate and inspire new directions in human-based science!