Aging phenotype in AD brain organoids: Track to success and challenges

Alzheimer ’ s disease (AD) poses a complex challenge, with abnormal protein accumulation in the brain causing memory loss and cognitive decline. Traditional models fall short in AD research, prompting interest in 3D brain organoids (BOs) from human stem cells. These findings hold promise for unveiling the mechanisms of AD, especially in relation to aging. However, an understanding of the aging impact of AD remains elusive. BOs offer insight but face challenges. This review delves into the role of BOs in deciphering aging-related AD and ac-knowledges limitations. Strategies to enhance BOs for accurate aging modeling in AD brains are suggested. Strengthened by molecular advancements, BOs have the potential to uncover the aging phenotype, advancing AD research.


Introduction
Alois Alzheimer, the distinguished German psychiatrist, is credited as the pioneering force behind the discovery of Alzheimer's disease (AD) (Hippius and Neundörfer, 2022).This neurodegenerative disorder is characterized by the accumulation of abnormal protein deposits in the brain, specifically beta-amyloid plaques and tau tangles, leading to impaired daily activities, abnormal neurobehavior, and, most notably, dementia (Shoghi-Jadid et al., 2002).AD primarily affects the brain regions responsible for thought, memory, and language.The primary risk factor for neurodegeneration is brain aging (Yankner et al., 2008), which involves intricate cellular and molecular processes that eventually result in cognitive decline (Bartzokis, 2004;Hullinger and Puglielli, 2017).Approximately 90% of elderly individuals are highly susceptible to developing the disease, alongside the prevalence of other forms of dementia(de Sousa et al., 2023).Numerous studies also indicate a strong association between AD and aging since the disorder predominantly affects individuals aged 65 and above, with its prevalence doubling every five years (Ulaganathan and Pitchaimani, 2023).As a result, researchers worldwide have prioritized exploring aging mechanisms and interventions to target the aging process in the pursuit of AD treatments.
Currently, AD animal models can be categorized into three types: aging models, transgenic models, and models induced by AD-inducing agents (Bi et al., 2021).However, these animal models fail to fully replicate the pathophysiology observed in human patients, making it challenging to effectively evaluate the efficacy and mechanisms of AD drugs (Fig. 1).This limitation has led to the disappointment of potential AD drugs that initially showed promising therapeutic effects in early animal studies but failed in phase III clinical trials (Doody et al., 2014;Salloway et al., 2014).Similarly, in vitro models of AD are also challenging due to the complexity required by existing cell culture systems and the inaccessibility of brain tissue.Specifically, the extracellular deposition of amyloidogenic peptides cannot be fully mimicked in induced pluripotent stem cell (iPSC)-derived neurons grown as a monolayer (Xu et al., 2023).Consequently, traditional animal and cell culture models fall short in completely reflecting the aging-associated physiological and pathological states of humans (Cenini et al., 2021) (Fig. 1).
Unlocking the mysteries of the human brain and aging has long been an elusive quest for AD researchers and scientists alike.However, a promising avenue for research lies in 3D brain organoid (BO) systems generated from human pluripotent stem cells (hPSCs).BOs are 3D stem cell-derived tissues that mirror the intricate cellular composition and structural complexity of the human brain itself (Lee et al., 2017).Intriguingly, these BOs replicate the key hallmarks of AD pathophysiology, boasting amyloid plaques and neurofibrillary tangles, offering an innovative platform to scrutinize and gauge the effectiveness of potential pharmacological agents in tackling the relentless progression of AD (Kang and Cho, 2021) (Fig. 2).Previous attempts to model human organ biology, such as the differentiation of human stem cells in 2D, with or without a 3D matrix, bio printing of human cells, and the use of microfluidic devices ("organ-on-a-chip"), have shown some potential for drug screening and human disease research (Jorfi et al., 2018).However, these methods fell short in many aspects.Venturing further into the depths of innovation, current research encounters the awe-inspiring patient-derived organoids (PDOs) -exquisite 3D tissue cultures shaped from stem cells (Cordella et al., 2022).These PDOs mirror organ-like structures with a combination of diverse cell types, orchestrated into a symphony of biological harmony.A groundbreaking feat, these PDOs allow us to delve into the intricate workings of human physiology and pathology, enabling studies that would be difficult to conduct in human subjects (Fig. 1).Therefore, to a certain extent, the organoid system holds promise as a more dependable and comprehensive aging research model (Box 1).The quest to unravel the mysteries of AD demands a holistic understanding of the aging process.Brain organoids, although valuable, may not be the ultimate solution due to their inherent limitations in replicating the complexity of aging-related changes and the disease's multifaceted nature.Nonetheless, they serve as a stepping stone in our journey toward unraveling the enigma of AD, inspiring researchers to further explore and develop advanced models that can better represent the intricacies of the aging brain in health and disease.
In this review, we reverberate the profound significance of aging BOs as model systems, where the mechanisms of aging and aging-related AD unfold for AD researchers in both mechanistic and translational research by pointing out key challenges that need to be addressed.With continuous scientific advancements, we are also closer to deciphering the secrets behind aging-related AD, ultimately paving the way for potential therapeutic interventions to alleviate its burden on aging populations worldwide.

Tackling age-related challenges: implications for AD brain organoid research
Aging, the natural process of becoming older, plays a pivotal role in the development of AD, particularly in the case of sporadic Alzheimer's disease (SAD) (Cataldo et al., 2000).Throughout the aging process, a multitude of genetic alterations occur, leading to significant changes in the overall cellular transcriptional profile (Zahn et al., 2007).To better understand AD, researchers have turned their attention to brain organoids (BOs) as a potential modeling tool.However, as potent as they are, brain organoids have limitations in accurately representing the complex pathology of AD and aging (Fig. 3).Brain organoids are derived mainly from early-stage embryonic-like cells, which may not be ideal for studying late-onset AD, the most prevalent form of the disease.Understanding late-onset AD is crucial because its manifestation is strongly associated with aging.In addition, brain organoids fall short in fully replicating the intricate and dynamic environment of the human brain.
Aging is an intricate process influenced by genetic, epigenetic, and environmental factors (D'Aquila et al., 2013;Khan et al., 2017).The challenge lies in replicating this entire process within the simplified structure of brain organoids compared with the complex human brain.Although they may display some features of aging (Box 2), they might not encompass the entirety of age-related changes, especially given the multifactorial nature of AD that involves multiple cell types and intricate Traditional cell-based models, while providing insights into AD pathogenesis, fall short in capturing the complete clinical spectrum of AD pathology.Likewise, animal models, although informative, display disparities in mimicking human-specific AD phenotypes, both physiologically and pathologically.The reproducibility of data is another issue that plagues these models.However, the emergence of the hPSC-based AD model, particularly the 3D culture conditions of AD brain organoids (AD-BO), holds promise to overcome these challenges.
M.K. Hossain et al. cellular interactions.Moreover, the cells present in brain organoids often remain in an immature state, leading to differences in gene expression and functionality compared to adult brain cells (Quadrato et al., 2016a).This immaturity can influence disease-related pathways and may not fully capture the aging-related changes observed in the brains of AD patients.Another critical aspect of AD pathology is the disruption of cerebral vasculature (Bell and Zlokovic, 2009) and the integrity of the bloodbrain barrier (Erickson and Banks, 2013).Unfortunately, brain organoids lack a complete vascular network, which hampers their ability to accurately model the vascular aspects of AD and its connection to aging-related changes.
In AD, a rich cast of characteristics beyond neurons takes center stage, including microglia and astrocytes-nonneuronal cells with pivotal roles in neuroinflammation and disease progression.However, brain organoids often lack these essential cell types or merely offer limited representation (Birbrair, 2021).This deficiency poses a formidable challenge in fully unraveling the intricate contributions of nonneuronal cells to the mysteries of AD pathogenesis and aging.
Regrettably, brain organoids are somewhat short-lived, as they can only be sustained for a brief period-typically a few months at best.Their shortened existence arises from the lack of a functional blood supply and the constraints of other physiological factors (Fuchs and Chen, 2013).AD, a slow-burning epic, thrives in chronicity, and the short experimental timeframe of brain organoids may falter in capturing the grandiose, long-term changes that manifest during aging in AD patients.The subtle spectacle of senescence, a hallmark of aging, can be challenging to observe in organoids, particularly given their abbreviated lifespan (Keshavarz et al., 2023).Adding to the complexity, the numerous performances of brain organoids exhibit considerable variability (Sivitilli et al., 2020;Velasco et al., 2019)-varying in size, shape, and cell composition across different laboratories and experiments.This disarray disrupts the harmony of reproducibility in research findings and complicates the cross-study comparison, leaving researchers in search of a standard protocol.The protocol of aging involves a precisely arranged interplay of gene expression and epigenetic modifications.However, in their mimicry, brain organoids may lose their gene expression patterns and epigenetic marks that are not quite aligned with those observed in the aging brain.
Furthermore, brain organoids lack exposure to the external environment (Sarieva and Mayer, 2021), including interactions with other organs, immune cells, and external stimuli, which can significantly orchestrate the symphony of brain aging and AD pathology.This scheduled existence could falter in replicating the intricate interplay between the brain and the rest of the body during aging.Ethical and technical challenges are a very important and crucial part of organoid research.The use of brain organoids derived from human stem cells raises ethical concerns related to the creation and study of human brain-like structures outside the body.While these organoids are not conscious entities, ethical considerations regarding their use for research must be carefully addressed.The fine line between exploration and responsibility must be carefully etched to honor the delicacy of this endeavor.Brain organoids, depicted in this figure, stand as a potential gateway to unravel the complexities of AD.These 3D cerebral constructs hold the potential to emulate aging characteristics and closely replicate the aging AD phenotype, facilitating comparative studies with normally functioning brains.Such organoids, when mimicking AD pathology, can serve as invaluable tools for enhancing predictive capabilities in preclinical trials.Moreover, brain organoids offer an expedient platform for efficient AD drug screening.

Bridging the gap: refining the BO system to induce an aging AD phenotype
Exploring the intricacies of the aging process can offer valuable insights into the advancement and evolution of AD.While brain organoids cannot fully match the intricate complexities of the human brain, they present an invaluable and ethically responsible platform to investigate disease mechanisms in a controlled environment.Unraveling the enigma of aging, with the aim of inducing aging phenotypes in AD brain organoids, demands a collaborative and multidisciplinary approach including genetics, epigenetics, cell biology, and neuroscience.In this study, we delved into crucial areas of research and examined scientific evidence that holds relevance in unlocking the secrets of aging to induce aging phenotypes in AD brain organoids, as depicted in Table 1 and Fig. 3.

Selection of iPSC source
The process of aging continues to stand as the predominant and prevalent risk element for AD conditions.Presently, cerebral organoids are cultivated employing either embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).While ESCs inherently denote youthful and undeveloped cellular entities, research indicates that iPSCs undergo revitalization during the reprogramming process (Grenier et al., 2020).Therefore, specified patient-derived iPSCs themselves might serve as valuable tools to study aging and neurodegenerative processes.Miller et al. employed progerin overexpression, a variant of lamin A linked to Hutchinson-Gilford progeria syndrome (HGPS), in iPSC-derived dopaminergic neurons to induce aging effects (Miller et al., 2013).This approach triggered aging-related traits such as neuromelanin buildup.Intriguingly, introducing progeria into iPSC-derived dopaminergic neurons from Parkinson's disease patients replicated disease characteristics such as Lewy-body precursor inclusions, enlarged mitochondria, tyrosine hydroxylase loss, and dendrite degeneration.This underscores the potential of using aging strategies with iPSCs to emulate neurodegenerative pathology and examine the entire aging spectrum.Meanwhile, Zhang et al. revealed that aged-cell-derived iPSCs manifest reduced expression of cell-specific glucose transporter 3 (GLUT3) compared to young cells, leading to compromised glycolysis and hindered oxidative phosphorylation regulation.Reactive oxygen species (ROS), apart from causing direct DNA damage, intensify age-related attenuation of the DNA damage response, hastening aging in rejuvenated cells (Zhang et al., 2017).Consequently, iPSCs from patients with progeroid syndromes offer a valuable avenue for studying aging processes.

Induction of Cellular Senescence
Cellular senescence stands as a prominent hallmark of aging, encompassing the decline in cellular division and functionality.Senescent cells substantially contribute to age-related maladies, including neurodegenerative conditions.The orchestration of cellular senescence entails multiple pathways, notably the activation of the p53 and p16INK4a pathways, culminating in cell cycle arrest and the secretion of proinflammatory agents, collectively known as the senescenceassociated secretory phenotype (SASP) (Chinta et al., 2018).Investigative findings have underscored escalated cellular senescence markers in aged organoids, contrasting their youthful counterparts.The induction of cellular senescence within brain organoids offers an avenue to scrutinize the repercussions of senescent cells on AD pathology and potential interventions.Several approaches, as depicted in Table 1, have been connected to provoke cellular senescence in brain organoids.A study revealed that exposing brain organoids to ionizing radiation resulted in heightened senescence markers, augmented SASP factors, and diminished cell proliferation, mirroring age-associated alterations (Grenier et al., 2020;Oyefeso et al., 2023;Schielke et al.).Other study also attributed brain organoids to oxidative stress through ROS-inducing agents, prompting elevated senescence-related genes and the excretion of proinflammatory cytokines (Nzou et al., 2020;Oyefeso et al., 2021), which is similar to senescent cell traits.

Epigenetic modification
The findings of two studies by De Jager et al. and Lunnon et al. indicated a connection between AD and alterations in DNA methylation.In particular, a distinct methylated area within the ankyrin 1 (ANK1) gene was identified as being linked to AD neuropathology (De Jager et al., 2014;Lunnon et al., 2014).Additionally, a more recent report by Zhao et al. established a correlation between AD and 5-hydroxymethylcytosine (5hmC) modifications (Zhao et al., 2017).These studies

Box 1
Brain Organoids as a Window into AD Model.
Researchers have explored various methods to study AD using 3D neuronal cultures.In 2013, Lancaster et al. conducted the pioneering work of utilizing human induced pluripotent stem cells (hiPSCs) to differentiate whole-cerebral organoids (Lancaster et al., 2013).Subsequently, Dang et al. reported that 30-day-old human cerebral organoids exhibited gene expression patterns similar to those found in the fetal brain during 8-9 weeks of pregnancy (Dang et al., 2016).Since then, various protocols have been developed to generate brain organoids (BOs) with distinct characteristics, representing different regions of the human brain, including the cortex, basal ganglia, hippocampus, choroid plexus, thalamus, retina, striatum, hypothalamus, midbrain, cerebellum, and human spinal cord.Both whole-brain and forebrain organoids have proven valuable for modeling AD (Esmail and Danter, 2021).Advancements in culturing techniques have enabled the generation of BOs that accurately represent specific brain regions affected by AD, such as the hippocampus and cerebral cortex.By using iPSCs derived from AD patients or those carrying relevant genetic mutations, researchers have successfully created organoids that mimic the pathological features observed in the brains of affected individuals (Barak et al., 2022;Chang et al., 2020).These organoids spontaneously develop AD-associated pathologies, including amyloid aggregation, hyperphosphorylation of Tau proteins (pTau), endosome abnormalities, and cellular apoptosis, features that are challenging to reproduce in traditional 2D culture models.Vazin et al. and Pavoni et al. demonstrated that neural cells derived from AD-free hiPSCs could be induced to develop AD phenotypes through chemical induction with Aβ-42 oligomers or Aβ-42 inducers, such as aftin5, albeit with some neuronal cytotoxicity (Pavoni et al., 2018;Vazin et al., 2014).Lee et al. generated cortical organoids from AD patient-derived induced pluripotent stem cells (iPSCs), successfully creating β-amyloid plaques, which could be reduced by inhibiting β-secretase and γ-secretase (Lee et al., 2016).Amiri et al. used cerebral organoids to track brain development through RNA sequencing (Amiri et al., 2018).A group confirmed the formation of AD-related hallmarks in iPSC-derived brain organoids, which could be reversed using inhibitors (Raja et al., 2016).Other group revealed abnormal endosome morphology.iPSCs combined with CRISPR technology were utilized to study AD-related APOE alleles' effects.APOE4 increased gene expression in lipid metabolism, immune responses, and synapse formation, leading to altered cellular functions, including increased amyloid β42 secretion (Cataldo et al., 2000).collectively highlight the role of epigenetic changes in AD development (Box 3) and shed light on potential avenues for further research and therapeutic interventions.Manipulating the genetic composition of brain organoids to encompass aging-linked genes or mutations stands as a plausible avenue to induce aging phenotypes.An illustration lies in the introduction of genes integral to cellular senescence or the DNA damage response (Ponnappan and Ponnappan, 2011).Epigenetic modifications exert a pivotal influence on aging and AD.DNA methylation, histone modifications (Gräff et al., 2012;Zhang et al., 2012), and chromatin remodeling (Feser et al., 2010) are prominent epigenetic mechanisms orchestrating gene expression during aging.Brain organoids have revealed age-associated shifts in DNA methylation patterns, affirming exploration into the molecular basis of aging in AD (Ximerakis et al., 2019).Cheng et al. employed brain organoids derived from AD patient-derived induced pluripotent stem cells (iPSCs) to search for DNA methylation shifts linked to AD, unveiling specific alterations pertinent to disease pathogenesis (Cheng-Hathaway et al., 2018).A study by Yang et al. connected brain organoids to unravel the interconnection between histone acetylation and AD pathology (Yang et al., 2017).Their findings proposed a correlation between histone modifications and AD phenotypes, as curtailing a histone deacetylase enzyme mitigated AD-related pathologies.Furthermore, exploration of noncoding RNAs within brain organoids may shed light on their influence on aging and AD progression.Furthermore, brain organoids derived from AD iPSCs uncovered altered microRNA expression profiles associated with AD-related processes.

Mitochondrial dysfunction
Mitochondrial dysfunction is a hallmark of aging cells and is also implicated in neurodegenerative diseases such as AD.Brain organoids derived from aged individuals or genetically modified to replicate mitochondrial dysfunction can help researchers study the interplay between mitochondrial dysfunction and AD pathogenesis (Quadrato et al., 2016b).Creating a brain organoid model with mitochondrial dysfunction to study aging and AD phenotypes is a complex task that requires careful manipulation of various cellular and molecular factors.Here, we provide some general information on how this can be approached (Table 1).It is essential to acknowledge that the field of organoid research is continuously evolving, and specific protocols may vary based on the current state of scientific knowledge.Mitochondrial DNA is susceptible to mutations due to its proximity to reactive oxygen species (ROS) generated during oxidative phosphorylation.Researchers can engineer organoids with specific mtDNA mutations associated with mitochondrial dysfunction and aging, such as deletions or point mutations.For example, some studies have utilized CRISPR-Cas9 technology to introduce mtDNA mutations in organoids (Yang et al., 2019).In addition, mitochondrial quality control mechanisms play a crucial role in maintaining mitochondrial health (Coelho et al., 2022).Impairing these mechanisms can lead to mitochondrial dysfunction.One approach is to overexpress proteins that interfere with mitochondrial dynamics and mitophagy, such as dominant-negative forms of mitofusin 1 and 2 (Mfn1 and Mfn2) or Pink1 (Coelho et al., 2022).This could lead to the accumulation of damaged mitochondria and contribute to the aging phenotype.Moreover, mitochondrial dysfunction is often associated with increased oxidative stress, leading to the accumulation of ROS.Exposing brain organoids to oxidative stress-inducing agents such as hydrogen peroxide or paraquat can mimic the age-related oxidative damage observed in AD patients (Pamies et al., 2022).Modulating the expression of key regulators of mitochondrial biogenesis and metabolism can also lead to mitochondrial dysfunction in brain organoids.For example, researchers could downregulate peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a master regulator of mitochondrial biogenesis, to impair mitochondrial function (Sun et al., 2016).

Protein aggregation and neuroinflammation
Protein aggregation, including amyloid-beta and tau protein, is a hallmark of AD.Aging is associated with increased protein misfolding and aggregation.Brain organoids can be engineered to overexpress amyloid-beta and tau to study their impact on neuroinflammation and AD-related pathology (Gerakis and Hetz, 2019).A study published in Stem Cell Reports in 2017 treated brain organoids with preformed Aβ aggregates, resulting in the development of AD-like pathologies, including tau hyperphosphorylation and synaptic dysfunction (Krencik et al., 2017).Researchers can also introduce amyloid-beta or tau aggregates into brain organoids to induce oxidative damage and study the effects of these pathological proteins on cellular function (Grenier et al., 2020).

Vascular dysfunction
Aging is linked to vascular changes and reduced blood flow to the brain, which can contribute to AD pathogenesis.By integrating vascular components into brain organoids, researchers can investigate the role of vascular dysfunction in AD development and test potential interventions (Cakir et al., 2019).Studying vascular dysfunction in brain organoids to develop an aging AD phenotype is a challenging but promising approach for modeling AD in the lab.To create brain organoids, researchers typically follow established protocols involving the differentiation of human pluripotent stem cells (hPSCs) into neural stem cells (NSCs).These NSCs then self-organize into structures resembling the brain's early development (Lancaster and Knoblich, 2014a, b).Researchers can either co differentiate endothelial cells (ECs) with neural stem cells during organoid formation or vascularize existing organoids by exposing them to endothelial cells or angiogenic factors (Pham et al., 2018).To induce vascular dysfunction, the vascular system can be manipulated within organoids, mimicking AD-associated changes in bloodbrain barrier (BBB) integrity, cerebral blood flow, and vessel abnormalities (Qian et al., 2016).Various techniques can be employed to assess vascular dysfunction and AD-related phenotypes in brain organoids, such as immunofluorescence staining, gene expression analysis, ELISAs, and functional assays to evaluate BBB integrity and neuronal function (Vatine et al., 2019) Box 2 Brain Organoids with certain AD-related features.
Brain organoids carrying mutated apolipoprotein E gene (APOE) also exhibit AD-associated phenotypes (Lin et al., 2018), such as increased α-synuclein, impaired synaptic functions, decreased glucocerebrosidase levels, and accumulated lipid droplets (Sun et al., 2023).Lentiviral transduction or CRISPR-Cas9-mediated genomic editing are employed as strategies to induce overexpression or expression of mutant APP, PS1, PS2, and APOE4 proteins in healthy hiPSCs, allowing researchers to capture the AD phenotype.While the majority of hiPSC-based AD models employ two-dimensional (2D) or embryoid body (EB) differentiation methodologies to generate various types of neurons, including forebrain, cortical glutamatergic, GABAergic, and cholinergic neurons, these models have limitations in reproducing all aspects of AD pathophysiology.However, they do show certain AD-related features, such as intracellular accumulation of soluble Aβ species, aggregation of insoluble Aβ species, and tau hyperphosphorylation (Papaspyropoulos et al., 2020).Choi et al. achieved the first successful attempt to model AD using organoids.They genetically manipulated human neuronal progenitor cells to overexpress mutant PS1 and APP, leading to the simultaneous presence of β-amyloid-and tau-related features in a single 3D model system.These 3D structures carrying familial AD (FAD) mutations exhibited increased detergent-resistant accumulations of phosphorylated tau and filamentous tau (Choi et al., 2014).More recently, a sophisticated AD cerebral organoid model was generated from FAD patients.The brain organoids displayed progressive accumulation of amyloidogenic Aβ peptides, accompanied by the development of structures strongly resembling amyloid plaques and neurofibrillary tangles (NFTs) (Gonzalez et al., 2018).Moreover, a novel 3D human tri-culture model consisting of neurons, astrocytes, and microglia was developed to model AD using microfluidics (Fig. 2).This model exhibited critical features of AD pathology, such as β-amyloid aggregation, tau hyperphosphorylation, neuroinflammatory activity, microglial recruitment, axonal cleavage resulting from neurotoxic activities, and release of nitric oxide (NO) with deleterious effects on AD neurons and astrocytes (Kadlecova et al., 2023).Cocultures of APOE4 microglia with patient-derived iPSC organoids showed impaired phagocytosis and increased TREM2 levels(ElAli and Rivest, 2016).Park et al. introduced microglia into 3D co cultures to investigate neuroinflammation in AD.They identified chemokines attracting AD-associated microglia and pro-inflammatory cytokines including Il-6, Il-8, TNF-α, MIF, and PAI-1.Moreover, Microglial migration and pro-inflammatory actions were dependent on TLR-4 and IFN-γ, demonstrating the potential of 3D cultures to study intricate cellular interactions in AD context (Park et al., 2018).In gist, the utilization of brain organoids derived from hiPSCs has provided valuable insights into the complex pathophysiology of AD and offers a promising platform for drug discovery and development, as well as personalized medicine approaches for AD patients.

Table 1
Some strategies and approach with possible outcome and some challenges to induce aging phenotype in AD using brain organoids.

Strategies Approaches Possible Outcome Challenges/Limitations
Selection of iPSCs source: • Using SAD patients-derived iPSCs

Extended culture time
Allowing brain organoids to grow and develop for an extended period might lead to some age-related changes.However, it is essential to carefully control the culture conditions, as prolonged culture can also result in other unintended consequences or cellular degeneration.Nevertheless, some studies and approaches have been proposed to improve the longevity and maturity of brain organoids, including those related to AD research.A study published in Nature Neuroscience in 2018 by Ayata et al. attempted to mimic how microglia clear dying cells and debris in the brain.They found that in the adult brain, this microglial cleanup is regionally controlled and relies on the rate of neuronal loss in the culturing environment (Ayata et al., 2018).Several researchers also found that long-term culturing of brain organoids resulted in age-related transcriptional changes, displaying gene expression patterns reminiscent of aging in the human brain.Extending the culture time of brain organoids allows them to undergo more extensive developmental processes, leading to increased cellular maturation and the appearance of age-related features.While standard brain organoid protocols usually last for a few weeks (approximately 2-3 months), an extended culture can be up to 9-12 months or more (Lancaster and Knoblich, 2014a, b).However, they also noted that these changes were not entirely identical to the aging human brain, indicating the challenges in fully inducing an aging phenotype.One approach to extend the culture time of brain organoids involves using 3D bioprinting techniques to create more complex and mature structures.A study published in Nature Biotechnology in 2020 demonstrated the use of a 3D bioprinting technique to generate brain organoids with improved cellular organization and increased longevity compared to traditional methods (Fig. 3).This technique allowed for better nutrient and oxygen diffusion throughout the organoid, leading to enhanced growth and functional maturation (Skylar-Scott et al., 2020).Several studies have investigated the use of various factors and compounds to enhance the maturation of brain organoids and prolong their culture time.For example, the application of small molecules, such as 3D2 and CHIR99021, significantly increased the maturity and survival of human brain organoids, making them more suitable for long-term studies (Zhang et al., 2023).The formation of vascular-like structures and improving oxygen supply within brain organoids has been suggested as another strategy to enhance their longevity.A study published in Cell Stem Cell in 2020 demonstrated that implanting human brain organoids into the brains of live mice allowed for the development of blood vessels within the organoids, improving nutrient delivery and extending their culture time (Mansour et al., 2018;Mansour et al., 2020).Using bioreactors, which are specialized devices designed to provide controlled and dynamic culture conditions, is another potential approach for extending the culture time of brain organoids.Bioreactors can improve nutrient exchange and waste removal, enabling organoids to survive and mature for longer periods.While this approach is promising, specific research references may vary, as it is a developing field with ongoing studies.

Coculture with other cell types
One important aspect of brain organoids is their ability to model different cell types found in the brain.However, in their basic form, brain organoids may lack certain components that are critical for • Difficulty in Long-Term Monitoring

Stimulation of Immune-Driven Brain Aging
• By introducing aged monocytes into the human cortical organoids cultivated within the microphysiological analysis platform (MAP) • Promoted the expression of agingrelated markers, such as elevated expression of p16  Their study utilized CRISPR/Cas9 to create a mouse model of AD with one copy of the single nucleotide polymorphism (SNP) encoding the R47H variant in murine Trem2.Another study explored chromatin accessibility shifts during neuronal differentiation, extracting dynamic alterations appropriate to aging and neurodegeneration (Yeo, 2020).Recent advances in epigenetic editing technologies, exemplified by CRISPR-based systems, empower researchers to target specific epigenetic marks, prompting or reversing aging-related changes in brain organoids.While the realm of epigenetic editing remains nascent, its application in diverse cell types hints at potential to rectify disease-associated epigenetic shifts.Notably, a study from (Raja et al., 2016) spotlighted the successful generation of brain organoids derived from patients bearing familial AD mutations within the APP gene.In this research organoids from people with familial Alzheimer's disease (fAD) carrying certain genetic mutations, such as amyloid precursor protein (APP) duplication or presenilin1 (PSEN1) mutation, these issues are seen more as individuals age, compared to control groups.Furthermore, these organoids showed elevated Aβ production and mirrored prototypical AD features, furnishing profound insights into the molecular mechanics underpinning the disease.Overall, these methods and scientific evidence showcase the potential of brain organoids as a valuable tool to study epigenetic changes associated with aging and AD phenotypes.However, it is important to note that the field of epigenetics and organoid research is rapidly evolving, and ongoing research is required to fully comprehend the complexities of AD development and aging in brain organoids.
M.K. Hossain et al. modeling age-related pathologies observed in the human brain.Coculturing brain organoids with other cell types (Box 4), such as immune cells or astrocytes, glial cells, microglia, or vascular cells, can provide a more representative and disease-relevant model to study AD phenotypes and aging processes.This technique is used to mimic the complex cellular interactions that occur in the brain and can be particularly relevant for studying aging and neurodegenerative diseases such as AD.Through a meticulous examination of the temporal progression, Quadrato et al. unveiled a progressive amplification of cellular heterogeneity over the course of the culture period.Employing a comprehensive approach involving single-cell RNA analysis, an expansive dataset encompassing over 80,000 cells derived from 31 distinct organoids was meticulously scrutinized, exhibiting heightened expression of mesenchymal markers.Remarkably, the investigation illuminated a remarkably diverse reservoir of cell precursor subtypes within these organoids, notably encompassing clusters exhibiting heightened expression of mesenchymal markers.Interestingly, specific neural cell varieties, such as photoreceptors and astrocytes, only manifested after an incubation period surpassing 6 months (Quadrato et al., 2017).

Oxidative stress induction
Applying various cellular stressors, such as oxidative stress, endoplasmic reticulum stress, or proteotoxic stress, can lead to the induction of an aging-like phenotype in brain organoids (Rocha et al., 2021).Exposing brain organoids to oxidative stress conditions can mimic some aspects of aging.A study published in Cell Stem Cell in 2017 exposed brain organoids to oxidative stress by treating them with hydrogen peroxide.This treatment resulted in increased cell death and accelerated aging-like changes in the organoids, highlighting the role of oxidative stress in brain aging (Khan et al., 2016;Li et al., 2017).Metals such as iron and copper can catalyze the production of ROS through Fenton and Haber-Weiss reactions, leading to oxidative stress.Adding metal ions to brain organoid cultures can mimic the accumulation of metals observed in AD brains and contribute to oxidative damage (Zhang et al., 2020).Researchers can manipulate mitochondrial function in brain organoids by using specific inhibitors or genetic modifications targeting mitochondrial proteins (Yang et al., 2020).However, these approaches require careful regulation to avoid excessive damage to the organoid.

Environmental manipulation
Altering the culture environment by adjusting nutrient levels, growth factors, or other factors relevant to aging may influence organoid development.Exposing brain organoids to intermittent hypoxia, which mimics the age-related decrease in oxygen supply, has been shown to promote aging-like features.This approach involves reducing oxygen levels temporarily during the culture process (Renner et al., 2017;Yu et al., 2014).In addition, mimicking the physical and mechanical properties of aged tissues in the culture environment might also play a role in inducing aging phenotypes.Biomechanical stress, such as mechanical stretching or compression, can influence cellular behavior and tissue development in brain organoids (Cakir et al., 2019).For example, researchers used cerebral organoids derived from induced pluripotent stem cells (iPSCs) with trisomy 21 (Down's syndrome, a chromosomal abnormality associated with neurodevelopmental disorders) (Fertan et al., 2023).They observed that Chromosome 21 trisomy increases the monomeric Aβ concentration released by the organoids, which might be linked to biomechanical alterations in AD-like brain tissue.The three-dimensional (3D) microenvironment and ECM stiffness play crucial roles in brain organoid development and cellular behavior.Altering the stiffness of the ECM can affect cell adhesion, migration, and differentiation, which are processes relevant to AD pathogenesis.Sun, Y. et al. highlighted the importance of ECM stiffness in regulating cellular plasticity in tumor cells (Wang et al., 2014).Applying similar principles to brain organoids could potentially influence their maturation and phenotype.A study conducted by Stacpoole, S. R. et al. focused on CNS precursors and highlighted the importance of oxygen tension in neural cell development (Stacpoole et al., 2011).Modulating oxygen levels in brain organoids may influence their aging process and AD-like features.However, it is important to note that while these studies provide insights into how physical and mechanical cues can influence brain organoids, specific research directly focusing on inducing an aging AD phenotype using these cues might be limited.

Stimulation of immune-driven brain aging
Recent research has unveiled a groundbreaking development in the study of immune-driven brain aging.A team of scientists successfully established a human brain organoid microphysiological analysis platform (MAP) to gain insights into the dynamic process of this intriguing phenomenon.The results of this groundbreaking study demonstrated the influential role of aged monocytes in driving brain aging.By introducing aged monocytes into the human cortical organoids cultivated within the MAP, the researchers observed a notable increase in infiltration.This infiltration, in turn, promoted the expression of agingrelated markers, most notably elevated expression of p16 within the organoids.The heightened expression of p16, a known marker of

Box 4
Non neuronal cell interaction implicated in promoting AD features.
Reactive astrogliosis, a phenomenon observed by Alois Alzheimer himself, is a recognized phenotype of AD.However, its potential benefits or harms remain unanswered.In AD, astrocytes undergo alterations in glutamatergic and GABAergic signaling, potassium buffering, and various signaling pathways such as cholinergic, purinergic, and calcium signaling (Osborn et al., 2016).Furthermore, a specific subtype of reactive astrocytes, prompted by activated neuroinflammatory microglia, transitions from their normal functions to acquiring neurotoxic capabilities.This subtype is prevalent in various human neurodegenerative disorders, including AD (Liddelow and Barres, 2017).Several researchers explored the potential of using aged astrocytes to induce aging-like features in brain organoids.They found that incorporating aged astrocytes improved the maturation of organoids and promoted the formation of neural networks with age-associated characteristics (Nascimento et al., 2019;Quadrato et al., 2017;Renner et al., 2017).However, this method still does not fully replicate the complexity of aging.Another study that exemplifies the use of cocultures with other cells in brain organoids to develop aging AD phenotypes is the work by Abud et al. (Abud et al., 2017).In this study, researchers successfully differentiated human microglial-like cells (iMGLs) from induced pluripotent stem cells (iPSCs), enabling the investigation of their role in Alzheimer's disease (AD).The iMGLs exhibited development patterns similar to microglia in vivo, demonstrated high transcriptomic similarity to cultured adult and fetal human microglia, and functionally responded to inflammatory stimuli in brain organoids.Moreover, transplanted iMGLs in transgenic mice and human brain organoids closely resembled in vivo microglia.The study also highlighted that iMGLs cultured in 3D brain cultures actively migrated, tiled, and encompassed the volume of the BO, extending processes reminiscent of early microglia development.Furthermore, they observed that the presence of microglia accelerated the neurodegenerative process in the organoids, providing crucial insights into the contribution of glial cells to AD pathology.cellular senescence, provides valuable evidence that aged monocytes may actively contribute to the aging process in the brain.The findings of this study pave the way for further research into the specific mechanisms underlying immune-driven brain aging.Understanding the role of aged monocytes in this process opens new avenues for therapeutic strategies that target age-related neurological disorders (Ao et al., 2022).

Concluding remarks and future directions
In conclusion, the creation of AD brain organoids with specific aging phenotypes represents a challenging (See Outstanding Questions) and evolving area of research that necessitates a deep understanding of the underlying biology and precise experimental techniques.Although brain organoids have revolutionized the study of neurological diseases such as Alzheimer's disease, achieving a complete aging phenotype remains a formidable task.Despite their limitations in fully recapitulating the complexity of an aging human brain, brain organoids offer valuable insights into certain aspects of AD research, including fundamental cellular mechanisms, early disease events, and potential therapeutic interventions.However, it is crucial to acknowledge that brain organoids should be utilized in conjunction with other complementary approaches, such as animal models and postmortem human brain analyses, to gain a comprehensive understanding of aging-associated AD.
Unlocking the secrets of aging to induce aging phenotypes in AD brain organoids necessitates a multidisciplinary approach encompassing genetics, epigenetics, cell biology, and neuroscience.As the field of brain organoid research continues to advance, researchers are tirelessly working to address the limitations of these models and improve their relevance in modeling AD and aging phenotypes.Efforts include refining organoid protocols, incorporating additional cell types, introducing vascular components and developing co-culture systems.However, researchers should exercise caution in interpreting the observed phenotypes in brain organoids and rigorously validate their findings using alternative approaches, such as immunohistochemical and transcriptomic analysis of primary human brain tissue and in vivo experiments.Additionally, ethical considerations must be taken into account when conducting extended culture experiments on human brain organoids.However, brain organoid models of aging are still in the early stages of development, and further research is indispensable to refine and validate these protocols for accurately studying aging-related phenotypes.Despite the challenges, the knowledge gained from our studies holds promise in elucidating the molecular mechanisms underlying AD and potentially guiding approaches to induce aging AD phenotypes, thus paving the way for combatting this devastating disease more effectively in the future.As the field progresses, advancements in organoid technology and a deeper understanding of aging processes will undoubtedly contribute to the continuous improvement of brain organoid models and their application in AD research.Through collaborative efforts, the integration of multiple lines of evidence will offer a more holistic understanding of age-associated AD.This endeavor aspires to accelerate progress aimed at achieving this pivotal objective while also bringing us closer to the creation of innovative treatments and interventions against AD.

Outstanding Questions
1. What molecular and analytic tools could be employed to achieve the refinement of 3D brain organoid model to more accurately represent the aging-related events observed in AD brains? 2. How can interdisciplinary collaboration between neuroscientists, stem cell biologists, bioengineers, and computational biologists help overcome the challenges and limitations of using 3D brain organoid models to study agingrelated Alzheimer's disease? 3. Considering the potential of 3D brain organoid models to revolutionize AD research, what are the key milestones that need to be achieved in the next decade to fully capitalize on this approach's promise?
(continued on next column) (continued ) 4. To what extent can brain organoids accurately model the intricate pathology of late-onset Alzheimer's disease, considering their derivation from early-stage embryonic-like cells? 5. What factors contribute to the immaturity of cells in brain organoids, and how might this immaturity affect the study of disease-related pathways and agingrelated changes?6.How does the lack of representation or limited presence of essential nonneuronal cells like microglia and astrocytes in brain organoids affect our ability to comprehend the role of these cells in Alzheimer's disease pathogenesis and aging?7. How accurately do brain organoids replicate gene expression patterns and epigenetic marks observed in the aging brain, and what are the potential consequences of any misalignment?8. How can the selection of iPSCs from patients with progeroid syndromes be optimized to better emulate aging-related changes in AD brain organoids?9. How can brain organoids be effectively validated and complemented with other experimental approaches, such as animal models and postmortem human brain analyses, to ensure the reliability of the observed aging-related phenotypes?

Fig.- 1 .
Fig.-1.Comparative Analysis of Animal, Cell, and hPSC-Based Models for Alzheimer's Disease (AD) Research.Traditional cell-based models, while providing insights into AD pathogenesis, fall short in capturing the complete clinical spectrum of AD pathology.Likewise, animal models, although informative, display disparities in mimicking human-specific AD phenotypes, both physiologically and pathologically.The reproducibility of data is another issue that plagues these models.However, the emergence of the hPSC-based AD model, particularly the 3D culture conditions of AD brain organoids (AD-BO), holds promise to overcome these challenges.

Fig.- 2 .
Fig.-2.Harnessing Brain Organoids to Illuminate Alzheimer's Disease Research.Brain organoids, depicted in this figure, stand as a potential gateway to unravel the complexities of AD.These 3D cerebral constructs hold the potential to emulate aging characteristics and closely replicate the aging AD phenotype, facilitating comparative studies with normally functioning brains.Such organoids, when mimicking AD pathology, can serve as invaluable tools for enhancing predictive capabilities in preclinical trials.Moreover, brain organoids offer an expedient platform for efficient AD drug screening.

Fig. 3 .
Fig. 3. Strategies and hurdles in inducing aging phenotypes using iPSCs for AD research.The prospective avenues and challenges associated with generating aging phenotypes using induced pluripotent stem cells (iPSCs) for AD investigation.The multifaceted approaches include the following: A. Choosing iPSCs derived from pertinent sources for accurate modeling.B. Extending the culture duration to induce cellular longevity and mimic aging.C. Employing agents that promote telomere shortening and DNA damage to induce cellular senescence.D. Utilizing DNA methylation and histone modifications to replicate age-related changes.E.Inducing oxidative damage through mitochondrial dysfunction.F. Mimicking the loss of BBB integrity, a hallmark of aging, to replicate an aged brain microenvironment.G. Replicating cellular interactions by introducing diverse cell types in coculture setups.H. Promoting ROS production to mimic aging-related oxidative stress.I. Introducing factors such as intermittent hypoxia and nutrient deprivation in 3D culture media to induce cellular stress.and J. Incorporating aged monocytes to elicit expression of aging-related markers and neuroinflammation.Despite these promising strategies, this figure also acknowledges the formidable challenges posed by each approach.It is through a meticulous balance of innovative techniques and rigorous experimentation that researchers aspire to unravel the intricacies of aging in the context of AD using iPSCs.
systems as a prominent tools implicated in AD research.The malleability of DNA accessibility to transcription factors and regulatory elements pivots on chromatin structure.Evaluation of chromatin accessibility in brain organoids extends insights into the influence of epigenetic shifts on aging and AD traits.Research conducted by Cheng-Hathaway et al. proposes that the R47H variant of the Triggering Receptor Expressed on Myeloid cells 2 (TREM2 R47H) increases the risk of Alzheimer's disease (AD) by causing a loss of TREM2 function and worsening neuritic dystrophy around plaques (Cheng-Hathaway et al., 2018).