Amyotrophic Lateral Sclerosis (ALS) is a lethal neurodegenerative disease of the central nervous system characterized by the progressive and relentless loss of both upper and lower (cortical and spinal) motor neurons. First described in 1860 by Jean-Martin Charcot, its currently clinical presentation is well defined by a plethora of signs and symptoms. Typically, it starts with focal muscle weakness and atrophy in the distal limbs, which later progresses until it involves bulbar muscles, ultimately leading to paralysis of respiratory muscles and resulting in patient’s death within approximately 3–5 years from the onset of the disease.

Standardized global ALS incidence by meta-analysis is 1.68 per 100,000 person-years, but varies by region [1] and the average age of onset is 64 years. The most common cases (about 90%) are sporadic forms sporadic amyotrophic lateral sclerosis (sALS), which occur spontaneously without a well-identified cause, while a small portion (approximately 10%) are familial forms (fALS) [2], defined for patients with a positive family history, for which currently over 40 genes have been identified in the pathogenesis of the disease. The most studied and well-defined disease-causing genetic variant in experimental models is the point mutation of the SOD1 gene that encodes for the enzyme superoxide dismutase. Discovered in 1993 [3], it causes the accumulation of misfolded protein and increased oxidative stress in neurons, hyperexcitability and mitochondrial dysfunction leading to cell death [4]. Another widely described genetic variant, in cases of fALS, is the hexanucleotide GGGGCC expansion in the intron of chromosome nine open reading frame 72 (C9orf72) gene. Transcription of the aberrant gene leads to the formation of pathological RNA, which accumulates in the nucleus and hinders gene transcription [5].

This gene has also been found in cases of ALS associated with Frontotemporal Dementia (FTD): these two pathologies indeed share neuropathological and clinical features. In fact, approximately 40% of FTD patients and 97% of ALS patients exhibit an accumulation of the TDP43 protein. Furthermore, a clinical overlap has been highlighted, with about 5% of diagnosed ALS patients showing behavioral alterations and executive dysfunctions similar to the behavioral variant of FTD. These findings suggest possible common therapeutic targets between the two neurodegenerative conditions [6, 7].

Other mutations associated with ALS include TARDP and FUS. However, the etiology of ALS remains unknown in the majority of cases, and its pathogenesis is multifactorial [7, 8, 9].

Currently, ALS remains incurable. Treatment is focused on using therapies that might slow disease progression. Moreover, coordinated multidisciplinary treatment represents the standard of care, as it guarantees an increase in survival and improvement in quality of life [9, 10, 11].

Some countries have approved a few medications to help the slowing down of disease progression, which include Riluzole and Edaravone [9].

Riluzole is an anti-glutamatergic agent and improves survival by few months [12].

Edaravone, a free radical scavenger and antioxidant, decreases the effects of oxidative stress in ALS and shows some efficacy in post hoc analysis of the first phase 3 trial [13]. The trial design, however, does not permit the result of the study to be generalized to all ALS patient population. Questions remain about the drug’s effectiveness, which have been raised after post post-marketing observations [14, 15].

Tofersen, a novel antisense oligonucleotide (ASO) drug, has recently been approved by the FDA, in April 2023. This drug was approved for the treatment of ALS patients with SOD1 mutation. In a double-blind, placebo-controlled clinical trial, treatment with Tofersen has shown reduced levels of SOD1 proteins in the cerebrospinal fluid by 36%, in patients with this type of ALS. Earlier treatment with Tofersen appeared to provide better measurement outcomes [16].

The combination of sodium phenylbutyrate and taurursodiol (Relyvrio) has been shown to be effective in reducing neuronal cell death and oxidative injury by reducing mitochondrial dysfunction and oxidative stress in the endoplasmic reticulum. The efficacy of Relyvrio was demonstrated in a randomized, placebo-controlled, multicenter, double-blind, parallel-group study [17]. The study reported that the patients treated with Relyvrio experienced a slower rate of decline in the assessment of daily activities when compared with those treated with placebo.

However, Relyvrio and Tofersen must be further studied, because data are lacking to validate their effectiveness [18].

The past failures of various therapeutic strategies to modify the pathology natural history and the ongoing strong urgency to find new ones, are due to the lacking of precise disease models to conduct research on.

Over the years, numerous in vitro and in vivo models have been proposed, with the aim of understanding the mechanism of motor neuron degeneration in patients with ALS. Despite significant progress, especially in demonstrating the key role of non-neuronal cells, such as astrocytes and glial cells, in inducing a proinflammatory environment that triggers and accelerates motor neuron death, the pathophysiological process of the disease has not been fully elucidated yet [19].

The difficulty in well understanding the disease arises from its etiological heterogeneity, which is also shared with other neurodegenerative diseases. Among the causes, both genetic and environmental factors are recognized, demonstrating a multifactorial process. Additionally, the complex interactions between neurons and other cells types within the microenvironment, makes it challenging to establish reliable in vitro models.

Traditionally, researches, in the context of neurological diseases, have centered around the utilization of animal models, predominantly rodents. Animal models have been a crucial tool in understanding the pathogenesis of ALS too, discovering the cellular and molecular interactions present within the neuromuscular tissue. However, there are lots of constraints associated with employing animal models for studying the disease, which are, for instance, different epigenomic background between humans and other species, different cellular subtypes comprising the brain, and different neuronal networks within the brain. The considerable distinction in brain size is noteworthy, given that human neurons must extend longer axons, transport vesicles and protein machinery across greater distances, and, on average, establish a significantly higher number of synapses per neuron compared to mouse neurons. Moreover, these animal models do not completely replicate the pathology observed in human diseases and, consequently, frequently exhibit limited predictability of drug efficacy in clinical trials [20, 21, 22]. In ALS studies, the majority of murine models have relied on the induced overexpression of SOD1 mutated gene, focusing preclinical studies on a model that represents only a small slice of ALS cases and does not molecularly replicate TDP-43 aggregates, defined as a recurrent pathological marker in ALS. Furthermore, there has been observed a poor correspondence between therapeutic results obtained in the preclinical trials on animals and those achieved in humans, suggesting interspecies variability and different patterns of connections in the motor system that make these models less reliable and poorly translating from animals to humans [23].

In fact, despite hundreds of clinical trials based on the discoveries made on animal models, none have allowed for the detection of a significant therapy able to alter the course of ALS.

Another aspect to consider, which may explain why past efforts have been futile, is the challenge of obtaining human nerve cells. For centuries, the study of the cerebral nervous system has indeed been limited by the inability, except through highly invasive and risky procedures, to collect material for analysis.

Examining post-mortem tissue from ALS patients offers insight into the full range of pathological phenotypes, even if it only shows a static moment at the disease terminal stage. Not all motor neurons (MNs) respond equally to degeneration in ALS, and the surviving cells observed during autopsy may represent groups relatively resilient to the disease progression. Examination of post-mortem tissue samples from ALS patients and murine ALS models has identified the activation of astrocytes and microglia, that confirms that neuroinflammation plays a pivotal role in ALS [24].

More than ten years ago, with the advent of induced pluripotent stem cell technology from adult somatic cells, there has been a revolution in the field of Biology and Medicine. They could be an opportunity to circumvent and overcome these issues and provide the crucial answer to the need for more robust preclinical models in neurodegenerative diseases, including ALS. Thanks to their ability of multipotency, self-renewal and genetic memory, they seem to be the ideal candidate to address the lack of representative ALS disease models [25].

This review aims to providing a comprehensive overview of induced pluripotent stem cells use in ALS, starting from the development of differentiation protocols into various cellular subtypes, and then showing the potential clinical-therapeutic applications discussed and experimented in the literature.

This review focused on the molecular aspects and clinical implications of iPS in motor neuron diseases. An extensive literature search was conducted through a comprehensive query to identify relevant and original reports describing the clinical application of induced pluripotent stem cells in ALS. The major databases for medical literature were searched (MEDLINE, Web of Science, Cochrane Library, Scopus, EMBASE).

According with the research enquiry, the inclusion criteria were defined as follows: 5-year time range (2018 to 2023), only studies presenting clinically relevant applications of induced pluripotent stem cells were selected, including original research consisting of clinical trials, prospective cohort studies, case series, articles and reviews. Only full-length publications were considered for this research using the search string “induced pluripotent stem cells” AND “motor neuron disease” (64 results) or “stem cells” AND “ALS” (124 results) or “iPSC” AND “Amyotrophic Lateral Sclerosis” (83 results). Some articles were also derived from bibliography of the chosen articles. Papers that were not in English, Italian or French language, outside the scope of this review and duplicates, were excluded. A minimum of two of the authors reviewed each study for relevance. Data extraction and synthesis will be performed to provide a cohesive overview of the current state of knowledge in this field.

Over the years, induced pluripotent stem cells (iPSCs) have been recognized as a valuable resource for analyzing the pathogenetic mechanisms underlying neurodegenerative diseases. They have provided a deeper understanding of the molecular and cellular dysfunctions that manifest in diseased tissues. Moreover, iPSCs have become an invaluable tool in drug experimentation, not only for assessing efficacy and side effects, but also for discovering potential new therapeutic targets.

The applications of stem cells in the field of neurodegenerative diseases, specifically in motor neuron diseases, are manifold [26, 27, 28, 29]. They include delving into the pathogenesis by studying different disease phenotypes and genotypes at various stages of progression. Stem cells are crucial tools in identifying new therapeutic targets, testing novel treatments [30], stratifying disease forms based on therapeutic response rather than clinical parameters, and ultimately creating patient-tailored therapies in the realm of precision medicine, often referred to as “Disease in a Dish”.

iPSCs possess the unique ability to self-renew, making them an endless source of cellular material. Additionally, they can differentiate into various cell subtypes, depending on the chosen culture medium and growth conditions. In contrast to embryonic stem cells (ESCs) derived from 4–5-day blastocysts, iPSCs are obtained from adult somatic cells of the patient, which are reprogrammed and returned to a pluripotent status through the ectopic expression of embryonic transcription factors. This approach reduces ethical issues and significantly lowers the risk of immunological rejection when transplanted in vivo. Furthermore, these cells retain the genetic background of the donor, revolutionizing the study of neurodegenerative diseases, particularly neuromuscular disorders, by allowing the in vitro reconstruction of the genotypic and phenotypic variability of affected patients [31, 32].

However, challenges still persist in this technology. In comparison to ESCs, iPSCs may exhibit more frequent epigenetic and genetic aberrations due to the reprogramming process. The considerable variability among cell lines, maintaining the individual genetic heritage of a specific patient, makes it challenging to standardize culture techniques and makes various study models less comparable [33, 34].

To obtain pure cell lines independent of genetic variability, one possibility is to use CRISPR/Cas9 technology. It allows for editing the genome of stem cells and precisely modifying specific DNA sequences. The system uses guide RNA to direct the Cas9 enzyme to the target sequence in the DNA, where it induces cuts or repairs mutations. This enables the generation of isogenic cell lines, meaning they share the same modified gene sequences. This genetic manipulation allows for obtaining cell lines with similar and easily identifiable phenotypes, making studies more comparable within the same cell culture or on cell lines obtained from parallel differentiations [35].

Over a decade ago, Takahashi and his team achieved a revolutionary milestone as they successfully, using four embryonic transcription factors (OCT4, SOX2, KLF4, and c-MYC), converted mouse fibroblasts into induced pluripotent stem cells (iPSCs). Since then, reprogramming techniques and cellular differentiation models have evolved, and today it is possible to obtain pluripotent stem cells (PSCs) from three germ cell types, originating from each embryonic germ layer: fibroblasts (mesoderm-derived), hepatocytes (endoderm-derived), and keratinocytes (ectoderm-derived). While fibroblasts have traditionally been the preferred cellular source for obtaining stem cells, considering the invasive nature of skin biopsy, there is a growing inclination towards more easily accessible cells that do not require invasive procedures for collection. Given these considerations, keratinocytes, urine cells, and blood cells represent valid alternatives [36, 37].

Over the years, there has been a development in cellular reprogramming techniques. The initial studies on iPSCs used retroviral or lentiviral vectors, which, by integrating into the cell genome, manipulated gene expression. However, this procedure often led to genomic alterations with aberrant results. Therefore, new transfer systems have been introduced that do not require the integration of foreign gene sequences into the cell DNA. Through the use of non-integrating vectors (such as the Sendai virus or episomal vectors), self-eliminating vectors, and non-viral vectors (such as small bioactive molecules, microRNAs, conventional plasmids, recombinant proteins), it is now possible to reprogram cells without irreversibly modifying their genetic heritage [37, 38, 39, 40, 41].

iPSCs can be cultured in feeder-free systems using specialized culture media and extracellular matrix substrates. However, to prevent spontaneous differentiation and maintain pluripotency, a defined culture medium is essential. This medium is characterized by the precise composition of specific chemical components, including growth factors and inhibitor agents. To ensure the quality of iPSC lines, pluripotency markers such as Oct4, Nanog, and SSEA-4 are often detected. Additionally, in vivo assessments involve inducing teratoma formation in mice to confirm the iPSCs’ ability to differentiate into cells of the three germ layers. To guarantee the stability of the iPSCs’ genome, karyotype analysis is conducted. This analysis serves to demonstrate the chromosomal integrity and overall genomic stability of the iPSC lines. These combined measures contribute to the robust maintenance and standardized characterization of iPSCs for research and therapeutic purposes.

Enhanced physiologically relevant 3D models could potentially mimic and present characteristics that are more closely and physiologically related to the human disease process [42]. Concerning animal models, both ethical and technical reasons have confined developmental biology research to selected animal models, which include chicken, frog, zebrafish and mice [42]. This animal studies have all greatly contributed to enhancing the understanding of biological processes and mechanisms. Nevertheless, transcriptional factors and epigenetic regulators function differently in human development. Furthermore, the human genome contains numerous non-coding regulatory elements that could be essential for human embryos and structures to develop and differentiate [42]. For these reasons, a suitable model could include hPSC-derived 3D cultures. These cells can self-organize in cell aggregates that can undergo developmental stages to generate the pseudo-organ of interest (i.e., the spinal cord) [43]. Moreover, spinal cords organoids assessed with this research models could be a key element to help uncover the disease mechanism that could remain otherwise unnoticed and/or not reproducible in 2D models [43].

ALS represents the perfect paradigm for using induced pluripotent stem cells (hiPSCs) as disease modelling. In fact, hiPSC, retain the genetic background of donor cells and they can differentiate into different cell types. The latter feature is crucial, since multiple cell types are related to the pathogenesis and the progression of ALS, such as MNs, astrocytes, oligodendrocytes and microglia.

Different studies have investigated transcriptomic ALS signatures in human iPSC models and profiled cultures in bulk [44] or transgenically labeled motor neurons [45]. These studies were performed when neurite degeneration and other phenotypes of overt disease emerged. It is, thus, not possible, in these models, to distinguish early transcriptomics events from secondary transcriptomic events. In trying to overcome those limitations, Ho and colleagues [46] developed a methodology using single-cell RNA sequencing analysis in motor neurons from healthy, familial ALS, sporadic ALS, and genome-edited iPSC lines. These were obtained from multiple patients, batches, and platforms, accounting for genetic and experimental variability to identifying unified and reproducible ALS signatures. The study by Ho et al. [46] reported that, by 18 days, iPSC-derived cells showed transcriptional states that globally resemble fetal hindbrain and spinal cord tissue at Carnegie stage 17, or about 42 days of in vivo development.

Stem cell technology has significant applications in therapeutic contexts, enabling the development of new therapeutic approaches and providing models for testing the efficacy and safety of emerging drugs.

In conclusion, in this review, we aim to describing the potential of hiPSC (induced pluripotent stem cell) technology, which enables the generation of infinite and renewable pools of ALS (Amyotrophic Lateral Sclerosis)-relevant cell types. The genetic heritage of a given patient is preserved during cell reprogramming in vitro, and this allows to reproduce the inter-individual heterogeneity and to study the numerous disease-causing mutations and the complexity of interactions between different cell subtypes.

New differentiation protocols have recently been introduced, and cell reprogramming technology continues to evolve. Through in vitro models, the key role of non-neuronal cells in ensuring the proper functionality of neuromuscular tissue has been discovered. All these elements contribute to making the use of stem cells a real diagnostic and therapeutic hope for motor neuron diseases in the not-too-distant future. However, there are still many challenges to overcome in order to achieve satisfactory results and to transfer the models in vivo. It is necessary to standardize cell differentiation technology to make scientific evidence more concrete, replicate the signs of cellular aging typical of neurodegenerative diseases that are lost during reprogramming into pluripotent cells, and finally, expand the data on ALS phenotypes, especially for sporadic forms, which are currently less described compared to familial forms.

One of the critical problems encountered in using patient-specific iPSC-derived from clinically relevant phenotypic cells as in vitro disease models is represented by the fact that the majority of these iPSC-derived phenotypic cells exhibit immature functional characteristics. These features are similar to their respective embryonic or fetal phenotypic cells. Moreover, they also contain a heterogenous mixture of varying proportions of phenotypic subtypes, giving rise to a variety of differentiations [33].

The development of a reliable disease model with defined cellular characteristics and a homogenous population of the phenotypic cells of interest is a crucial pre-requisite for developing therapeutic applications [33].

iPSC-based disease modelling has been widely used for early-onset diseases and proved to be successful model for this kind of diseases, such as spinal muscular atrophy.

For late-onset conditions, the disease models are prone to fail considering that the iPSC-derived relevant phenotypic cells lack the adult maturation characteristics to exhibit the disease phenotype [33].

There have been several approaches to induce the maturation of these primitive iPSC phenotypic cells, which include treatment with mitochondrial stress inducers and inhibitors of protein degradation, 3D co-culture to enhance paracrine-mediated stimulation of ageing and maturation, improved formulation of cultured/conditioned medium and derivation of iPSCs from adult aged patients [33]. Cell maturation obtained tends to be modest, despite all these efforts.

An alternative approach could be the direct conversion of somatic cells such as fibroblasts into clinically relevant phenotypic cells, to preserve the cellular ageing of markers.

Data and Materials pertinent to the study are all provided in the manuscript.

LC, LV and GP designed the study, wrote the manuscript editied the paper and approved the final version; MV, TI, CS, LS and MZ assisted in the research, editing, preparation and approval of the final version of the article. All the authors contributed to editorial changes in the manuscript. All the authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Not applicable.

Not applicable.

This research received no external funding.

The authors declare no conflict of interest.