Alzheimer’s disease (AD) is a multifactorial disorder without a cure. In 2021, the yearly cost of AD was anticipated to reach $355 billion, with a steep rising curve [1]. An estimated 6.2 million adults over 65 years of age are now living with this devastating disease, with a projected 2-fold increase in patient number by 2050 [1]. These numbers ardently necessitate the development of therapeutics that could halt disease progression. A commensurate amount of effort is being employed in this field. Novel underlying AD mechanisms have been unearthed, forcing researchers to question the role of amyloid-β (Aβ) and phosphorylated tau (pTau) protein as major culprits in AD pathogenesis ^[2][3][4][5][2,3,4,5]. Controversial ^[6][7][6,7] results of clinical trials on reducing Aβ ^[8][9][8,9] or pTau levels ^[3][4][5][3,4,5] refocused AD drug development. Increasing importance is now being given to inflammation, synaptic dysfunction, altered metabolism, neurogenesis, and epigenetics [10]. In fact, 126 clinical trials in 2021 targeted mechanisms other than Aβ and pTau [11]. Multiple new and repurposed compounds focused on early disease mechanisms are currently in preclinical trials. The availability of models that accurately recapitulate disease phenotypes in a patient-specific manner could play a critical role in successful translation of experimental approaches to human trials. Historically, animal models were extensively used in AD research to test preclinical candidate compounds. AD phenotypes in these models were mainly recapitulated by the overexpression or ablation of genes of interest [12]. Particularly, transgenic mouse models with the overexpression of human mutant amyloid precursor protein (APP) or presenilin 1 (PSEN1) protein, both involved in Aβ production [13], were used the most [12]. However, these genetic mutations are found only in rare familial variants of AD (FAD). The most prevalent form of AD, sporadic late-onset AD (LOAD) [14], is associated with multiple risk factors, including age, female sex, and the presence of the apolipoprotein E4 (apoE4) allele, which makes modeling of LOAD in mice very difficult. While new National Institutes of Health (NIH) programs (e.g., MODEL-AD [15]) were initiated to develop animal models of LOAD, there is a necessity for complementary models that recapitulate LOAD phenotypes for screening of new compounds at an early stage of the drug development pipeline.

The recent emergence of stem cell technology power orovides an outstanding opportunity to address this need. The 2012 Nobel Prize–winning work of Shinya Yamanaka, MD, PhD, showed that human fibroblasts can be reprogrammed to become pluripotent [16]. Though embryonic stem cells have been studied for 2 decades [17], the use of human induced pluripotent stem cells (HiPSCs) lies in not only removes source restrictions but also mitigates ethical concerns [18]. While modeling AD, fibroblasts can be easily obtained from patients by minimally invasive superficial skin biopsy. HiPSCs are generated from their aese fibroblasts by overexpressing pluripotency factors using cell reprogramming [16]. HiPSCs are then directed by appropriate growth factors [19,20], small molecule cocktail [21,22], or lentiviral overexpression of transcription factors [23,24] to differentiate into various types to rof neurons, astrocytes, or microglia [25]. Alternatain uniely, just like in animal models, known genes involved in FAD could be overexpressed in these HiPSCs using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein 9 (CRISPR/CAS9). Cells obtained from differentiating these modified HiPSC lines have FAD-associated mutations. These cells with AD traits, either directly from the patient or by forced expression, are then cultured in different setups that span from simple 2-dimensional (2D) cultures to organoid and complex microfluidic device–based cocultures.

The power of HiPSCs lies in their ability to retain unique genetic traits of an individual. This technology also can capture the acquired epigenetic modifications associated with environmental exposure. These advantages make HiPSC technology exceptionally suited to study the mechanisms of LOAD and to establish patient-specific therapeutic development platforms. Fibroblasts collected from individuals using skin biopsy are converted into HiPSCs by an overexpression of 4 transcription factors (octamer-binding transcription factor 4, Krüppel-like factor 4, SRY-box transcription factor 2, and c-myelocytomatosis oncogene product) [16]. HiPSCs can be almost indefinitely expanded and passaged to inexpensively obtain large numbers of cells. Multiple methods have been developed to differentiate mature and functional neurons ^[17][26], astrocytes ^[18][27], and microglia ^[19][28] from these HiPSCs. Initially, these differentiation protocols were formulated by mimicking embryonic development. Dual SMAD (an acronym from the fusion of the Caenorhabditis elegans and Drosophila genes, Sma and Mad [mothers against decapentaplegic]) inhibition is one of the most well-established protocols. SMAD is a group of proteins that are crucial for cell development and growth. By systematically inhibiting unwanted germ layer (mesendoderm and trophectoderm) growth using proteins [20] and/or small molecule cocktail ^[21][29], the HiPSCs are forced to assume neuroectodermal fate. Tailored growth factor combination is then used to differentiate these precursor cells into selective subtypes of neurons (e.g., glutamatergic or dopaminergic) and glial cells. These methods are especially useful where brain development is under scrutiny.

Brain-like organoids were also developed using this technique ^[22][30]. Though constantly improved by ongoing studies, this method does not yield a pure population of cell types and requires the use of advanced purification systems, such as fluorescence-activated cell sorting (FACS). This makes analysis of specific cell type attributes difficult. Multiple transcription factor or micro-RNA–based methods have also been developed to yield a pure population of desired brain cells (e.g., cortical neurons [23] or astrocytes [24]) from HiPSCs. Tetracycline-inducible expression of transcription factors can be used to rapidly stimulate neuronal [23] or astrocytic [24] differentiation in a controlled manner. Antibiotic purification using puromycin, hygromycin, or blasticidin yields a pure population of desired cell types. Striatal medium spiny neurons have been differentiated from fibroblasts using a similar method ^[25][31]. These striatal cells, when differentiated from cells from patients with AD, are reported to retain host genetic traits and express AD-like phenotypes. Alternately, clustered regularly interspaced short palindromic repeats CRISPR associated protein 9 (CRISPR/CAS9)/CAS9 ^[22][30] can be used to introduce AD-related mutations in HiPSCs. Cells differentiated from these modified HiPSCs could also model AD in vitro.

These cells with AD-related traits are then cultured separately or together in different setups, ranging from simple 2D to complex 3-dimensional (3D) cultures and brain organoids. Enhanced brain mimicry and freedom over mechanical cues in these cultures allow a closer look into mechanisms of AD in systems with human origin. Instead of generalizing an AD phenotype for all patients, these approaches aim to understand AD in a patient-specific manner, contributing to individualized medicine approaches. In general, these in vitro techniques to study AD could be divided in 3 categories: (1) HiPSC-derived 2D models, where monolayer cells are cultured on a flat surface; (2) HiPSC-derived organoid models, where cell (HiPSC) aggregates are differentiated in a controlled environment to yield 3D constructs comprising multiple cell types; and (3) engineered 3D models, where differentiated brain cells are engineered by mechanical cues to create 3D constructs with predefined attributes. Herein, we describe the advantages and limitations of these models.

AD is marked by initial episodic memory loss and gradual severe cognitive decline ^[26][32]. Historically, AD progression and cognitive decline have been best correlated with neurodegeneration and neuronal dysfunction ^[27][28][33,34]. This observation has set the initial focus of AD models towards neurons. After the initial establishment of HiPSC technology in 2007, most of the HiPSC-based AD models adopted a minimalist 2D approach where patient-derived stem cells are differentiated to yield 2D monolayer neuronal cultures. When derived from patients with FAD, these cultures present consistent AD-related phenotypes, including early endosomal dysfunction ^[29][35], elevated levels of Aβ and pTau ^[29][35], increased Aβ₄₂/Aβ₄₀ ratio in culture medium ^[30][31][36,37], and activation of glycogen synthase kinase-3β (GSK3β) ^[29][35]. However, these phenotypes have only been shown to emerge in cells derived from a subset of patients with LOAD ^[29][30][35,36]. Since the development of LOAD can be influenced by epigenetic factors ^[32][38], the conversion from the patient’s fibroblasts to neurons may not retain these traits. Another possibility includes failure to recapitulate delayed onset of LOAD phenotypes in immature (21–180 days) cultures. This also can be true for FAD cultures. In one study, endoplasmic reticulum and oxidative stress ^[30][36] were not observed in 21-day-old FAD cultures but were found in 180-day-old cultures. Regarding the generation of Aβ, studies show that the β-secretase inhibitor, β-site APP cleaving enzyme 1 (BACE1), can reverse adverse Aβ pathology ^[30][31][33][36,37,39]. Interestingly, endosomal dysfunction in neuronal cultures with FAD mutations are shown to be mediated by this amyloidogenic processing of APP (β-C-terminal fragment [β-CTF]) but not Aβ itself ^[34][40]. These Aβ pathologies are prominent in FAD-derived but inconsistent in LOAD-derived cultures. However, the identification of the AD risk factor apoE4 allele has been shown to improve the consistency of the outcomes ^[35][41] contributing to the reconsideration of a causative role of Aβ in AD ^[36][42].

Accumulation of pTau, another hallmark of AD, was consistently found in neuronal cultures derived from patients with FAD and patients with LOAD ^[29][37][35,43]. Neurons differentiated from HiPSCs of patients with apoE4 allele (apoE4⁺) and LOAD using lentiviral overexpression of transcription factor neurogenin-2 [23] demonstrated increased levels of pTau and activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). Interestingly, this tauopathy was ameliorated by gene substitution from apoE4 to apoE3. These pure neuronal cultures were more sensitive to Aβ oligomers or hydrogen peroxide (H₂O₂) -induced oxidative stress compared to their healthy patient–derived counterparts [23]. However, similar to previous findings, the Aβ₄₂/Aβ₄₀ ratio was elevated only in cultures derived from patients with FAD but not LOAD. While some studies claim there is a correlation between Aβ and pTau ^[29][35], pTau has been independently correlated with cholesteryl esters, the storage product of cholesterol excess ^[38][44]. This study also identified 2 different pathways through which cholesteryl esters affect Aβ and pTau (cholesterol binding site in APP and proteosome, respectively). This model was instrumental in identifying compounds that could alleviate tauopathy by targeting cholesterol metabolism pathways. Furthermore, docosahexaenoic acid, an omega-3 fatty acid, was shown to be effective in reducing Aβ oligomer-induced oxidative stress in these cultures ^[30][36]. These results support the importance of abnormal lipid homeostasis in AD pathogenesis and demonstrate the utility of 2D neuronal models for therapeutic development.

Endocytosis and transcytosis of low-density lipoproteins were impaired in neurons differentiated from healthy HiPSCs with mutant PSEN1 introduced using CRISPR/CAS9 ^[39][45]. Guanosine triphosphate hydrolase Ras-related protein encoding gene Rab11, an important regulator of vesicle recycling and transcytosis ^[40][46], was found to be downregulated in axons and upregulated in the soma of these neurons ^[39][45]. Ras-related protein 5a encoding gene Rab-5, an early endosome enlargement marker, was also found in abundance in neurons from APP and PSEN1 knock-in HiPSCs ^[34][40]. These studies conform to previous works ^[34][39][40][40,45,46] demonstrating that endosomal dysfunction in neurons is mediated by amyloidogenic processing of APP (β-CTF fragments) but not Aβ itself ^[39][45] and application of BACE1 inhibitor improves endosomal abnormalities. These results emphasize the role of nonamyloid pathways in AD pathophysiology, the involvement of endosomal and axonal trafficking abnormalities in particular.

Mitochondria were recently stated as a viable target for therapeutic development for AD ^[41][42][43][47,48,49]. Mitochondrial dysfunction ^[42][48] is found in AD brain prior to the emergence of Aβ or pTau pathology ^[44][45][50,51]. Neuronal cultures derived from patients with LOAD recapitulated this trait ^[46][52]. Aberrant reactive oxygen species production was observed in LOAD cultures, which correlated with DNA damage but not Aβ and pTau accumulation. These neurons exhibited altered oxidative phosphorylation (OXPHOS) and reduced resistance to H₂O₂ injury ^[47][53]. Mitophagy, a process involved in the removal of damaged mitochondria, was also shown to be affected in these cultures, leading to the accumulation of defective mitochondria ^[48][54]. An additional study showed an increase in dysfunctional lysosomes, defects in OXPHOS, and aberrant mitophagy in neurons derived from patients with FAD with PSEN1 A246E mutation ^[49][55]. These results corroborate previous findings and establish mitochondria as a potential therapeutic target in AD ^[50][51][56,57].

In early stages of AD, human neurons exhibit hyperexcitability ^[52][58]. The recapitulation of this phenotype in vitro requires a presence of functional synaptic networks. Neurons differentiated from HiPSCs with AD-related mutations in APP or PSEN1 (introduced using CRISPR/CAS9) yielded this phenotype ^[53][59]. These neurons exhibited increased frequency of spontaneous action potentials, evoked activity, altered action potential shape, and shorter neuritic processes. Supporting hyperexcitability in AD, ɣ-aminobutyric acid–mediated (GABAergic) neuron-specific degeneration was also found in cortical ^[53][59] and forebrain interneurons ^[54][55][60,61] differentiated from apoE4⁺ LOAD HiPSCs. FAD HiPSC-derived GABAergic neurons also showed altered functionality, enhanced levels of pTau, activation of stress response pathways, and upregulation of neurodegenerative pathways ^[56][62]. Possible deficiencies in chloride exporter KCC2 (SLC12A5) in these neurons hindered the achievement of chloride ion reversal potential, leading to deficient neuronal inhibition. Both gene substitution from apoE4 to apoE3 and small molecule structure correctors were effective in ameliorating GABAergic neuron-specific degeneration ^[55][61]. Cholinergic neurons derived from patients with LOAD also showed increased susceptibility to glutamate-mediated cell death ^[57][63], increased Aβ₄₂/Aβ₄₀ ratio ^[58][64], and altered calcium ion (Ca²⁺) flux ^[58][64].

Taken together, this body of evidence establishes 2D monolayer cultures as an excellent tool to assess a single cell-specific neuronal AD phenotype. These cultures are relatively simple and produce network level insights relevant to neuronal activity. However, the absence of glial cells, essential for synaptic maturation ^[59][65] and inflammation, can be marked as one of the major limitations of these systems.

Though neuron-focused models can capture multiple aspects of AD, other cell types, including astrocytes ^[60][61][66,67] and microglia ^[62][68], also actively contribute to disease development and progression. Microglia and astrocytes are key players in neuroinflammation, which is a contributing factor to AD pathogenesis ^[63][64][69,70]. In the systems described in Section 2.1.1, astrocytes derived from patients with LOAD or FAD were not used. Evidently, these approaches overlooked the important astrocyte-specific disease phenotypes. This gap is primarily due to the difficulty in differentiating astrocytes using conventional methods where neural progenitor cells (NPCs) are obtained from HiPSCs by dual SMAD inhibition [20]. This inhibition directs the NPCs towards astrocytic fate. NPCs are then expanded until gliogenesis ^[65][66][71,72]. This process is time consuming (requiring more than 6 months ^[67][73]) and yields a heterogenous mixture of different cell types. Additional FACS is often required to isolate cells of interest ^[68][19].

Regardless of the aforementioned difficulties, the importance of astrocytes in the pathogenesis of AD was demonstrated in the HiPSC-derived cocultures of patients with early-onset FAD where astrocytes exhibited altered metabolism, increased oxidative stress, and disturbed Ca²⁺ signaling in the endoplasmic reticulum ^[69][70][74,75]. In line with AD-related defects in neuronal lipid metabolism ^[71][76], 2D astrocyte cultures from patients with FAD showed impaired fatty acid oxidation ^[72][77], altered morphology ^[73][78], and nonstimulated release of soluble inflammatory mediators ^[73][78]. Fatty acid oxidation impairment in 2D astrocyte cultures was corrected by synthetic peroxisome proliferator activated receptor-β and -δ (PPARβ/δ) agonist GW0742 ^[72][77]. PPARβ/δ is a key player in brain energy homeostasis and metabolism. Further studies showed that, in astrocyte-neuron cocultures, these astrocytes provide reduced support towards neuronal survival and synaptogenesis, exhibit reduced glucose uptake ^[74][75][79,80], and oversupply cholesterol to neurons causing neuronal lipid raft expansion and Aβ₄₂ production ^[76][81]. Altered bioenergetic metabolites and transcriptomes were also reported in these astrocytes ^[75][80].

Taken together, astrocyte-focused models of AD establish astrocytes as key players in the pathogenesis of AD, which supports multiple neuronal AD phenotypes, including a decreased resistance to H₂O₂ injury and defects in lipid metabolism. An inflammatory AD phenotype, absent in neuron-only models, was recapitulated in the presence of astrocytes. However, as mentioned earlier, these models were mainly based on dual SMAD inhibition of HiPSC-derived NPCs and time-consuming gliogenesis. Inherent heterogeneity of cell types makes these systems unreliable and difficult to control.

The involvement of microglia in AD neurobiology has been well established ^[77][82]. However, the elusive embryonic origin of microglia has impeded the development of reliable protocols for their differentiation from HiPSCs. Due to this, past studies were limited to rodent, hard-to-obtain ex vivo human microglia ^[78][83], or immortalized cell lines, yielding cultures with substantially different characteristics ^[79][84]. Recently, primitive myeloid progenitors were identified as the origin of microglia ^[80][85], differentiating these cells from other tissue-resident macrophages originating from yolk sac–derived erythromyeloid progenitors ^[81][86]. This ontogenetic advancement has recently led to the development of the first microglia differentiation protocol ^[82][87]. 2D cocultures from apoE4⁺ neuron [23], astrocyte ^[83][88], and microglia ^[82][87], derived from patients with LOAD [23], were developed using this method ^[84][89]. Concurring with neuron-only models, neurons in this triculture setup exhibited an increased number of synapses and elevated neuronal Aβ₄₂ secretion. Astrocytes exhibited impaired Aβ uptake and cholesterol accumulation, which was shown to increase astrocytic cholesterol secretion, leading to neuronal lipid raft expansion and Aβ₄₂ production ^[76][81]. Importantly, microglia exhibited altered morphology, impaired Aβ phagocytosis ^[84][89], and mutual astrocyte-microglia activation ^[85][90].

Recent development of small molecule–guided ^[86][91] microglial differentiation has further accelerated studies with cocultures ^[87][92]. Microglia differentiated from HiPSCs derived from patients with LOAD and FAD using small molecules exhibited apoE4 genotype–induced aggravated microglial inflammatory response, decreased microglial metabolism, phagocytosis, and migration. Counterintuitively, microglia generated from HiPSCs with FAD-related mutations in APP and PSEN1 showed only a slight decrease in proinflammatory cytokine release and increase in chemokines, indicating a senescent-like state ^[87][92], while microglia derived from HiPSCs from patients with LOAD with apoE4 allele assumed a more proinflammatory state ^[87][92]. These results indicate the possibility of mechanistic differences between LOAD and FAD ^[87][92].

Advancement in stem cell differentiation techniques and automated culturing setups have further allowed the development of a high-throughput and automated HiPSC-derived cell culturing platform ^[88][93]. Using this platform, highly replicable 2D neuron-astrocyte-microglia tricultures were created in 384-well plates ^[88][93]. Application of soluble Aβ₄₂ in these tricultures resulted in Aβ plaque formation with surrounding dystrophic neurites ^[88][93]. The apoE4/3 microglia were found to internalize, exocytose, and package soluble Aβ₄₂ as plaque structures ^[88][93]. Hence, microglia not only surrounded these plaques, but participated in plaque formation. Multiple other AD-related phenotypes, such as synapse loss, dendritic retraction, axonal fragmentation, pTau accumulation, and neuronal cell death, were also reliably recapitulated in these cocultures. The highly automated and precise nature of this setup made it a convenient tool for high-throughput compound screening. Seventy neuroprotective small molecule compounds (4 concentrations in at least 2 independent cultures for each compound) were screened in this platform, using the improvement in dendritic (microtubule-associated protein 2 positive) area, axonal (βIII-tubulin positive) area, number of synapses (synapsin 1/2 positive puncta count), and cell survival (cut like homeobox 2 positive cell count) as the therapeutic index. Nine hits were identified in this screening, including inhibitors of well-known active kinases in AD, such as DLKi27 ^[89][94], indirubin-3′-monoxime ^[90][95] (GSK3β and CDK5 inhibitor), AZD0530 ^[91][96] (Fyn inhibitor), and demeclocycline HCl ^[92][97] (calpain inhibitor), along with luteolin ^[93][94][98,99] and curcumin ^[94][99] and its derivative J147 ^[95][100]. Involvement of the DLK-JNK-cJun pathway in AD was further confirmed by validating the neuroprotection of VX-680 ^[96][101] (a different DLK inhibitor), GNE-495 ^[97][102] (MAP4K4 inhibitor upstream of DLK45), PF06260933 (a different MAP4K4 inhibitor), and JNK-IN-8 (JNK1/2/3 inhibitor) ^[88][93]. Neuroprotective effects of anti-Aβ antibody were also validated using this setup. These results show the potential and capacity of a full-fledged, high-throughput system that uses HiPSCs and automation technologies.

The addition of the extra dimension to conventional 2D cultures started with spheroid cultures. Spheroid cultures are cell aggregates embedded in an artificial ECM. This cellular aggregation can be induced by a low attachment substrate ^[98][109] or confinement ^[99][100][110,111]. The primary hurdle of this technology is core necrosis. Since organoids are not vascularized ^[101][112], nutrients cannot reach deep inside cell clusters. Cellular waste also cannot be efficiently removed from the deep layers of cell aggregates. As a result, there is upward cellular waste and downward nutrient gradient from the spheroids’ outer layer to the center, resulting in a necrotic core which limits culture size. Advanced biocompatible ECM or scaffolds partially allow nutrients to enter deep inside the cultures and help to overcome core necrosis. Cells are generally embedded in these scaffolds and then differentiated. Recently emerged bioreactor ^[102][103][113,114] technology employs highly controlled rotating walls, creating optimum medium flow that negates gravitational pull to create microgravity or weightlessness. Scaffold-embedded cells are then differentiated under these conditions, which promote aeration, uniform cellular aggregation, and enhanced nutrient diffusion. In the case of HiPSC-derived spheroids, cells in these aggregates are differentiated into their fates determined by supplied growth factors. Interestingly, during the differentiation process, the gradually changing microenvironment can influence cells to assume brain-like laminar positions. These spheroids are termed brain organoids and are defined as 3D structures derived from either pluripotent stem cells (embryonic stem cells or HiPSCs) or neonatal or adult stem/progenitor cells, in which cells spontaneously self-organize into properly differentiated functional cell types and recapitulate at least some function of the organ ^[104][115].

Existing 3D HiPSC-based AD models use ECM to hold cells in their 3D locations. NPCs or HiPSCs embedded in Matrigel (Corning Life Sciences, Bedford, MA, USA) can be differentiated to yield homogeneous cortical neuronal spheroid cultures ^[105][116]. Neuron-specific adeno-associated viral overexpression of P301L (tau mutation most frequently observed in patients with frontotemporal dementia and parkinsonism ^[106][117]) and application of recombinant human tau (K18) yielded tauopathy in a Matrigel-embedded setup ^[105][116]. In the same system, immortalized human NPCs (ReNcell VM, ReNeuron Group plc) with FAD-related mutations in APP and PSEN1 yielded homogeneous neuronal cultures with extracellular deposition of Aβ plaques, Aβ₄₂/Aβ₄₀ ratio–correlated tauopathy, and cell death ^[107][118]. Interestingly, Aβ plaques were found in 2D neuron-astrocyte-microglia triculture only after application of synthetic Aβ oligomers ^[88][93], while in FAD 3D cultures they appeared spontaneously. Compared to 2D cultures, induced neurons also showed enhanced maturation in this 3D setup ^[107][108][118,119]. However, these spheroid cultures did not show any brain-like cellular organization or layers.

Further progress in brain organoid technology has produced methods to mimic corticogenesis (the developmental process of the cerebral cortex) in a dish and create neocortex-like 3D cultures from HiPSCs ^[109][120]. In these systems, HiPSCs are first self-aggregated in low-adherent, concave (V- or U-bottom) wells to generate embryoid bodies. These embryoid bodies are maintained long term in the media with rho kinase inhibitor, low concentration Matrigel, and other growth factors ^[109][120]. Next, the transforming growth factor β and wingless/Int-1 signaling pathways are chemically blocked to induce neuroepithelium differentiation on a low-adherent substrate. This method yielded spherical structures (spheroids) that exhibited brain-like cortical regions, including progenitor zones and ventricular zones, termed cerebral organoids (COs). HiPSC-derived COs ^{[110][111][112][113]}[121,122,123,124] from patients with FAD and LOAD ^[84][89] were characterized with Aβ plaques, elevated pTau, mislocalized mutant tau protein, and dysfunctional axons. These phenotypes were cross-validated in 2D cultures, COs, cerebral spinal fluid, and ex vivo brain tissue from the same patient with an FAD-linked APP mutation ^[114][125]. Mitochondrial axonal transport ^[115][126] was impaired in COs generated from patients with familial frontotemporal dementia with R406W (known cause of frontotemporal dementia with parkinsonism ^[116][127]) tau mutation. This phenotype was absent in previous systems. Moreover, microtubule stabilization rescued axonal transport deficiency, indicating a contribution of mitochondrial motility to the AD mechanism. Furthermore, the process of neuronal differentiation was impaired by the repressor element 1-silencing transcription factor (REST) gene in COs derived from patients with LOAD ^[117][128]. This impairment was unaffected by Aβ production, BACE1, and γ-secretase inhibitors ^[117][128]. These observations shed light on possible developmental defects in AD and reestablishes REST, a regulator of aging brain stress response, as a viable marker of AD ^[118][129]. Hippocampal organoids were also developed by chemical differentiation of HiPSC spheroids in suspension ^[119][120][130,131]. Similar to COs, these hippocampal organoids showed increased Aβ₄₂/Aβ₄₀ ratio and decreased levels of synaptic proteins ^[119][120][130,131].

The 3D, spheroid, and organoid models of AD have added a new dimension to conventional 2D models. Notably, the hallmark of AD, the formation of Aβ plaques, was readily observed in these models. Organoids also exhibited AD-related defects in axonal trafficking. Both traits cannot be mimicked in 2D cultures without the application of synthetic Aβ peptides. Since AD is a multifactorial disease, efficacious therapeutic strategies would be expected to improve multiple pathways. Spheroids and especially organoids present a wholesome accumulation of almost all known AD phenotypes in a single model. This makes them a valuable tool for drug discovery compared to conventional 2D cultures. While 2D cultures continue to be the most convenient model to assess single cell attributes, organoids are rapidly recognized for their ability to provide input on a systems level. Interestingly, in this 3D system, organoids were able to recapitulate AD phenotypes in months, which takes years to develop in a human brain ^[114][125].

Despite their promises, brain organoids face some crucial problems that hinder their use in high-throughput drug screening applications. Since organoids are differentiated from HiPSCs or NPC aggregates without any purification step, cellular composition in these cultures cannot be exactly predefined ^[121][122][132,133]. Probably because of these discrepancies, neural activity also is not well defined in organoids ^[123][134]. In addition, culture morphology cannot be designed with these suspension setups ^[124][135]. Cellular and morphologic heterogeneity result in substantial culture-to-culture variations ^[114][125]. Scaffold-based optimized variants of these cultures, called mini brain ^[125][136], develop brain-like regions. However, these regions also vary considerably in shape and location among cultures. These problems require more controlled and defined 3D culturing techniques.