Table 1.

Summary of relevant findings from primary literature reviewed in text organized by section.

Reference	Section(s) of Review	Model Type(s)	Summary of FXS-iPSC-Relevant Findings
Urbach et al. (2010) [46]	Generation of human iPSC-derived cellular models of FXS Characterization of FXS-iPSCs	FXS-iPSCs	In FXS-iPSCs, FMR1 was inactive. FMR1 hypermethylation was present. FMR1 contained histone modifications associated with a heterochromatin state. FMRP expression was absent.
Sheridan et al. (2011) [47]	Generation of human iPSC-derived cellular models of FXS Characterization of FXS-iPSCs Characterization of neurodevelopmental and electrophysiological abnormalities of iPSC-derived cell models of FXS	FXS-iPSCs FXS-iPSC-derived neurons	# of CGG repeats in FXS-iPSCs may not equal the # of CGG repeats in fibroblasts of origin. Determined the methylation status of FMR1 and FMRP expression. FXS-iPSC-derived neurons exhibited underdeveloped neurites.
Doers et al. (2014) [48]	Generation of human iPSC-derived cellular models of FXS Characterization of FXS-iPSCs Characterization of neurodevelopmental and electrophysiological abnormalities of iPSC-derived cell models of FXS	FXS-iPSCs FXS-iPSC-derived neurons	FMR1 repeat expansion and hypermethylation of the promoter is retained following the reprogramming of FXS patient fibroblasts to iPSCs. Axonal growth cones of FXS-iPSC-derived neurons have reduced motility and rates of extension compared to controls.
Esanov et al. (2016) [49]	Generation of human iPSC-derived cellular models of FXS Characterization of FXS-iPSCs	Primary neurons from postmortem FXS patient brain tissue Primary fibroblasts from FXS patients Immortalized lymphocytes from FXS patients FXS-iPSCs FXS-iPSC-derived neurons FXS-ESC-derived neurons	FMR1 promoter 5hmC enrichment was present in primary FXS neurons but is absent in primary FXS fibroblasts, lymphocytes, FXS-iPSCs, FXS-iPSC-derived neurons, and FXS-ES-derived neurons.
De Esch et al. (2014) [50]	Generation of human iPSC-derived cellular models of FXS Characterization of UFM iPSCs	UFM iPSCs	The reprograming process from fibroblasts to iPSCs caused silencing of the fully mutated FMR1 gene in UFM iPSCs.
Brykczynska et al. (2016) [51]	Generation of human iPSC-derived cellular models of FXS Characterization of UFM iPSCs	UFM iPSCs	FMR1 in UFM iPSCs could be silenced when the gene contained CGG repeats above the threshold of around 400.
Halevy et al. (2015) [52]	Gene expression and translation in the 2D hiPSC-derived cell models of FXS	FXS-iPSC-derived neurons	RE-1-silencing transcription factor (REST), which suppresses neuronal differentiation and axon guidance genes, was upregulated, and hsa-mir-382, which represses REST, was downregulated in FXS-iPSC-derived neurons. Overexpression of hsa-mir-382 significantly upregulated REST-mediated genes in FXS-iPSC-derived neurons.
Utami et al. (2020) [53]	Gene expression and translation in the 2D hiPSC-derived cell models of FXS Characterization of neurodevelopmental and electrophysiological abnormalities of iPSC-derived cell models of FXS	FXS-iPSC-derived NPCs & neurons Isogenic FMR1-KO hESCs FMR1-KO hESC-derived NPCs & neurons	RNA-seq revealed that genes related to kinase activity, amino acid transport, and RNA methylation were upregulated in FXS-iPSC-derived neurons. Genes related to axon guidance, neuron differentiation, transsynaptic signaling, and messenger RNA splicing were downregulated in FXS-iPSC-derived neurons. Significantly smaller neural rosettes were formed by FXS-iPSCs compared to controls. Proliferation was increased in FXS-iPSC-derived NPCs compared to controls. Neurite outgrowth was decreased in FXS-iPSC-derived neurons compared to control neurons.
Lu et al. (2016) [54]	Gene expression and translation in the 2D hiPSC-derived cell models of FXS	FXS-iPSCs FXS-iPSC-derived neurons	RNA-seq revealed upregulated genes related to neuronal differentiation and neural development and downregulated genes encoding potassium channels in FXS-iPSC-derived neurons.
Boland et al. (2017) [55]	Gene expression and translation in the 2D hiPSC-derived cell models of FXS Characterization of neurodevelopmental and electrophysiological abnormalities of iPSC-derived cell models of FXS	FXS-iPSC-derived NPCs & neurons	REST target genes were dysregulated in FXS-iPSC-derived NPCs and neurons. Many DEGs associated with developmental signaling and cell migration were identified in FXS-iPSC-derived neural cells. Immature neurons derived from FXS-iPSCs exhibited increased neurite lengths compared to control neurons.
Sunamura et al. (2018) [56]	Gene expression and translation in the 2D hiPSC-derived cell models of FXS	FMR1-KO hiPSC-derived NPCs	FMR1-null NPCs exhibited: Elevated expression of glial fibrillary acidic protein (GFAP), which was corrected by reintroduction of FMRP via a lentiviral vector. Reduced spontaneous calcium bursts Both of the above abnormalities were corrected by treatment with protein kinase inhibitor LX7101.
Raj et al. (2021) [21]	Gene expression and translation in the 2D hiPSC-derived cell models of FXS Modeling FXS with human brain organoids	FXS-iPSC-derived NPCs Isogenic FMR1-KO iPSC-derived NPCs FXS-iPSC-derived cortical organoids at differentiation day 28	The authors developed a flow-cytometry-based high-throughput single-cell assay to measure translation and proliferation markers specific to neural cell type. FXS NPCs expressed higher levels of markers of proliferation than those of neurogenesis. An inhibitor of a catalytic subunit of PI3K ameliorated the protein synthesis defects in FXS NPCs. FXS organoids showed increased NPC proliferation. Transcriptome analysis of FXS organoids identified significantly upregulated genes to be related to proliferation and significantly downregulated genes to be related to neuronal fate specification, migration, differentiation, and maturation.
Kurosaki et al. (2021) [57]	Gene expression and translation in the 2D hiPSC-derived cell models of FXS	FXS-iPSCs FXS-iPSC-derived neurons	FMRP represses nonsense-mediated mRNA decay (NMD). Small molecules that inhibit NMD restored the aberrant expression of neurodifferentiation markers and increased the neurite growth in FXS-iPSC-derived neurons.
Li et al. (2020) [23]	Studies of FMRP function and targets	Dorsal and ventral forebrain NPCS & neurons derived from FMR1-FLAG tagged and FMR1-KO hPSCs (iPSCs and ESCs)	CLIP-seq identified FMRP targets common to all four types of cells as well as cell type-specific targets. An integrative analysis of CLIP-seq and transcriptomic data revealed FMR1-regulated pathways essential in human neurodevelopment. FMRP bound preferentially to longer RNA targets and to coding regions of mRNAs.
Goering et al. (2020) [58]	Studies of FMRP function and targets	FXS-iPSC-derived neurons FMRP-KO mouse CAD neuronal cells	FMRP regulates the localization of certain gene transcripts to neurites within neurons, and these FMRP-localization target genes were enriched with G-quadruplex structures in their 3′ UTRs. Localization targets of FMRP differed from translation targets of FMRP.
Niedringhaus et al. (2015) [90]	Characterization of neurodevelopmental and electrophysiological abnormalities of iPSC-derived cell models of FXS	FXS-iPSC-derived neuronal cultures using a micro-raft array	FXS-iPSC-derived neurons had a significant decrease in synaptic vesicle recycling and an increase in unloading of synaptic vesicles compared to control neurons.
Zhang et al. (2018) [60]	Characterization of neurodevelopmental and electrophysiological abnormalities of iPSC-derived cell models of FXS	FXS-iPSC-derived neurons FXS-ESC-derived neurons	Baseline amplitude and frequency of miniature excitatory postsynaptic current (mEPSC) and inhibitory equivalent (mIPSC) did not differ between FXS neurons and controls. FXS NPCs exhibited impaired retinoic-acid-mediated regulation of synaptic strengths, which was rescued by the recovery of FMR1 expression by excision of the expanded CGG repeats using CRISPR/Cas9.
Das Sharma et al. (2020) [61]	Characterization of neurodevelopmental and electrophysiological abnormalities of iPSC-derived cell models of FXS	FXS-iPSC-derived neurons	FXS-iPSC-derived neurons fired shorter and more frequent spontaneous action potentials than FMR1-expressing controls. Voltage-gated sodium (Na+) channel activator treatment increased the duration and reduced the frequency of action potentials in FXS neurons, normalizing the firing patterns to resemble those in controls. In contrast, treatment of control lines with a persistent Na+ current (Nap) blocker and a calcium (Ca2+)-activated potassium (KCa) channel blocker both altered the firing patterns to resemble those in FXS neurons.
Danesi et al. (2018) [63]	Characterization of neurodevelopmental and electrophysiological abnormalities of iPSC-derived cell models of FXS	FXS-iPSC-derived NPCs Fmr1-KO and dMT mouse NPCs	Intracellular calcium release signaling in response to membrane depolarization and the expression of L-type Cav channels were increased in both human and mouse NPCs lacking FMR1 expression at an early stage of differentiation.
Achuta et al. (2017) [62]	Characterization of neurodevelopmental and electrophysiological abnormalities of iPSC-derived cell models of FXS	FXS-iPSC-derived NPCs Fmr1-KO mouse NPCs	FXS-hiPSC-derived NPCs and Fmr1-KO mouse NPCs both exhibited larger intracellular calcium release amplitudes in response to the group I mGluR (mGluR1 and mGluR5) agonist (S)-3,5-dihydroxyphenylglycine (DHPG) compared to controls. MPEP affects neural development in a species-dependent and cell-type-dependent manner.
Achuta et al. (2018) [59]	Characterization of neurodevelopmental and electrophysiological abnormalities of iPSC-derived cell models of FXS Maturation of excitatory transmission in FXS	FXS-iPSC-derived NPCs Fmr1-KO mouse NPCs	FXS-iPSC-derived NPCs and Fmr1-KO mouse NPCs express a higher level of Ca2+-permeable AMPA receptors (CP-AMPARs) lacking the GluA2 subunit than controls. Naspm, a Glu2-lacking CP-AMPAR inhibitor, reduced the lengths of neurites in both FXS and control neurons so that their lengths were not significantly different from each other.
Brighi et al. (2021) [24]	Characterization of neurodevelopmental and electrophysiological abnormalities of iPSC-derived cell models of FXS Maturation of excitatory and inhibitory transmission in FXS Modeling FXS with human brain organoids	FMRP-KO-hiPSC-derived neurons FMRP-KO-hiPSC-derived organoids	FMR1-null NPCs had elevated expression of glial fibrillary acidic protein (GFAP). FMR1-null NPCs exhibit increased spontaneous electrophysical network activity and depolarization in response to GABA. FMRP-KO organoids were bigger in size and had an increased number of glial cells, presumably astrocytes.
Bar-Nur et al. (2012) [64]	FMR1 reactivation—Pharmacological rescue	FXS-iPSCs FXS-iPSC-derived neurons	5-azaC treatment Reactivated FMR1 expression in both FXS-iPSCs and FXS-iPSC-derived neurons. Reduced the methylation of the FMR1 promoter region in FXS-iPSCs and neurons in a concentration-dependent manner. Led the histone H3 acetylation and H3K4 methylation levels to be comparable to those in controls.
Kaufmann et al. (2015) [65]	FMR1 reactivation—Pharmacological rescue	FXS-iPSC-derived NPCs	A high-content imaging assay was developed and used to screen compounds that could reactivate the expression of FMR1. Several compounds that led to a weak expression of FMRP were identified.
Kumari et al. (2015) [67]	FMR1 reactivation—Pharmacological rescue	FXS-iPSC-derived neural stem cells	A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was developed and used to screen compounds that could reactivate the expression of FMR1. Six compounds that could partially reactivate FMR1 were identified.
Li et al. (2017) [68]	FMR1 reactivation—Pharmacological rescue	FXS-iPSCs	Human iPSC reporter line with a CRISPR/Cas9-based knock-in of a Nano luciferase (Nluc) gene (FMR1-Nluc reporter line) to detect FMR1 expression was created.
Vershkov et al. (2019) [69]	FMR1 reactivation—Pharmacological rescue	FXS-iPSCs FXS-iPSC-derived NPCs FXS-iPSC-derived NPC transplants in mice	FMR1 reactivation in FXS-iPSCs and FXS-iPSC-derived NPCs by 5-azadC was enhanced by the addition of 3-deazaneplanocin A (DZNep). FMR1 reactivation in response to 5-azadC was verified in vivo in FXS-iPSCs injected into immunocompromised mice and FXS-iPSC-derived NPCs transplanted into mouse brains.
Kumari et al. (2020) [66]	FMR1 reactivation—Pharmacological rescue	FXS-iPSC-derived neural stem cells & neurons	Chaetocin, a fungal toxin that inhibits mammalian histone methyl-transferases, had a synergistic effect with 5-azadC in reactivating FMR1 in neural stem cells and neurons derived from FXS-iPSCs. Chaetocin, DZNep, and BIX01294 delayed the re-silencing of 5-azadC-activated FMR1 expression.
Park et al. (2015) [70]	FMR1 reactivation—Gene editing	FXS-iPSCs FXS-iPSC-derived neurons	Expanded CGG repeats in FMR1 were excised by CRISPR/Cas9 in FXS-iPSCs, which led to the near-complete demethylation and expression of FMR1, and FMRP expression in FXS-iPSCs and CGG-repeat-edited-FXS-iPSC-derived neurons.
Xie et al. (2016) [71]	FMR1 reactivation—Gene editing	FXS-iPSCs Somatic cell hybrids containing a human FXS chromosome	Expanded CGG repeats in FMR1 were excised by CRISPR/Cas9 in FXS-iPSCs, which led to the demethylation and expression of FMR1, and FMRP expression in FXS-iPSCs and FXS somatic hybrid cells.
Liu et al. (2018) [72]	FMR1 reactivation—Gene editing	FXS-iPSCs FXS-iPSC-derived neurons FXS-iPSC-derived NPC transplants in mice	Expanded CGG repeats were demethylated using CRISPR/dCas9-Tet1 in FXS-iPSCs. Targeted methylation editing led to an active chromatin state of FMR1 promoter, normalization of electrophysiological abnormalities in neurons derived from methylation-edited FXS-iPSCs. FMR1 reactivation was sustained in methylation-edited neuronal engrafts in mice.
Graef et al. (2020) [73]	FMR1 reactivation—Gene editing	FXS-iPSCs Isogenic FMR1-KO iPSCs FXS-iPSC-derived neurons	5% of normal FMRP expression was enough to rescue the elevated spontaneous activity in an FXS mosaic neuronal culture. Moreover, a neuronal culture in which greater than 20% of cells express FMRP had a normal electrophysiological phenotype.
Kang et al. (2021) [22]	Modeling FXS with human brain organoids	FXS-iPSC-derived forebrain organoids	FXS organoids at differentiation day 56 (D56) exhibited reduced NPC proliferation, premature neural differentiation, altered cortical layer formation, and disrupted differentiation of GABAergic interneurons. FXS organoids at D56 showed accelerated synapse formation and hyperexcitability FXS organoids had altered gene expression profiles and aberrant cell-type-specific developmental trajectory. PI3K inhibitors but not mGluR5 antagonists rescued NPC proliferation defects and synaptic formation deficits in FXS organoids. A large number of human-specific FMRP targets were identified via eCLIP-seq, including chromodomain helicase DNA-binding protein 2 (CHD2)

FXS = fragile X syndrome, iPSCs = induced pluripotent stem cells, FXS-iPSCs = iPSCs derived from primary cells from individuals with FXS, ESC = embryonic stem cell, NPC = neural progenitor cell, UFM = unmethylated full mutation, MPEP = 2-methyl-6-(phenylethynyl)pyridine, 5-azaC = 5-azacytidine, 5-azadC = 5-aza-2′-deoxycytidine or decitabine, CRISPR = clustered regularly interspaced short palindromic repeats, eCLIP-seq = enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing.