Patient-derived induced pluripotent stem cells: Tools to advance the understanding and drug discovery in Major Depressive Disorder

Major Depressive Disorder (MDD) is a pleomorphic disease with substantial patterns of symptoms and severity with mensurable deficits in several associated domains. The broad spectrum of phenotypes observed in patients diagnosed with depressive disorders is the reflection of a very complex disease where clusters of biological and external factors (e


Introduction
In 2019, over one billion people lived with mental or addictive disorders, with depressive and anxiety disorders being the main contributors.Depression was considered the leading mental health contributor to the Global Burden of Disease and affected approximately 300 million people (latest report from 2019 Vos et al., 2020).The last years of the COVID-19 pandemic and the intensification of large-scale conflicts and recession have substantially increased the burden of depressive disorders, as proven by surveillance reports from different countries (EUROSTAT, 2023; Health at a Glance: Europe 2022, 2022; Vos et al., 2020).
Clinically, major depressive disorder (MDD) is an etiologically and phenotypically heterogeneous disorder whose diagnosis relies on the convergence of assessment methodologies aiming to explore patients' emotional responses, functioning, physical cues and symptoms following structured tools such as the Diagnostic and Statistical Manual of Mental Disorders 5th edition or the International Classification of Diseases and Related Health Problems (ICD-10) (American Psychiatric Association., 2013).
The symptomatology is predominantly characterised by an unremitting depressed mood and/or anhedonia (mandatorily required for the diagnosis) and, combinations of cognitive, behavioural, affective and vegetative symptoms impacting somatic and psychic functioning every day and lasting for at least 2 weeks.Substances of abuse (e.g., alcohol, opioids, amphetamines), drugs and medical conditions can be associated with the induction of depression.Furthermore, the symptomatology of some medical conditions may overlap with symptoms of depression which hurdles the differential diagnosis (e.g., neurodegenerative and neurological disorders, oncological diseases, diabetes mellitus) (American Psychiatric Association., 2013;Otte et al., 2016).
MDD is associated with increased morbidity and mortality, particularly among patients who display suicidal ideation and an increased susceptibility to developing or worsening comorbid disorders.Many patients do not achieve full symptomatic remission and are prone to relapse.However, it is uncertain whether this issue arises due to a partial response to treatment, resistance to treatment or antidepressants are less effective for being selected according to medical guidelines and empirically prescribed without the support from biological readouts (e.g., biomarkers) to help adjust the active principle and dosage for each patient (Gorwood et al., 2008;Penninx et al., 2013;Scott et al., 2023).
The knowledge generated over the last decades is owed to extensive preclinical research on animal models of depression, human postmortem, neuroimaging and genetic studies.Despite being highly valued and honed, animal models of depression are grounded on modelling organic or behavioural features of the disorder (thoroughly reviewed (von Mücke-Heim et al., 2023)).However, the induction and assessment of depressive-like phenotypes in conformity with the heterogeneous and cyclic nature of the disease and genetic and epigenetic landscape akin to humans remain challenging (Bale et al., 2019;Gururajan et al., 2019;Kaiser and Feng, 2015).
Despite being technically difficult and financially demanding, strides have been made towards the discovery of human-specific molecular signatures through iPSCs-derived models.

iPSCs-derived neuronal cells: tools to uncover human pathophysiology
iPSCs, discovered by Shinya Yamanaka, have become an important tool for researching human diseases, screening drug candidates and repurposing drugs.iPSCs provide vital information about humans' distinctive genetic and epigenetic landscape, which helps circumvent the ethical and technical challenges of obtaining human tissues (Takahashi and Yamanaka, 2006).
These cells can be obtained from the reprogramming of somatic cells, frequently fibroblasts and peripheral blood mononuclear cells, through mRNA, episomes, minicircles, piggyBac transposons, recombinant proteins and Sendai virus containing recombinant viral vectors expressing four reprogramming factors Oct4, Sox2, Kfl4 and c-Myc.Therefore, iPSCs can be differentiated into cells sharing the same genetic background as the donor, exhibiting disease-relevant phenotypes, recapitulating healthy and pathological tissues, organ development, molecular, and pathophysiological pathways that can drive the discovery of novel molecular targets for drug development.Co-cultures or more complex structures, including brain organoids, assembloids and organoids/ organs-on-a-chip have been used to develop more realistic disease models (Mollinari et al., 2018a;Soliman et al., 2017).
Several strategies, which include improvements in the reprogramming processes and gene-editing tools such as CRISPR/Cas9 gene editing to introduce homozygous or heterozygous modifications, have been employed to improve the scalability and clinical relevance of these cells enabling an unlimited source of accessible human cells for research (Bassett, 2017;Lee et al., 2020).
These efforts enable the development of more functional, personalized and unique models representing the molecular, biological, and human nature of the disease.To achieve this, endeavours are being made to improve the scalability of iPSCs differentiation into more functional, mature neuronal cells expressing markers of specific brain regions known to be at the core of psychiatric diseases.These improvements have also aided in surpassing limitations concerning purity and instability in long-term storage (Chehelgerdi et al., 2023) In what concerns the central nervous system (CNS), the cellular environment can be manipulated and enriched by patterning signal molecules to induce neural differentiation into region-specific neurons, such as Wnts, retinoic acid and Sonic Hedgehog (SHH).Recently, research teams have successfully induced neuronal differentiation of feeder-free iPSCs into neural progenitor cells (NPCs) expressing markers of specific brain regions.These NPCs were differentiated into forebrain cortical neurons, forebrain interneurons, midbrain dopaminergic neurons and spinal motor neurons.In addition, a successful differentiation into NPCs similar to those found in the hippocampal dentate gyrus (DG) was achieved through the supplementation of the cellular environment with antagonists of the SHH-signalling pathway, WNT, transforming growth factor β, bone morphogenetic proteins, Noggin pathways and DG-CA3 neurons from patients diagnosed with Schizophrenia were generated and co-cultured to explore intrinsic neuronal activity (Comella-Bolla et al., 2020;Imaizumi et al., 2015;Kiecker and Lumsden, 2012;Kirkeby et al., 2012;Moya et al., 2014;Sarkar et al., 2018;Sato et al., 2021;Yu et al., 2014).
The evaluation of the functional properties of iPSCs-derived neuronal cells is fundamental for the validation of these models.Electrophysiology assays are routinely performed to examine hallmarks of neuronal functionality and maturity, including threshold, rise time, amplitude, half-height width and after-hyperpolarization amplitude, calcium, potassium and sodium currents, and ligand-gated GABA receptors, among others.Alternatively, automated patch clamp systems enable the electrophysiological analysis of suspended cells or in platforms containing multi-/micro-electrode array (MEAs) with planar chipbased devices that analyze 10-700 cells at the same time (reviewed (Bell and Fermini, 2021)).The installation of MEAs systems on neural cultures has the advantage of following the spontaneous activity, and maturity of cells and searching for dynamic changes in receptors/channels upon exposure to a panel of drugs and/or neurotransmitters over time.Both techniques allow the detection of anomalies in ion channels, neural receptors in disease substrates and test the dynamic interaction between drug-ion channels, drug binding/unbinding, protonation/deprotonation and the extent to which the mechanistic modulation of ion channels by drugs is of relevance for the targeted disease (Lukacs et al., 2021;Obergrussberger et al., 2018).

Application of iPSCs-derived models in psychiatry
Undeniably, animal models of depression have contributed massively to the understanding of MDD and pharmacokinetics and pharmacodynamics of antidepressants however, the high demands to reduce, refine, replace animal models and offer more therapeutical options to humans has sparked the interest to seek opportunities to get access to human cells without relying on post-mortem tissue.
Unlike other psychiatric diseases, research on MDD is lagging and there are only a few studies that have been performed on patient-derived iPSCs (Table 1).The lack of research on this matter may be conditioned by the misdiagnosis of some psychiatric diseases whose symptoms can vary markedly within the same diagnosis and overlap between different diagnoses (e.g., MDD and Bipolar Disorder), inequitable global distribution of funding, barriers in enrolling in research projects ranging from the unacceptance of the diagnosis, stigma, lack of interest, rewards and accessibility for participants (Castelpietra et al., 2020;Hazo et al., 2017;Nestsiarovich et al., 2021;Woodall et al., 2010).
We hypothesise that besides these factors, the biomedical research involuntarily or voluntarily tends to focus on other severe mental illnesses that are polygenic, have a high risk of causing harmful thoughts and behaviour, treated with antipsychotics that can lead to metabolic dysregulation and a spectrum of side-effects that can compromise treatment adherence.As a result, more efforts have been made to identify new pharmacological options for these disorders (Jawad et al., 2018;Kishi et al., 2022;Kishimoto et al., 2021;Rao et al., 2021).
However, MDD shares an increased risk of unfavourable illness trajectory with high rates of societal costs and failure to achieve symptomatic and functional remission which highlights the need to explore neurobiological substrates to the fullest and deliver new pharmacological options (Berlim and Turecki, 2007).
Even so, some studies have explored these substrates from patient-derived models.Patient-derived forebrain neurons from a MDD patient cohort that had their previous pharmacological treatment suspended and were enrolled in an 8-week trial with citalopram or escitalopram, as part of the Pharmacogenomic Research Network Antidepressant Medication Pharmacogenomic Study whose scope involves the assessment of clinical outcomes and genetic factors of patients diagnosed with MDD, treated with these SSRIs antidepressants.Non-respondent patients and patients with a very successful response to the treatment were subjected to a skin biopsy to generate neurons.The research team found that neurons from SSRIs-resistant patients exhibited altered postsynaptic serotonergic neurotransmission, 5-HT (5-hydroxytryptamine) induced hyperactivity via upregulated 5-HT2A and 5-HT7 receptors (implicated in MDD).In addition, the group found longer serotonergic neurites and lowered expression of the protocadherin gene (PCDHA6/A8) in SSRIsresistant patients compared to healthy controls and MDD patients who responded to the treatment.The authors suggested that these changes in serotonergic wiring may contribute to maladaptive circuitry, leading to SSRIs resistance (K.C.Vadodaria, Ji, Skime, Paquola, Nelson, Hall-Flavin, Fredlender, et al., 2019, 2019).Furthermore, 5-HT2A and 5-HT1A receptors are involved in depressive and anxiety-like behaviours, and the transcriptional dysregulation of the 5-HT1A receptor, potentially induced by Freud-1, may also play a role in SSRIs resistance (Albert et al., 2014;Ślifirski et al., 2021;Vahid-Ansari et al., 2017).
Protocadherins have a high molecular diversity and are important for the establishment of neuronal connectivity.A deletion of the pcdhα cluster disturbs serotonergic axons leading to inadequate terminal arborization and increased depressive-like behaviour.Within the pcdhα cluster, pcdhac2 has been recognized as essential for the wiring of serotonergic axons (Jia and Wu, 2020).An aberrant regulation of PCDH9 transcription was found to be a mediator in the increased genetic risk induced by SNP rs9540720 or its linkage disequilibrium SNPs in MDD (Xiao et al., 2018).As alluded to, MDD is enigmatic and the scrutiny of these molecular pathways and their correlations with MDD are crucial.
Recently, researchers obtained GABAergic interneurons and ventral forebrain organoids from patient-derived iPSCs diagnosed with MDD and suicidal behaviour.The GABAergic interneurons from MDD patients had decreased expression of 5-HT2C which was reversed by trazodone,  A., et al., 2018, Cell Stem Cell (Sarkar et al., 2018).
Brain organoids from persons diagnosed with Bipolar Disorder and persons without psychiatric disease (control group).
iPSCs-derived forebrain neurons from a MDD patient cohort enrolled in an 8-week trial with citalopram or escitalopram.
Longer serotonergic neurites and lowered PCDHA6/A8 levels in SSRIs-resistant patients compared to healthy controls and MDD patients that responded to the treatment.Lu K., et al., 2023, EMBO Molecular Medicine (Lu et al., 2023).
iPSCs from patients diagnosed with MDD were differentiated into GABAergic interneurons and ventral forebrain organoids.
Compared with controls, GABAergic interneurons from patients had morphological changes, increased neural firing and weakened calcium signalling propagation.Results from transcriptomic sequencing indicated a decreased expression of 5-HT2C which may cause defects in neuronal activity in MDD.These defects were reverted by pharmacological and genetic approaches.Anorexia Nervosa Negraes, P.D., et al., 2017, Translational Psychiatry (Negraes et al., 2017).
iPSCs-derived cortical neurons from patients diagnosed with Anorexia Nervosa (restricted to females).
The expression of TACR1 was significantly upregulated in iPSCs-derived cortical neurons.
Evaluation of the hepatic metabolism-dependent cardiotoxicity caused by clomipramine (tricyclic antidepressant).Exposure to a low concentration of clomipramine (1 μM) heightened cardiotoxicity with negative repercussions on cell viability, cardiac contractility and calcium flux.Exposure to a high dose of clomipramine (10 μM) reduced the synthesis of urea and affected liver function.Cavalleri L., et al., 2017, Molecular Psychiatry (Cavalleri et al., 2018).iPSCs-derived dopaminergic neurons.
Psilocin increased synaptogenesis, BDNF upregulation and excitability, as well as an increase in postsynaptic current frequency and amplitude.
A. Vaz et al. an antidepressant that is an agonist of 5-HTC2 receptors.Trazodone enhances neural differentiation, inhibits PERK/eIF2α-P, and restores the decreased expression of 5-HT2C.Additionally, these cells exhibited increased neurite arborization and neural firing and decreased calcium signalling compared to the control group.Interestingly, these morphological changes (increased neurite branches) were also observed in iPSCs-derived serotonergic neurons from MDD patients.Weakened calcium signalling and hyperactivity could be considered putative early disease markers, which could be misleading in advanced stages due to the progressive loss of GABAergic neurons.However, genetic and pharmacological approaches with trazodone and lentivirus reversed deficits in neuronal activity measured by calcium signalling and electrophysiology (Lu et al., 2023).Organoids are 3D in vitro structures whose gene, protein expression, tissue architecture and metabolic function share similarities with the human organ intended to generate.Pluripotent stem cells such as embryonic stem cells or iPSCs, differentiate into the native organ cells and maintain the capacity to renew and restock the stem cell poll that selforganizes and proliferates resembling the native organ.One of the major challenges that still lie ahead is the intensive labour, heterogeneity and high cost, however, it is consensual that these are valuable platforms for the study of neurodevelopment, neuropsychiatric and oncological diseases (Bian et al., 2018;Giandomenico et al., 2021;Stachowiak et al., 2017).
Specific brain regions resembling the neocortex, cerebellum and midbrain were generated in brain organoids (Kadoshima et al., 2013;Muguruma et al., 2015;Qian et al., 2016Qian et al., , 2018)).iPSCs-derived brain organoids from monozygotic twins (one pair discordant for schizoaffective disorder, bipolar type and two pairs of monozygotic twins discordant for Schizophrenia) were subjected to cRNA-seq analysis, functional genomic analysis and exhibited enhanced GABAergic specification, defective Wnt/β-catenin signalling pathway in neuronal cells, reduced progenitor proliferation and excess network inhibition during cortical development.Interestingly, brain organoids from patients diagnosed with Bipolar Disorder were compared with brain organoids from persons without psychiatric diseases (control group) and transcriptomic differences were found, particularly in genes involved in neurodevelopment, cell adhesion, synaptic plasticity and upregulated genes involved in signalling of immune responses.Functional studies have shown that organoids from patients diagnosed with Bipolar Disease exhibit physiologically normal baseline firing patterns and anomalies in response to stimulation/depolarization (Kathuria et al., 2020;Sawada et al., 2020).
A recent neurodevelopmental model encompassing BrainSpheres (deemed as more reproducible, stable in size, with higher homogenous cellular populations and less necrotic centres) allowed the scrutiny of differentiation and maturation of neurons, astrocytes, oligodendrocytes, neurite outgrowth, synaptogenesis and myelination upon exposure to paroxetine (SSRIs, 20 and 60 ng/mL).The authors detected deleterious consequences on neurite outgrowth, a decrease in synaptic marker expression and a reduction in oligodendrocyte population, suggesting the need for further investigation.Thus, the lines CLR-2097 (commercial line) and iPS2C1 (provided by another researcher) exhibited a slightly different response to 20 ng/mL of paroxetine, which implies different cell lines may respond differently.These decreases in neurite outgrowth upon paroxetine exposure are congruent with the role of serotonin in shaping neuroplastic events during neurodevelopment and the putative disruption of the maturation processes of oligodendrocytes (Fan et al., 2015;Rojas et al., 2014;Zhong et al., 2020).
Similar to BrainSpheres, bioengineered neuronal organoids (BENOs) consist of iPSCs-derived glutamatergic and GABAergic neurons supported by astrocytes, oligodendrocytes and capable of establishing neural networks.To the best of our knowledge, there hasn't been any research published yet on the application of BENOs in MDD (Zafeiriou et al., 2020).
Neurexins are presynaptic neuronal adhesion molecules that regulate the properties of synapse transmission and plasticity.Depletions in neurexins have drastic consequences ranging from reduced synapse numbers, decreased Ca 2+ influx, decoupling of Ca 2+ channels with synaptic vesicles and for that reason, they are thought to exert various modulatory functions on specific synapses whose current research hints towards dependence on certain isoforms and splicing.These consequences have long been linked to Bipolar Disorder, Schizophrenia, and Autism Spectrum Disorder.However, only a restricted number of studies explored the pathological consequences of disease-associated NRXN1 variants on iPSCs patient-derived neurons, with a clinical diagnosis of Schizophrenia and Autism Spectrum Disorder.The main findings were changes in calcium signalling, cell fate and impaired synaptic function (Avazzadeh et al., 2019;Castronovo et al., 2019;Chen et al., 2017;Hu et al., 2019;Jenkins et al., 2014;Lam et al., 2019;Lett et al., 2011;Luo et al., 2020;Pak et al., 2015;Reichelt et al., 2012;Südhof, 2017).
Until recently, a synaptic biding partner, LRRTM4 (post-synaptic leucine-rich repeat transmembrane) was found to be related to an increased risk of suicide attempts among patients diagnosed with Bipolar Disorder and altered brain expression of NRXN1 was also found in persons diagnosed with Bipolar Disorder and Schizophrenia whom died from suicide.Both symptomatology and genetic background crosscut between Bipolar Disorder and MDD.However, the absence of the aforementioned studies on MDD/treatment-resistant MDD cannot establish such a link (Reichman et al., 2020).
The investigation of biological/genetic predictors of MDD through these models could be particularly informative to define biomarkers with the potential to improve diagnosis and increase the effectiveness of treatments.Future studies could focus on the sex dimorphism (females and males) in MDD and the neurobiological factors that could be implicit and the development of more therapeutical options for pregnant and lactating women.

The contribution of iPSCs models in the elucidation of the mechanism of action of psychotropics
iPSCs can support the study of key events concerning the differentiation and maturation of cells in disease substracts and can also, be used to investigate antidepressant mechanisms of action.
Ketamine, a N-methyl-d-aspartate receptor (NMDAR) antagonist and rapid-acting antidepressant, has shown promising results in subgroups of patients diagnosed with MDD and Bipolar Disorder who are not benefiting from first-line antidepressants due to the time lag until therapeutic effects manifest (Bartoli et al., 2017;Yang et al., 2019).
In iPSCs-derived dopaminergic neurons (known to be impaired in mood disorders), ketamine stimulated structural plasticity, more precisely, increased dendritic outgrowth and soma size by the recruitment of BDNF and a protein kinase mechanistic target of rapamycin and upstream activation of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors.This drug is dependent on the dopamine D3 receptors (Cavalleri et al., 2018).
In another study, iPSCs from healthy donors were differentiated into DA neurons and exposed to (2R,6R)-hydroxynorketamine (HNK), a ketamine metabolite that produced analogous effects (dendrite outgrowth) at doses and exposure times compatible with clinical data on the pharmacokinetics of ketamine infusions.GABAergic and glutamatergic neurons were also present in these cultures and the authors decided to exclude the analysis of these neurons.However, the analysis of these neurons could have provided new insights into the effects of ketamine as in vivo data suggests that ketamine reduces the inhibitory input from GABAergic neurons and may cause homeostatic synaptic plasticity and increased synaptic drive on glutamatergic neurons.Still, it is not clear whether GABAergic interneurons, the first ones to be inhibited, trigger the disinhibition of the activity of glutamatergic neurons (Collo et al., 2018;Duman et al., 2016;Gerhard et al., 2020).
More recently, iPSCs-derived dopaminergic neurons from a healthy donor were used to understand structural changes induced by glucocorticoids.An exposure to an array of high doses of cortisol (10-50 μM) to mimic a putative in vitro "acute" and "chronic" stress for 24, 48, 72 and 96 h, caused hypotrophy on cortical dendrites and alterations in the soma size compared to controls.
The impaired neuronal structural plasticity induced by glucocorticoids was reversed by ketamine and HNK which underscores the need to leverage how ketamine and its metabolites modulate neurotransmission in MDD (Cavalleri et al., 2024).HNK doesn´t lead to psychotomimetic effects and abuse potential as ketamine and both upregulate glutamatergic neurotransmission via increased expression of AMPARs and hippocampal BDNF.Even though molecular targets of HNK in different brain areas remain obscure, it regulates glutamatergic and cholinergic neurotransmission (Guhathakurta et al., 2024).
A recent proof-of-concept study, developed iPSC-derived liver-heart organoids on-chip device with compartmentalized chambers where liver organoids grew in the upper multi-well chamber and heart organoids in the bottom micropillar array concurrently, were used to evaluate the hepatic metabolism-dependent cardiotoxicity caused by clomipramine (tricyclic antidepressant).Exposure to a low concentration of clomipramine (1 μM) heightened cardiotoxicity with negative repercussions on cell viability, cardiac contractility and calcium flux.Moreover, exposure to a high dose of clomipramine (10 μM) reduced the synthesis of urea and affected liver function which is consistent with current evidence.In addition, the integration of brain organoids from MDD patient-derived iPSCs in an organoids-on-a-chip device may elucidate not only the pathophysiology of MDD but also, increase the robustness of in vitro preclinical models (Yin et al., 2021).
Investments in iPSCs-derived neural cultures from patients diagnosed with MDD will potentially bolster the improvement of pharmacological approaches by underlying genetic and physiological factors impacting the onset and course of the disease.

High-throughput screening and high-content screening in iPSCs-based models: facts and future perspectives for MDD research
Drug development pipelines for psychiatric disorders haven't evolved much since the 1950s when the first antidepressants were identified.iPSCs-derived neurons can be used in high-throughput screening analysis (HTS) where panels of bioactive molecules, drugs, and drug candidates, usually from compound libraries, are tested (Fantuzzo et al., 2020;Sherman and Bang, 2018).
These neurons have been preferentially used as single populations which, do not capture the complex interactions with glial cells known for their well-recognized role in psychiatric diseases.A recent HTS device with a microfluidic plate-based system coupled with a microscopy platform aims to explore the neurocircuitry function and crosstalk between distinct neuronal subpopulations to open a new avenue for personalized psychiatry (Kirihara et al., 2019).
Also, two in vitro scaffolds combining iPSCs-derived brain organoids anchored in a photolithographically defined (SU-8 photoresist) structures named cerebral organoid connectivity apparatus, were developed to capture the 3D brain's microenvironment and cytoarchitecture whilst avoiding outbursts of tissue growth and ensuring optimal media perfusion, maintenance and easier handling.The development of these sophisticated devices offers new opportunities to surpass some technical and functional limitations including the impossibility of maintaining functional connectivity and viability in long-term cultures (Cullen et al., 2019;Lago et al., 2021;Robles et al., 2021).
Phenotypic drug screening can be exploited in patient-derived iPSCs models (disease vs. control groups with isogenic lines or healthy donors) to identify molecules/prospective hits able to reverse pathophysiological mechanisms.To achieve it, iPSCs-derived cells can be cultured with ligands (e.g., CNS agonists, cytokines, growth factors, antigens, etc.) and each response can be evaluated through multiple functional readouts (e. g., phosphorylation of cell signalling proteins or mRNA expression) including single-cell high-content screening (e.g., flow and mass cytometry, high-content microscopy, single-cell RNA sequencing).The resulting ligand-readout-cell subtype combination is considered a cellular response "node" that combined with multiple nodes of each donor, provides a cellular profile and response in case multiple doses of ligands are being applied.An array of nodes from different groups (e.g., patients and control groups) can be compared (Rosenbaum et al., 2020).
The development of solid high-throughput readouts for neural network activity is challenging, and as a result, only a few methods have been developed thus far.However, progress in this field will have a positive impact on the discovery of new hit compounds and genes suitable for pharmacological interventions in MDD.

Technical prospects and hurdles of iPSCs-based models in psychiatric diseases
The number of participants and respective collection of biological data is still a matter of debate as some researchers may opt for a smaller set of participants and increase the number of clones/lines whilst others decide to use a single iPSCs line for each patient (Hoffman et al., 2017;Rosenbaum et al., 2020).
Some authors suggested that iPSCs derived from cohorts of 20 to 80 participants may be sufficient to capture the effects of individual regulatory variants (e.g., SNPs) with moderately large effect sizes that would also benefit from improved differentiation protocols with reduced variability.In contrast, large-scale iPSCs studies are beginning to emerge but still, statistical power and inter and intra-donor variability are factors that should be taken into account when designing studies.Improved biological coherence can be achieved through the improvement of neuronal differentiation or induction protocols (Bonder et al., 2021;Marcatili et al., 2020;Schwartzentruber et al., 2018).
It can be challenging to determine the developmental age of certain neuronal and glial cells derived from iPSCs, as they may be less mature than their human brain counterparts.This makes it difficult to identify biomarkers that can accurately pinpoint their developmental age.Consequently, it remains a challenge to extrapolate in vitro findings to in vivo phenotypes (Mertens et al., 2016).
During the reprogramming and differentiation processes, genetic and epigenetic errors may occur (e.g., epigenetic erasure).These events can be influenced by the donors' age, and cell type and some researchers suggest executing neural transdifferentiation approaches: 1) somatic cells are directly reprogrammed into neurons, yielding smaller samples of functional cells; 2) derive induced neural progenitor cells with feasible applications for higher sample sizes.In transdifferentiation, somatic cells are exposed to transcription factors, miRNA, patterning molecules and other molecules that regulate signalling pathways.Fibroblasts are widely used as the main source of patient-derived iPSCs and many transdifferentiation protocols focus on these cells.The constrained capacity of self-renewal following 50 or more passages hampers the use of these cells for transdifferentiation (Han et al., 2012;Hermann and Storch, 2013;Hofer and Lutolf, 2021;Mollinari et al., 2018b;Vadodaria et al., 2016).
Some psychiatric diseases can have a highly polygenic nature which demands the handling of complex models where human neural cells interact mimicking the in vivo, physiological and/or pathological microenvironment while preserving the epigenetic signatures/genetic networks.As such, these complex models (e.g., organoids) have been perfected to mitigate some major technical caveats that include the lack of control over stochastic matrix interactions, heterogeneity between batches and standard protocols to provide maximized robustness, rentability and stronger biological significance to put forward regenerative and personalized psychiatry (Huch et al., 2017;Mulero-Navarro et al., 2015;Xinaris et al., 2015).

Lessons and perspectives from iPSCs-derived models applied in brain diseases that would benefit clinical psychiatry
The majority of studies strive to develop realistic and reproducible disease models, whilst others aspire to bring iPSCs into the clinics.iPSCs can be integrated into clinical practice as clinical tools to stratify the risk of developing neurodegenerative and neuropsychiatric diseases.In fact, this strategy of stratification and monitoring the risk of developing diseases within families with a clinical history has been done in oncology (Ramazzotti et al., 2018).The stratification and interpretation of clinical data can be aided through the integration of machine learning algorithms and therefore, clinicians may have the additional support to evaluate the risk and choice of drugs (Mandai et al., 2017).
On a different end, iPSCs-derived cell transplants have already been exploited to treat age-related macular degeneration and Parkinson's Disease in patients, without a control group for compassionate use.A 4year follow-up of the autologous iPSC-derived retinal pigment epithelium revealed the survival of these cells with a discrete proliferation of the pigmented area without adverse events (Takagi et al., 2019).iPSC-derived midbrain dopaminergic progenitor cells phenotypically identical to substantia nigra pars compacta neurons were grafted into the putamen, without immunosuppression.The patient showed modest clinical improvement 18 and 24 months after the transplant.Despite ongoing pharmacological treatment, a 6 % reduction in levodopa was prescribed (Schweitzer et al., 2020).Still, the authors suggested the survival of the grafted cells may had been restricted and no additional follow-up reports have been published thus far on the evolution of the symptoms, patient quality of life, pharmacotherapy and emotional, cognitive and functional integrity.
These authors have performed cell transplants in rodents which initially had repercussions on neuroinflammation.The occurrence of a surgically-induced injury ("needle trauma") resulted in the infiltration of peripheral immune cells and death of iPSCs-derived dopaminergic neurons that were reverted by the intra-striatal co-transplantation of autologous T reg cells.The surgical procedure itself and "needle trauma" trigger a profound host response that is characterized by acute neuroinflammation, robust infiltration of peripheral immune cells and brain cell death.Some strategies may be implemented to improve engraftment: 1) optimized cell density of transplanted cells; 2) transplant cells at early stages of differentiation to enhance complete recovery, survival and fiber outgrowth (Hiller et al., 2022).
Psychiatric diagnoses rely solely on the clinical presentation and history of symptoms.The pursuit of more accurate biological/ physiological-based diagnosis, as well as risk prediction and treatment optimization according to individual needs and characteristics, has long been the premise of precision psychiatry.Over the years, the search for biomarkers with a potentially relevant role in the diagnosis and treatment of MDD has been one of the main priorities but, not sufficiently robust data has permitted its implementation in the clinical setting.
These biomarkers, sourced from biopsies and bodily fluids, have been extensively studied since the onset of the disease, during treatment, and recovery, and are classified according to their clinical applications (e.g., susceptibility/risk, diagnostic, monitoring, pharmacodynamic/ response, predictive, prognostic, safety, and risk biomarkers) (Cagney et al., 2018).
Extracellular vesicles (EVs) enclose DNA, metabolites, proteins and RNA known to mediate cell-to-cell communication through endocytosis, phagocytosis, membrane fusion and regulate downstream events in physiological (e.g., energy metabolism, neurogenesis, neuroinflammation) and pathological processes.These structures have arisen as important mediators of CNS pathologies including MDD and carriers of bioactive compounds in iPSCs-derived neurodegenerative models.The clinical applications are still premature but promising as diagnostic and treatment biomarkers, and putative drug carriers as they cross the blood-brain barrier (Li et al., 2023;Okano et al., 2020;Wang, 2021).iPSCs-based models have made important contributions to drug repurposing for amyotrophic lateral sclerosis.Ropinirole (an antiparkinsonian drug), retigabine (an antiepileptic drug), and bosutinib (an anti-chronic myelocytic leukaemia drug) were identified as interesting candidates for clinical trials (Okano et al., 2020).
The growing interest in iPSCs-derived models to survey MDD pathogenesis, drug development and cell therapy may fulfil a relevant role in the future of personalized psychiatry (Fig. 1).

iPSCs-derived cellular transplantation in MDD: A realistic approach?
Cell transplantation may be one of the biggest ambitions in stem cell research and even though preclinical data on cell transplantation in depressive disorders is scarce, new studies in this field may be implemented in the years to come.
As previously highlighted, MDD is intricate, and the conception and exploitation of iPSCs-derived neural cells and their therapeutic products (e.g., EVs) for transplantation are tied to challenges and uncertainties.Besides, biomarkers are not sufficiently robust to stratify patients and the clinical setting hasn't evolved sufficiently to scrutinize the impaired mechanisms and aetiology of each patient.Many questions arise concerning the use of cell transplantation as a treatment option for depressive disorders.These include: 1) which differentiation/commitment stage and type of cells should be transplanted to effectively treat the disease and reduce "needle trauma"?; 2) Where should they be transplanted and would they survive and integrate into their new microenvironment?; 3) Can cell transplants be a viable option for patients with treatment-resistant depression?If patients are already taking antidepressants, how will this affect the engraftment and clinical course after transplantation?; 4) Should patients continue taking antidepressants after the transplant as an augmentation strategy?; 5) Will the benefits of the transplant outweigh the surgical risks?; 6) What is the best drug delivery system to use?And finally, should the transplant be done using the patient's cells or cells from a donor?These are both practical and philosophical questions to which there isn´t a definite answer yet.These hypotheses must first be rigorously evaluated in preclinical models.As far as we know, only two studies have attempted to tackle these issues.In the first, the authors encapsulated mesenchymal stem cells (MSCs) and transplanted them into the striatum and lateral ventricle of rats (a model of treatment-resistant depression) to ascertain the effect on the neurogenic niche located at the subventricular zone and the DG of the hippocampus.The secretion of vascular endothelial growth factor, BDNF, fibroblast growth factor-2, and ciliary neurotrophic factor was satisfactory and stable after transplantation of MSCs (day 15 post-transplantation).Another research group transplanted mesenchymal stem cells on a rat model of depression (flinders sensitive line) that migrated mostly to the ipsilateral DG, CA1 and CA3 regions of the hippocampus (Kin et al., 2020;Tfilin et al., 2010).
Studying the longitudinal trajectories of transplanted animals would provide us with more comprehensive insights.However, such studies would require a large number of animals and resources.To conduct such research, we would need to allocate wild-type (control) animals and those exhibiting depressive-like behaviour into several groups that would be followed over time (for example, over the course of a year).These animals would be exposed to antidepressants to mimic the clinical reality and to evaluate the efficacy and clinical interest of the treatment.Such studies would provide us with a better understanding of depressive disorders.With the addition of optogenetics-based electrophysiology approaches, the analysis of differentiation patterns after transplantation, network integration of transplanted neuronal cells and development of long-distance projections and synapses would be elucidating.
Glial scar ablation and neuromodulation with electrical stimulation can be implemented to modulate outcomes in cell transplantation.These strategies have been leveraged in neurodegenerative disorders with conflicting results.
Treatment with cell transplantation coupled with electrical stimulation with the tail nerve electrical stimulation can improve cellular integration, differentiation, and functional recovery compared with the cell transplant alone as it can influence signalling pathways that regulate migration and differentiation of neural stem cells (Patil et al., 2023).
A marginally different approach based on focused ultrasound bloodbrain barrier opening in the hippocampus of rats, explored the potential of this technique to improve hippocampal neurogenesis.However, the results did not suggest any increase in neurogenesis.Future studies may clarify the isolated therapeutic potential of this procedure when used in conjunction with antidepressants, transplanted iPSCs-derived neurons, glial cells, or novel drugs (Meng et al., 2021;Mooney et al., 2018).

Conclusions and future perspectives
MDD is not exclusively associated with abnormal changes in independent brain regions but also with an extensive system dysfunction that affects the integrity of neural circuits.The integration of transdiagnostic perspectives, multidimensions of psychopathology, biomarkers, and data from iPSCs-derived neural cells could be relevant to tracking outcomes, including symptom progression and phenotype families/patients at higher risk of developing the disease and not achieving remission.
The wealth of iPSCs models holds onto the scrutiny of molecular, genetic data that can be integrated into databases comprising druggable molecular targets to aid the discovery of new hit drugs and drug repurposing.
By focusing on the analysis of gene networks and understanding the relationship between genomics and external risk factors, we can identify early disease signatures.Additionally, studying the interplay between oxidative stress, mitochondrial dysfunction and inflammation, which are known to be impaired in MDD, can help elucidate the mechanisms of the disease.
Although studying downstream mechanisms of action of antidepressants in patient-derived iPSCs models is not yet commonplace in academic and pharmaceutical industry settings, it will certainly elicit more robust evidence to guide clinical investigation in the future and implement personalized psychiatry on a broad scale.In the clinical setting, including outpatients and inpatients diagnosed with MDD of all Fig. 1.The potential of iPSCs-derived models to explore the neurobiological substrates of Major Depressive Disorder (MDD) and contribute to the understanding of genetic and epigenetic factors shaping the onset of the disease and sex differences is highlighted in this schematic overview.These models can serve as a valuable tool to test candidate compounds, drug repurposing, and identify molecular targets that can be used to design new drugs.In the clinical setting, these patient-derived cells can help accelerate the discovery and validation of biomarkers to aid diagnosis, clinical recovery, and remission.Created in BioRender.com.ages, sexes, and different degrees of severity, following cohorts longitudinally with standard procedures to collect blood samples and clinical data would improve experimental pipelines.This would generate wellpowered evidence to identify epigenetic changes and other molecular/ cellular factors that confer risk and/or resilience to MDD and generate progression biomarkers.It is important to avoid limiting the analysis to case-control cases and focus on looking for differences within cases upon concomitant or non-concomitant exposure to psychotropics as seen in clinical practice.This will broaden the understanding of the different dimensions of the disease.
Psychedelics such as lysergic acid diethylamide and psilocybin have shown promise as potential treatment options for MDD with long-lasting clinical improvements.Although it is not fully agreed upon whether psychedelics act as 5-HT receptor agonists (5-HT 1A , 5-HT 2A , and 5-HT 2C ) and the antidepressant effect of psilocybin may not be dependent on the activation of 5-HT 2A R, data from patients suggest that the psychedelic experience may be necessary for their clinical efficacy.Therefore, the role of psychedelic properties of 5-HT 2A R agonists in their antidepressant effects remains a matter of debate.Some synthesized 5-HT 2A R agonists and ergot-derived dopamine agonists void of psychedelic effects (e.g., lisuride) have been explored as pharmacotherapy options for mood disorders (Sekssaoui et al., 2024).
The expression of genes involved in synaptic function and neural plasticity induced by psilocin were tested on iPSCs-derived cortical neurons.The authors documented that a decrease in receptor surface density, which can be prevented by inhibiting the GTPase dynamin, may be associated with the internalization of cell surface-located 5HT 2A R. Additionally, they found increased synaptogenesis and BDNF upregulation in human cortical neurons after treatment with psilocin, along with evidence of protein kinase C activation and endocytosis, both of which contribute to 5HT 2A R internalization.These changes resulted in alterations in intrinsic neuronal properties and network function, including increased excitability, as well as an increase in postsynaptic current frequency and amplitude (Schmidt et al., 2024).
Deep phenotyping patients and participants in clinical trials with multidimensional data comprising psychological scales, genetic testing, neuroimaging data, iPSCs-derived models and matching it with data from postmortem and rodent-based studies and healthy, without family and clinical history of psychiatric disorders and isogenic controls could reduce the risks associated with novel drugs and drug repurposing.
On another note, stratifying responders and non-responders on first or multiple episodes; full remitters and partial remitters with residual symptoms; patients who experience recurrence and different degrees of severity and compare outcomes with other types of depressive disorders, would improve proof-of-concept, drug development and post-marketing studies.Optimizing experimental conditions by analysing EVs from different groups (in vitro) could identify plausible biological mechanisms determining gene and environment interactions.Tracking changes in the cargo and structure of EVs following in vivo and in vitro studies could be very insightful concerning the underpinnings of remission and recurrence.
Because GWAS studies encompass large sample sizes, they have provided valuable insights into the influence of common genetic variants in pathology.However, the hits generated are commonly from noncoding regions of genes and are considered functional proxies of other variants, with a low effect size.For this reason, the reprogramming of iPSCs into patient-derived specialised cells (from large cohorts of patients) allows the disclosure of the individual genetic background, genotype-phenotype interactions, pharmacogenomics and sex dimorphism with chances to identify genetic modifiers and improve the translation of GWAS data (Uffelmann et al., 2021).
Machine learning and artificial intelligence algorithms are being used to analyse arrays of multidimensional data to estimate the trajectory of the disease (Fröhlich et al., 2018).In the long run, complementing experimental/preclinical data from animal models and iPSCs-derived neural cells with longitudinal clinical data would empower the bench-to-bedside translation.However, it is necessary not to underestimate the importance of animal models in the assessment of pharmacokinetic and pharmacodynamic properties of drugs and for that reason, keeping in mind that iPSCs-based models will probably not replace them but could be an important complement to refining them.

Declaration of competing interest
The authors unequivocally declare that they have no conflicts of interest and assume full responsibility for ensuring the reliability and impartiality of the scientific content of this article.

Table 1
Studies highlighting iPSCs-based models to recapitulate psychiatric diseases and accelerate drug discovery.