Table 2 Disease modeling using 3D human brain organoids and challenges.

 Microcephaly

Mouse models do not mimic the severity of Microcephaly because of smaller brain and limited expansion

Cerebral organoids

Y-27632, N2 supplement, Heparin, Matrigel, B27 supplement without vitamin A, Insulin

Progenitor cells with decreased radial glial cells show premature neural differentiation

[9]

Microcephaly patients with Aspm mutations possess extremely reduced brain size, which is quite difficult to mimic in animal models

Cortical organoids

endo-IWR1, LDN-193189, SB431542, heparin, BDNF, GDNF, cAMP, matrigel

Patient-derived cortical organoids displayed proliferation deficient progenitors, less mature neurons and abnormal cortical lamination, neuronal dysfunction

[37]

 Lissencephaly (Miller Dieker Syndrome)

Absence of outer radial glial cells in developing rodent cortex leads to milder phenotypes in Pafah1b1+/− mice as compared to human patients

Cerebral organoids

Y-27632, WNT inhibitor, TGF-β inhibitor, FBS, Matrigel and heparin

Defective cell migration, massive apoptosis of founder neuroepithelial stem cells, Defective mitosis in outer radial glia

[146]

 Bipolar disorder

Mechanistic understanding of the Bipolar Disorder is unclear due to the scarcity of causal genes displaying robust effect sizes

Cerebral organoids with dorsal forebrain identity

STEMdiff Cerebral Organoid Kit & maturation kit with BDNF

Cerebral organoids derived from diseased and control iPSCs were not apparently different. Bipolar cerebral organoids show precise defects in response to stimulation and depolarization

[147]

 Bipolar disorder/schizophrenia

Underlying cellular mechanisms for the pathogenesis of Bipolar disorder are not clear due to heterogeneity and technical constraints on cellular complexity of the brain.

Cerebral organoids

SB431542, LDN-193189, Y-27632, Matrigel

Accelerated neuronal differentiation, increased GABAergic specification, decreased cell proliferation following reduced Wnt signaling

[148]

 Developmental and epileptic encephalopathies (DEE)

DEE is a heterogeneous disorder associated with intractable seizures, abnormal brain development, and functional abnormalities. Every patient has distinct genetic background and brain development, which cannot be modeled in a rodent model

Cerebral organoids

bFGF, Y-27632, Matrigel, Insulin, CHIR-99021, vitamin C

Defective DNA damage response, activation of Wnt pathway, abnormal cortical differentiation with disproportionate glutamatergic and GABAergic neurons and increased astrogenesis

[149]

 Timothy syndrome

In vitro modeling of migration of interneurons and their functional integration into human cortex during fetal development remains a challenge

Forebrain spheroids containing cortical (hCS) and subpallium spheroids (hSS)

hCS: dorsomorphin, SB-431542, FGF2, EGF, BDNF, NT-3 hSS: FGF2, EGF, IWP-2, SAG, NT-3, allopregnanolone, retinoic acid, BDNF

Increased saltation frequency led to defective migration of interneurons

[32]

 Tuberous sclerosis complex (TSC)

Highly heterogeneous developmental disorder, cortical tubers are not formed in rodent models

Cortical spheroid

Dorsomorphin, SB431542, EGF, FGF, BDNF, NT-3

Hypertrophic NPCs, enlarged and dysmorphic neuronal cells with enhanced glia production

[150]

 Angelman syndrome (AS)

AS mice models show synaptic dysfunction and impaired plasticity; however, mechanisms in AS patients remain unclear

Cortical organoids

Dorsomorphin, SB431542, B27 without vitamin A, EGF, FGF-basic, BDNF, GDNF, NT3, db-cAMP

Hyper-excitability and synchronous firing, UBE3A via degradation of calcium- and voltage-dependent big potassium (BK) channels inhibits neuronal hyper-excitability

[151]

 Rett Syndrome

Non-availability of embryonic or perinatal post-mortem tissues from Rett Syndrome patients

Cerebral organoids

Y-27632, SB431542, dorsomorphin, Matrigel, B27 without vitamin A, B27 with vitamin A

increased ventricular area, reduced radial thickness and massive increase of neural progenitor cells followed by abnormal migration of neurons and aberrant neurogenesis

[152]

Lack of studies on influence of MeCP2 mutation on human interneuron development and function

Human medial ganglionic eminence (MGE) and cortical organoids

LDN-193189, SB-431542, XAV-939, Y-27632, hMGEO: B27 supplement without vitamin A, recombinant SHH, purmorphamine hCO: B27 supplement with vitamin A, BDNF, ascorbic acid, cAMP

BET inhibitor, JQ1, improves the functional deficits of RTT interneurons in brain organoids by showing area-scale synchronization of calcium surges and rescuing the dysregulated transcriptome

[153]

RTT phenotypes are widespread and various kind of mutations present the disease phenotypes differently

Dorsal and ventral forebrain organoids

Dorsal Forebrain: Dorsomorphine, A83-01, CHIR99021, SB-431542, Heparin Ventral: SB-431542, LDN-193189, IWP2, SAG and Heparin. BDNF, GDNF, dibutyryl cAMP and ascorbic acid

Premature development of the deep-cortical layer with less proliferating neural progenitor/proliferative cells along with the impairments of interneuron’s migration, dysfunctional RTT neurons, defects in production of medial ganglionic eminence (MGE) progenitors in ventral organoids

[154]

Genetic mutation of MeCp2 gene impairs neurodevelopment, still therapeutic treatment for this syndrome is not available

Cortical organoids

SB431542, Dorsomorphin, Y-27632, FGF2, EGF, BDNF, GDNF, NT-3, ascorbic acid and dibutyryl-cAMP

In MECP2‐KO cortical organoids, two lead compounds rescued synaptic pathways and neural network function

[155]

 Down syndrome

Technically inaccessible pathological samples, presence of asynchronous and heterogeneous disease phenotypes in in vitro culture

Cerebral organoids

N2 supplement, Matrigel

Diminished neurogenesis, reduced proliferation and decreased expression of cortical neuronal markers in layer II and IV in the subcortical regions

[156]

Vague knowledge of role of Olig genes in GABAergic neuron generation and inconsistencies in recapitulating DS-related genotype–phenotype relationships and contradictory results from human samples

Ventral forebrain organoid

SB431542, Noggin, Matrigel, Laminin, B27-RA, FGF2, hLIF, CHIR99021, Y-27632, SHH, purmorphamine, BDNF, GDNF, dibutyryl-cyclic AMP and ascorbic acid

Overproduction of OLIG2+ Progenitors, disproportionate interneuron production, recognition memory disrupted in neuronal chimeric mice

[157]

 ASD

Phenotypic heterogeneity and absence of behavioral phenotypes of ASD in rodent models

Telencephalic organoids

B27 supplement without vitamin A, N2 supplement, 2-mercaptoethanol, Y-27632, FGF2, Noggin, rhDkk1, EGF, ascorbic acid, BDNF, GDNF and dibutyryl-cAMP

Elevated neuronal maturation and synaptic overgrowth, Massive inhibitory synapses in ASD-derived neurons, balance between the number of excitatory and inhibitory neurons in ASD-organoids was disturbed

[111]

Heterogeneous population of neurons expressing forebrain, midbrain, and hindbrain markers was present in monolayer neuronal culture system

Cerebral organoid

DKK-1, BMPRIA-Fc, SB431542, N2 supplement, laminin, and fibronectin, B27 and l-glutamine

Radial glia progenitor cells, GABAergic and glutamatergic neurons were present in cerebral organoids

[114]

To integrate the finding that DNA methylation patterns of GAD1 are dysregulated during development from patient’s post-mortem brain and rodent models

Cerebral organoids

Y-27632, Matrigel

Early development follows a diverse methylation patterns in GAD1 in ASD cerebral organoids

[158]

Lack of understanding of cell lineages and molecular pathways implicated in telencephalic development in ASD

single neural rosettes derived telencephalic organoids

N2 Supplement, Heparin, B27 Supplement with vitamin A, Dorsomorphin, SB431542, EGF, FGF, BDNF, GDNF and NT-3

Smaller size of SHANK3-/-organoids, decreased population of neurons with smaller nuclei sizes, decreased number of excitatory synaptic puncta

[159]

ASD

Mechanisms underlying valproic acid contribution to accelerating ASD risk in human are not known

Forebrain organoids

SB431542, LDN193189, Y27632, Heparin, CHIR99021, WNT-3A, Matrigel, insulin, Forskolin, Ascorbic Acid, BDNF, GDNF

Disturbed synaptic transmission in VPA treated organoids, differentially expressed genes enriched in calcium, and potassium signaling pathways, oxytocin signaling, synaptic transmission, and neural development

[160]

How interactions among gene–environment increases ASD risk are unknown

Brain organoids

FGF, EGF, GDNF, BDNF

Exposure of CHD8 +/− organoids with organophosphate pesticide resulted in disturbed Neurite outgrowth and imbalance of excitatory/inhibitory neurotransmitters

[161]

 Alzheimer disease

Two-dimensional in vitro models poorly demonstrate the aggregation of extracellular protein involved in Alzheimer disease

Neural organoids

Y-27632, SB431532, IWRe1, Dorsomorphin, heparin Matrigel, B27 supplement

Amyloid beta, tau pathology and endosome abnormality is mimicked in fAD organoids, which were responsive to drug treatment

[162]

Investigation of AD was hindered in absence of non-invasive method for harvesting brain tissue from living patients

Cerebral organoids

Y27632, Matrigel

Aβ deposits and Tau phosphorylation was observed in cerebral organoids

[163]

AD organoids should mimic the genetic background of patients and the functional features of AD brain

Cerebral organoid

Y-27632, Matrigel

AD organoids displayed a small size, increased Aβ42/Aβ40 ratio, disrupted calcium homeostasis, and enhanced neuronal activity

[164]

5-Hydroxymethylcytosine contribution to AD pathology has not been determined

Forebrain organoids

FGF, Matrigel, Dorsomorphin, A-83, Heparin CHIR99021,SB-431542, B27, GDNF, BDNF, TFGβ, cAMP

Forebrain organoids mimicked cellular and molecular phenotypes of AD brain. More 5hmC peaks in EBs compared to mature organoids and modulated in intragenic regions

[165]

 Parkinson

2D models do not mimic the neuron- glia interaction in a spatially organized cellular architecture

Midbrain-like organoids

SB-431542, LDN-193189, CHIR99021, SAG, Y-27632, ascorbic acid, Matrigel

Midbrain organoids comprised of dopaminergic neurons, which secrete dopamine. In patient organoids, number and complexity of mDANs was decreased

[36]

Immaturity and heterogeneity of Midbrain organoids (MO) architecture and less efficient protocol

Midbrain organoids

CHIR99021, Noggin, SB431542, Dorsomorphin, A83-01, LDN, FGF8, SAG, Matrigel

MOs comprised of homogenous distribution of dopaminergic neurons, astrocytes and oligodendrocytes

[126]

Mutation in DNAJC6 is associated with early-onset Parkinson’s disease, but role of DNAJC6 in PD pathogenesis was unknown

Midbrain-Like organoids (MLOs)

B27, SB431542, Noggin,CHIR99021, Y27632, purmophamine, SHH-C25II, FGF8, BDNF, GDNF, Matrigel, ascorbic acid

MLOs display death of dopamine neuron, aggregation of α-synuclein, dysfunction of mitochondria and lysosomes

[166]

 Frontotemporal dementia

Role of p25/Cdk5 in tauopathy using organoid model was not known

Cerebral organoids

SB431532, IWRe1,Dorsomorphin, heparin, FBS, matrigel

Tau phosphorylation was reduced and expression of synaptophysin increased in cerebral organoids derived isogenic iPSC lines

[167]

 Zika virus

Inefficient organoid differentiation protocols lead to enormous variability in brain organoids

Cortical organoids and ventral telencephalic organoids

Y-27632, 40% O2, B27 without vitamin A, Growth Factor Reduced Matrigel, heparin, Leukemia Inhibitory Factor

Functional neurons display network-like activities in organoids, Increased progenitor apoptosis leads to smaller organoids size, efficacy of different drug tested

[168]

 Schizophrenia

Functional role of interaction between DISC1 and Ndel1/Nde1 in brain development is difficult to delineate

Forebrain organoids

Dorsomorphine, A83-01, Matrigel, Insulin, WNT-3A, CHIR99021, SB-431542

Cell-cycle defects in radial glial cells in forebrain organoids resulted in reduced proliferation of neural stem cells in the ventricular zone and disturbed neurogenesis

[169]

 Prenatal hypoxic injury

Technical challenge in accessing human fetal brain tissues and inappropriate animal models have led to poor understanding of effects of hypoxia on progenitor steady state and developmental progression during early human brain development

Cerebral organoids

Matrigel, N2 supplement, B27 supplement without vitamin A, insulin

Immense apoptosis in cerebral organoids followed by greater loss of outer radial glia progenitors and differentiating neuroblasts/immature neurons. During hypoxia, NSCs shifted to symmetric division and replete their population

[170]

 Tuberous sclerosis/periventricular heterotopia

Partial recapitulation of human cortex features by mouse models have led to non-suitability of these models for understanding the mechanisms of neuronal heterotopia

Cerebral spheroids

Y-27632, N2 supplement, Heparin, Matrigel, B27 supplement without vitamin A, Insulin

Defective morphology of neural progenitor cells and altered neuronal migration in a subset of neurons

[171]

 Retinitis pigmentosa

Retinal organoids

Matrigel, G418 and/or Ataluren (PTC124)

Massive cell death of rod photoreceptor at day 150 followed by thinning of the outer nuclear layer by day 180 of culture in patient-derived organoids

[172]

 Brain tumor including glioblastoma and central nervous system primitive neuroectodermal tumor

Modeling of brain tumors is limited owing to genetic heterogeneity and non-suitability of animal models

Neoplastic cerebral organoid

bFGF, Y-27632, Matrigel, vitamin A

By using gene editing techniques and generating gain or loss of function phenotypes for several genes, tumorigenesis was established in cerebral organoids

[173]

 Glioblastoma

Inability to recapitulate the cellular and mutational diversity of parental tumors in in vitro models and longer development time of these models.

Glioblastoma organoids (GBOs)

Patient Glioblastoma Tissue, N2 supplement, B27 supplement w/o vitamin A, insulin

Cellular composition of parental tumors recapitulated in GBOs as they display enormous heterogeneity in cell identity and morphology

[95]

Limited donor availability for patient-derived xenograft (PDX) models and findings from mouse genetic models are not translatable to human clinical trials

Cerebral organoids

Y-27632, Heparin, N2 supplement, 1X B27 supplement w/o vitamin A, insulin, Matrigel

Invasive phenotype of genetically modified cells inside the organoid caused massive destruction of surrounding organoid structures, and display tumor pathology upon transplantation in mice

[174]

Limited GBM models due to the lack of a normal human microenvironment and the inability to recapitulate the GBM biology

Cerebral organoid glioma

Y-27632, Heparin, N2 supplement, 1X B27 supplement w/o vitamin A, insulin, Matrigel

regional proliferation followed by massive invasion accompanied by formation of tumor microtubes and phenocopy patient glioblastoma

[175]