Louise Forsberg et al.

Translational Psychiatry (2018)8:14

DOI 10.1038/s41398-017-0062-x Translational Psychiatry

ARTICLE Open Access

Epigenetics and cerebral organoids:

promising directions in autism spectrum
disorders
Sheena Louise Forsberg1, Mirolyuba Ilieva1 and Tanja Maria Michel1,2,3

Abstract
Autism spectrum disorders (ASD) affect 1 in 68 children in the US according to the Centers for Disease Control and
Prevention (CDC). It is characterized by impairments in social interactions and communication, restrictive and
repetitive patterns of behaviors, and interests. Owing to disease complexity, only a limited number of treatment
options are available mainly for children that alleviate but do not cure the debilitating symptoms. Studies confirm a
genetic link, but environmental factors, such as medications, toxins, and maternal infection during pregnancy, as well
as birth complications also play a role. Some studies indicate a set of candidate genes with different DNA methylation
profiles in ASD compared to healthy individuals. Thus epigenetic alterations could help bridging the
gene–environment gap in deciphering the underlying neurobiology of autism. However, epigenome-wide association
studies (EWAS) have mainly included a very limited number of postmortem brain samples. Hence, cellular models
mimicking brain development in vitro will be of great importance to study the critical epigenetic alterations and when
they might happen. This review will give an overview of the state of the art concerning knowledge on epigenetic
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changes in autism and how new, cutting edge expertise based on three-dimensional (3D) stem cell technology
models (brain organoids) can contribute in elucidating the multiple aspects of disease mechanisms.

Introduction individuals to extremely afflicted individuals in need of

Autism is characterized by a clinical triade of symptoms lifelong support and care (World Health Organization).
such as impairment of social interactions and commu- Autism is the psychiatric disorder with the highest her-
nication as well as restrictive and repetitive patterns of itability rate, the concordance rate for monozygotic twins
behaviors and interests. Lately, the Diagnostic and Sta- is >90%2, and many susceptibility genes have been iden-
tistical Manual of Mental Disorders 5th edition (American tified during the past decade. Moreover, there is evidence
Psychiatric Association, 2013)1 has classified infantile of environmental factors, such as, e.g., hypoxia during
autism, Asperger’s syndrome, childhood disintegrative birth, contributing3. There is a high prevalence of autistic
disorder, and pervasive developmental disorders not symptoms in syndromes with chromosomal aberrations,
otherwise specified (PDD-NOS) as autism spectrum dis- such as Fragile X syndrome and Tuberous Sclerosis4.
order (ASD), usually appearing before the age of 3 years. However, even in those disorders following the classical
The term “spectrum” denotes a variety of symptoms and genetic Mendelian rules, a penetrance of <50% has been
severity from mildly afflicted, highly functioning reported, indicating that epigenetic factors might play an
important role in explaining at least part of the neuro-
biology of autism5.
Correspondence: Mirolyuba Ilieva (milieva@health.sdu.dk) Epigenetic alterations are defined as non-permanent
1
Department of Psychiatry, Institute for Clinical Research University of Southern and potentially heritable changes that regulate the
Denmark Odense Denmark
2
Department of Psychiatry, Psychiatry in the region of Southern Denmark,
expression of genes through alterations to the shape and
Odense, Denmark configuration of DNA, rather than nucleotide sequence.
Full list of author information is available at the end of the article

© The Author(s) 2017

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Louise Forsberg et al. Translational Psychiatry (2018)8:14 Page 2 of 11

Zygote

Imprinng of parental
Placental dysfuncon genes
Maternal metabolism

Prenatal epigenec programming

Maternal nutrion Severe infecons

Toxic exposure
Oxidave stress

Fig. 1 Prenatal epigenetic programming. Several prenatal factors could lead to epigenetic dysregulation in ASD. In utero development could be
affected by maternal environmental conditions, such as maternal toxin exposure, severe infections, placental dysfunction and hormonal disruptions,
oxidative stress, maternal nutrition, and metabolic imbalance. This can result in activation of adaptation mechanisms for survival and growth
restriction, which in turn leads to alteration in cellular organization through disturbed proliferation/maturation balance; epigenome modification via
DNA methylation, histone modification, and RNA interference; and alteration in cellular metabolic pathways

This leads to changes in the ability of certain genes to be includes RNA silencing complexes that, e.g., bind to
transcribed—meaning, that the chromatin threads carry- mRNA and blocks the ribosome, thereby promoting gene
ing the genetic information can unwind, coil more tightly, silencing.
loop, and interact with other proteins to turn certain Epigenetic mechanisms are involved in regulating the
genes on or off. Epigenetic control can, besides from prenatal development by directing processes, such as cell
direct DNA modification6, also take the form of mod- proliferation and differentiation, as well as tissue specifi-
ifications in the three dimensional (3D) structure and cation9. Epigenetic alterations are partly due to environ-
packaging of DNA, histones, and noncoding RNA-related mental factors, which thereby affect the phenotype by
factors7. Chromatin is a dynamic structure. Its accessi- modulating gene expression7. Since the underlying causes
bility for transcription factors is determined by certain of ASD remain elusive, epigenetic alterations take the role
modifications, including DNA methylation as well as of the environment with regards to gene expression into
acetylation, phosphorylation, ubiquitination, and methy- account. Certain environmental conditions or fluctuations
lation of histones. They could modify histone proteins, can stimulate epigenetic changes of the genome (Fig. 1).
nucleosome movement, and even larger genomic regions. Therefore, epigenetic modulations are a promising can-
This change in histone modifications has been pointed to didate to explain the complex neurobiology leading to
as being the gene–environment interface along with DNA ASD. Research involving rodents showed that rat pups
methylation6, 8. In the case of DNA methylation, a methyl receiving limited maternal care resulted in the change of
group (-CH3) is added to cytosine, leading to gene the expression of stress-related genes. Those alterations
silencing. Chromatin remodeling could entail sliding of were also maintained in future generations10. Similarly,
the nucleosome cores by the disassembling/reassembling the effect of maternal care on gene expression has also
of the core and could either induce or repress expression. been seen in nonhuman primates11. Moreover, research
Histone modifications involves amino acids on the on human subjects has revealed that childhood abuse
terminal end that can bind to methyl, acetyl, phosphate, alters DNA methylation patterns in the brain, resulting in
or ubiquinone, and the most known effect being that of epigenetic changes to the gene expression12. Furthermore,
acetylation of lysine in which chromatin is opened and a dysregulation of growth factors has been found in a
transcription induced. RNA interference is a process that number of adults with ASD. These growth factors are

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Louise Forsberg et al. Translational Psychiatry (2018)8:14 Page 3 of 11

involved in neuronal growth, differentiation, proliferation, Although the epigenetic mechanisms involved in autism
and survival in the course of neurodevelopment and can are not yet fully understood, there are findings suggestive
also modulate axonal and dendritic outgrowth13–18. DNA of genome-wide dysregulation and epigenetic alterations
methylation of the brain-derived neurotrophic factor in ASD33. These studies point to DNA methylation as a
(BDNF) gene has for instance been linked to early life likely contributor in the development of the disorder34.
adversity, including autism13. Epigenetic processes and There are certain syndromes that have been linked to
the interaction with environmental conditions might help ASD. DNA methylation in connection to imprinting and
to explain the differences in gene expression patterns in X-chromosome inactivation could be relevant to the field
ASD patients. It is still not entirely clear which environ- of ASD research. X-chromosome inactivation is a process
mental factors are responsible for the epigenetic changes in which one of the copies of X chromosomes is inacti-
seen in ASD, but many have been suggested: environ- vated and this is also achieved through DNA methylation.
mental toxins, parental age (especially of fathers), mater- It might be associated with autism, as inactivation or
nal diet, and chemical exposure19–22. An environmental removal of inactivation could lead to genetic aberrations.
factor that has been suggested is the maternal use of An example for this is the formation of tiny “X rings” in
valproic acid (an anticonvulsant and potent histone dea- females. These rings prevent the inactivation of genes
cetylase inhibitor) in early pregnancy23. The maternal use within it resulting in an overexpression and could fur-
of valproic acid during pregnancy could affect the pro- thermore end in developmental abnormalities such as
duction of GABAergic neurons via the blocking of histone those seen in certain Turner syndrome cases—a minority
deacetylase24. A study also found that severe maternal with mental retardation and developmental delay35. The
viral infection in the first trimester of the pregnancy and deficits seen in this syndrome are most commonly related
bacterial infection in the second trimester was associated to social reciprocity and communication; in addition to
with a diagnosis of ASD in the child25. that, it is also associated with an increase in the risk of
Along similar lines to the gene–environment interaction autism especially in incidences in which the maternally
is the investigation of oxidative stress’ role in ASD. One inherited X chromosome was retained36, 37. The authors
recent study (total n = 189) including adults with ASD (n surmised that this was due to an X-linked imprinted gene
= 92) pointed to increased oxidative stress in the patient’s and the expression of which affecting the risk of autism36.
group, in that it found significantly increased levels of DNA methylation is also involved in long-distance chro-
superoxide dismutase 1 (although not superoxide dis- mosomal interactions, in which methylation signals
mutase 2 or xanthine oxidase (XO))26. Promising results interact with other chromosomes or faraway parts of the
suggest a major role of oxidative stress in the neurobiol- same chromosome to alter gene expression. This phe-
ogy of other neurodevelopmental and psychiatric dis- nomenon can be seen in chromatin looping, in which
orders, which are often comorbid with autism27–32 such as gene sequences are brought together by the formation of a
depression27, 29, 30 and schizophrenia28, 31. There have loop, enabling long-distance gene activation38.
been several reports connecting depressive disorders and Imprinting is a differential inhibition of gene expression
oxidative stress27. The pro-oxidant XO has been studied (imprinted genes are not expressed) depending on its
in this regard and been found to be elevated in both parental origin. In this way, imprinting controls the
schizophrenia28 and depression29. Another study investi- expression of either the paternal or the maternal gene in
gated whether oxidative stress could contribute to the the offspring39. Imprinting’s role in ASD has been sug-
brain structural changes seen in patients with recurrent gested in several instances36, 37, 40, 41, and it is interesting
depressive disorder30. This study, consisting of seven to note that the imprinted domain at 15q11-13 is
patients and seven healthy controls, then compared responsible for Angelman syndrome42 as well as
manganese (Mn) and copper/zinc (Cu/Zn) superoxide Prader–Willi syndrome43. In the case of Angelman syn-
dismutase (SOD) concentrations in postmortem pre- drome, children suffering from the syndrome also tend to
frontal cortex and hippocampal brain tissue. The study be diagnosed with ASD40, 41, 44. One of these studies
found that the concentration of Cu/Zn-SOD was investigated the prevalence of Angelman Syndrome in
increased in the prefrontal cortex although not in the school children and analyzed its comorbidity with autistic
hippocampus of the patients30. Another study investigated disorder and found that 4 out of the almost 49,000 con-
impaired oxidative stress defense in schizophrenia spec- formed to a diagnosis of Angelman syndrome and that all
trum disorder by determining the concentrations of Cu, 4 also met full behavioral criteria for a diagnosis of autistic
Zn and MnSOD in postmortem brain tissue31. This study disorder40. Another study investigated 23 Angelman
found significantly elevated levels of Cu, Zn and MnSOD syndrome patients and made a comorbid diagnosis of
levels in frontal cortex and substantia innominata of the ASD in 14 out of the 23 patients (61%)41. Angelman
samples31. syndrome is associated with a maternal deletion in the
chromosomal region 15q11-13 affecting UBE3A (related

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 Table 1 Epigenome-wide studies and findings

Study description Tissue Participants Findings

EWAS

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Ginsberg Genotyping and DNA methylation Brain: cerebellar and occipital tissue 9 ASD individuals and 9 Found no changes in DNA methylation between the groups. Found
et al.103 (DNAm) sequencing controls downregulation of genes of MOP and protein translation
Ladd-Acosta DNAm Brain: DLPC, temporal cortex and 19 autism cases and 21 controls Found 4 significant DMRs in: TSPAN32, PRRT1, ZFP57, SDHAP3
et al.48 cerebellum
Nardone et al.49 DNAm Brain: nterior cingulate gyrus and ACG: 11 ASD cases and 11 Found 5329 DMPs with significant methylation changes compared to
prefrontal cortex controls controls. Found brain regions less epigenetically varied than the
controls. Implicated genes HDAC4and C11ORF21
PFC: 12 ASD cases and 12
controls
Peripheral tissues
Nguyen et al.104 Global methylation profile of LCLs Lymphoblastoid cell line 3 pairs of twins discordant in 2 candidate genes, BCL-2 and RORA, exhibited hypermethylation of
Louise Forsberg et al. Translational Psychiatry (2018)8:14

autism specific CpG sites (gene silencing)

105
Wang et al. DNAm in PBCs Blood 5 ASD children and 5 age/sex- Indicated ENO2 to a subset of autism and pointed to as potential
matched controls biomarker. This association has not been found since
Wong et al.50 Differentially methylated CpG sites Blood 50 pairs of monozygotic twins Genes found to be differentially methylated: NFYC, DUSP2
of whole blood samples
Similarities in PXDN and C11ORF1 in at least two discordant twins
MDPs in MGC3207, OR2L13, FAM181A
51
Berko et al. DNAm Buccal epithelium 47 ASD cases and 48 controls Found ASD-specific DMRs. A DMR in the OR2L13 gene promoter of the
ASD-specific DMP also found in peripheral blood
HAWAS
Sun et al.106 Histone acetylation population Brain: prefrontal and temporal 45 ASD samples and More than 68% of syndromic and idiopathic ASD cases share common
study cortex, cerebellum 49matched controls acetylome signature in the prefrontal and temporal cortex

DNAm DNA methylation, MOP mitochondrial oxidative phosphorylation, DLPC dorsolateral prefrontal cortex, DMR differentially methylated region, ACG anterior cingulate gyrus, PFC prefrontal cortex, DMP differential
methylated position, LCL B-cell derived lymphoblastoid cell lines, PBC peripheral blood cell, HAWAS histone acetylome-wide association study

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Louise Forsberg et al. Translational Psychiatry (2018)8:14 Page 5 of 11

to ubiquitine pathway) expression while the paternal allele cingulate cortex49. Tetraspanin 32 gene/chromosome 11
is silenced42. Prader–Willi syndrome is, on the other hand, open reading frame 21(TSPAN32/C11ORF21) has also
caused by the deletion of the parental copies of the been shown to have lower methylation levels in a DMR in
SNRPN located in the same region (15q11-13). At the the temporal cortex and cerebral cortex48, along with
same time, due to imprinting the maternally inherited lower methylation in the anterior cingulate gyrus and
copies are silenced. This is linked to psychotic episodes in prefrontal cortex49. Olfactory receptor family 2 subfamily
the cases of maternal uniparental disomy or imprinting L member 13 (OR2L13) is a special case having shown
abnormalities43. Also worth mentioning is that the 15q11- both significantly increased and decreased methylation in
13 imprinting region has been associated with the studies50, 51. Although they have provided insights, there
increased incidence of autism when maternally dupli- are also some associated issues with the EWAS studies
cated45. This study conducted microsatellite and methy- conducted on postmortem brain samples. The most
lation analyses of three children and their parents, in fundamental issue is the uncertainty in how many con-
which two of the children had autism and the third did clusions can be drawn from postmortem samples to a
not. It was found that the two individuals had a maternal disorder that arises through the course of development52.
inheritance of a 15q11-13 duplication while the unaf- The dependency on bio-banks also limits the number of
fected child did not have this duplication. Interestingly, a samples one can attain. All of the aforementioned studies
recent study conducted on mice might have discovered had fewer than 100 samples and should optimally include
the first proof of principle with regards to epigenetic- more for increased power of the study. There are only a
based therapy, in particular Prader–Willi syndrome46. The limited number of samples obtainable from bio-banks.
study found that two selective inhibitors (UNC0638 and Therefore, cell culture systems could offer a viable alter-
UNC0648) activated imprinted genes on the maternal native and will be discussed later on.
chromosome of cells usually repressed in those with the Even as genome-wide approaches seem to be the focus
disorder. Considering this discovery as a possible in most studies, candidate genes (see Table 2) and their
epigenetic-based treatment of Prader–Willi46, imprinting methylation status could still offer valuable insights into
might yet provide some clues to the etiology and possible the neurobiology of ASD. Although certain genes appear
treatments for ASD as well. to be involved in the disorder, investigating epigenetic
In summary, epigenetic processes are multifaceted and alterations will contribute to get a better understanding of
can be connected to multiple genes or genetic pathways. the complex mechanisms involved. Differences in
This could make it difficult to identify the essential targets methylation have been observed in different areas of the
and pathways involved in epigenetic changes. It is believed brain47, 53–59, and the CpG island (genomic regions >200
that epigenetic processes play a major role in brain bp in length with high CpG sites—in which a cytosine
development and neurodevelopmental disorders47. For all nucleotide is followed by a guanine nucleotide in the
the genetic insights that have been gained, we still do not linear sequence of bases) known to regulate the oxytocin
have a full picture of the etiological basis of ASD. It is only receptor (OXTRA) has presented increases in the DNA
recently that more emphasis has been put on environ- methylation status in the temporal cortex as well as in
mental factors and this means that epigenetic mechan- blood (plasma)47. Further studies of, e.g., methylation
isms should also be investigated to further ASD status in blood and other more easily obtained peripheral
knowledge. tissues could possibly result in finding helpful biomarkers
for the disorder. The biomarkers could assist the diag-
Epigenome-wide analysis and candidate genes in nostic process and improve the prognosis, as well as
ASD possibly identifying individuals at risk of ASD eventually
Several studies have focused on DNA methylation levels leading to preventive measures. Other epigenetic
on a genome-wide scale (or EWAS) and one very recent mechanisms, aside from DNA methylation itself, have not
study has investigated histone acetylation (see Table 1). been investigated nearly as closely and could be involved
These EWAS studies have identified potential bio- in the pathogenesis as well.
markers that could be useful in future studies and
implicated certain brain regions with signs of epigenetic Induced pluripotent stem cells and cerebral
dysregulation. Regarding proline-rich transmembrane organoids: models for studying epigenetics
protein (PRRT1), a study found lower methylation of a in vitro
differentially methylated region (DMR) in the regions of The neurobiology leading to ASD might already start
the temporal cortex, cerebellum, and prefrontal cortex48. early in the prenatal stages. Therefore, mimicking the
Similarly, a region related to zinc finger protein 52 prenatal development in vitro can give extremely valuable
(ZFP52) showed higher methylation of a DMR in the insight into the etiology of autism. In addition to that, the
temporal cortex48, as well as higher methylation of the challenges in research using postmortem brain tissue

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Table 2 Candidate genes and suggested mechanisms

Candidate Function of the gene Possible epigenetic mechanisms

gene

OXTR G-protein coupled receptor for oxytocin. Modulates: stress, Hypermethylation and silencing47
anxiety, social memory, maternal–offspring behavior, etc. Decreased OXTR expression

GABRB3 Responsible for a protein that is a part of the gamma- Dysregulation of imprinting or issues in pairing of the homologous
aminobutyric acid-A receptor. Regulates the neurotransmitter alleles108 via disruption of long-distanced chromosomal
gamma-aminobutyric acid (GABA) and plays a role in synaptic interactions109–111
107
function Decreased expression

UBE3A Known for its role in Angelman syndrome Loss of imprinting of one copy, and production of antisense RNA
that binds to UBE3A and mRNA Prevents translation112
Involved in the maintenance of synaptic plasticity and central
for experience-dependent modifications in the brain113
GAD1 Encodes an enzyme that catalyzes the decarboxylation of Increased hydroxymethylation and binding of MeCP2 (silencing) in
glutamate to GABA, the main inhibitory neurotransmitter GAD1 promoters53
Decreased expression
EN2 Encodes a homeodomain-containing protein and thought to Hypermethylation and hydroxymethylation54, 115

play a role in controlling pattern formation during Increased EN2 gene expression and translation
development of the central nervous system114

RELN Regulates neuronal migration and positioning in the Enriched levels of 5-hmC at RELN promoter, increased binding of
developing brain by way of cell–cell interactions MeCP2 to 5-hmC
Regulates synaptic plasticity by enhancement of the induction Reduced gene expression and translation53, 116, 117

and maintenance of long-term potentiation

MECP2 Encodes a methyl binding protein that binds methylated areas Several: decreased MECP2 expression119, 120
. Decreased MECP2hi
of DNA to silence genes. Has a role in synaptogenesis and long- protein cells associated with methylation of MECP2 promoter55,
term synaptic plasticity118 121–123

Inability to define methylation and X-inactivation borders56

Associated with Rett syndrome Unlcear X-inactivation role56, 124, 125

Other: MECP2 regulation of other genes via epigenetics:

recruitment of co-repressors, chromatin looping126, 127

OXTR oxytocin receptor, GABRB3 gamma-aminobutyric acid-A receptor, UBE3A ubiquitin-protein ligase E3A gene, GAD1 glutamate decarboxylase, EN engrailed 2, RELN
Reelin, MECP2 methyl CpG-binding protein 2

warrants the utilization of other viable disease models, of iPSCs, for example: keratinocytes61, epithelial bladder
such as cell cultures, and in vivo recapitulation of brain cells derived from urine62 as well as from hair follicles63.
development and related pathology on the basis of new, IPSCs and derived differentiated cells retain the genetic
cutting edge technology involving induced pluripotent signature of the individual that they have been taken from
stem cells (iPSCs). IPSCs refer to somatic cells that have and thus they represent a relevant disease model. IPSCs
been reprogrammed to a pluripotent stage and then can have also been utilized in ASD research64. The respective
be triggered to differentiate into all cell types in the study found increased rates of proliferation of neural
human body, including neurons. The technology is rela- progenitor cells and neuron numbers in the ASD indivi-
tively new; for the first time, it became clear about a dual IPSCs who also had an increase of brain volume as
decade ago that iPSCs could be generated via a set of seen on magnetic resonnce imaging. The increase in
transcription factors introduced to fibroblasts in vitro60. proliferation was due to dysregulation of the beta-catenin/
Other somatic cells have also been used in the generation BRN2 transcriptional cascade, and it seemed that the

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defects in neuronal networks could be rescued by insulin with in vivo settings, as well as increased drug sensitivity
growth factor 164. (due to monolayer) and exposed surface.
Along with the advantages, there are also some addi- The development of stem cell technology gave rise to
tional challenges regarding iPSCs. Since it is a new tech- the possibility of growing and differentiating iPSCs into
nique, there is limited availability of “bio-banks”. small 3D structures called organoids. Cerebral organoids
Additionally, the cells themselves may present different follow the in vivo timeline and can recapitulate the early
phenotypes in two lines derived from the same person65. (8–10 gestation week) to mid-fetal (22–24/35 gestation
A possible solution to this issue could be the generation of week) of human brain development. Depending on the
several clones of each patient iPSC line but at additional presence or absence of growth factors and morphogens,
time and cost. Reprogramming iPSCs has also been found different region-specific organoids can be formed such as
to introduce mutations of the genomic DNA as well as the forebrain, midbrain, cortex, and hypothalamus.
exogenous reprogramming genes66. iPSCs sustain an Moreover, brain organoids can exhibit a well-defined sub
epigenetic memory, essentially maintaining residual DNA ventricular zone (SVZ) and six-layered cortical archi-
methylation from the tissue of origin67. Most iPSCs are tecture typical for the human brain. However, the brain
derived from differentiated somatic cells, namely, fibro- cortex and SVZ in the mid-fetal development are the
blasts. This should be taken into account in the epigenetic main targets for modeling ASD. This makes cerebral
studies of central nervous system in which iPSCs are used organoids a valuable new tool for neurodevelopmental
as a model and transdifferentiation is considered prefer- disorders. The organoid gene expression program is
able to eliminate this challenge. There have also been highly similar to human fetal tissue78–84.
reports on prolonged culturing affecting the tran- The organoids more closely resemble the cells' natural
scriptomes and the phenotypes of cells68. Throughout the environment, demonstrating cellular variety and inter-
course of life, somatic cells (fibroblasts) accumulate cellular communication, cell–matrix interactions, and
somatic mutations. Individual clones from the same per- complex transport systems of nutrients85–91. 2D mono-
son could host different somatic mutations, as in the case layer cultures are not relevant in this aspect, leading them
of mitochondrial DNA in iPSC neurons69. The study to be poorer determinants in drug and toxicity studies92–
95
reports that somatic mutations could randomly occur in . 3D cultures are a relevant model of cell migration,
individual cells and those mutations of mitochondrial differentiation, and growth86–91, 96. Furthermore, 3D cell
DNA in iPSCs had a tendency to increase along with the cultures have a different gene expression compared to
age of the donor. Another study found that iPSC genomes those grown in 2D83, 87, 97. There seems to be a dysre-
potentially exhibit imperfectly rewired 3D folding linked gulation of morphology, response to environmental stress
to inaccurately reprogrammed gene expression70. How as well as cellular regulation in 2D cultures compared to
much this chromatin architecture is reorganized in the 3D cultures98. This could limit the use of 2D cell culture
process of reprogramming remains uncertain. As it was in the search for epigenetic regulation in ASD since
mentioned before, a possible way around the dis- comparisons would be harder to make at the interface of
advantages could be achieved by removing the iPSC step gene–environment.
as the midway, i.e., transdifferentiation71, 72. In this The 3D cell cultures offer several advantages such as:
method, fibroblasts are directly converted into induced the cytoarchitecture is similar to that found in vivo,
neural progenitor cells by way of timely restricted variety of cellular populations, organ specific functions,
expression of the following genes: Sox2, Klf4, and c-Myc and niche-like environment. Carrying the specific genetic
and limited and very strictly controlled expression of signature from the individual the cells were taken from,
Oct473. Another method based on forced expression of they can be utilized to compare the early brain structure
neurogenin 2 (Ngn2) turns skin fibroblasts into functional and composition with ASD to that of their healthy family
neurons74. Treatment of Ngn2-transduced fibroblasts members as well as healthy controls. This means that
with a mix of small molecules including SB431542 (a researchers to a certain extent are able to model neuro-
transforming growth factor-β inhibitor), CHIR99021 (a development in vitro and re-create the brain pathology
GSK3β inhibitor), and Noggin (a protein inhibitor of bone related to ASD. This could also aid in elucidating the
morphogenic protein signaling) increase conversion effi- interface of gene expression levels and epigenetic chan-
ciency up to 85%75. ges99–101. The disadvantages related to this method
Two-dimensional (2D) cell cultures of iPSC and derived include: diffusional transport limitations (oxygen, nutri-
neurons offer easier environmental control, cell manip- ents), culture-dependent alterations in gene expression,
ulation, and imaging; it is homogenic and has fair repro- technical challenges in manipulation and imaging, stan-
ducibility76, 77. Along with the advantages, there are also a dardization and reproducibility issues, as well as being
set of disadvantages, including an inability to depict traits time and labor consuming.
exhibited in vivo (e.g., gene expression), less compatibility

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ASD
Familial Idiopac Controls

Somac cells

iPSC

Brain organoids

Genomics
Epigenomics
Microenvironment
Transcriptomics Cellular organizaon
Oxidave stress
Proteomics and Communicaons
Hypoxia
Metabolomics

Molecular mechanisms of disease

Biomarkers

Diagnosc
Targeted individualized treatment
Prevenon
Fig. 2 Schematic presentation of a brain organoid-focused study, with groups consisting of familial ASD cases, idiopathic cases, and a
control group. Somatic cells (for example, fibroblasts) can be obtained from the participants and reprogrammed into iPSC and furthermore into
brain organoids specific for each individual. This could enable an in vitro multi-omics studies (genomics, transcriptomics, proteomics, epigenomics,
metabolomics), oxidative stress, cellular organization, etc., hopefully, unveiling a molecular mechanism of the disorder that in the future can lead to
better tools in diagnosis, prevention, and treatment

It should also be noted that the method still has an issue bioreactor-based batch effects as part of the explanation
of “batch syndrome” in which different batches of orga- for the organoid-to-organoid variability86.
noids demonstrate significant variability in quality and A recent study has conducted the first transcriptome
brain regions they produce102. A very recent study ana- and epigenome-wide sequencing of cerebral organoids by
lyzed 80,000 individual cells isolated from 31 human brain comparing them to human fetal brain, using genome-
organoids. In addition to finding that organoids could wide, base resolution DNA methylome and transcriptome
generate a broad diversity of cells, they could also be sequencing83. Particularly, organoid differentiation was
developed over an extended period of time leading to the able to recapitulate the transcriptomic dynamics of early/
formation of dendritic spines as well as spontaneously middle fetal development. DNA methylation of cerebral
active neuronal networks. Furthermore, this study also organoids found a new kind of mCH enrichment (cytosine
quantified variability among organoids and pointed to DNA methylation in non-CG contexts) that was indica-
tive of transcriptional repression of the later brain in vivo.

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Louise Forsberg et al. Translational Psychiatry (2018)8:14 Page 9 of 11

Hypermethylation of DNA methylation valleys, regions of Dahl Algot for technical support as well as Graham Dauncey for proof reading
low methylation of at least 5 kb in length (DMVs), served the article as a native speaker.

as a robust epigenomic signature of forebrain specifica- Author details

tion. Pervasive demethylation of pericentromeric repeats 1
Department of Psychiatry, Institute for Clinical Research University of Southern
were found in human neural cultures that are not Denmark Odense Denmark. 2Department of Psychiatry, Psychiatry in the
region of Southern Denmark, Odense, Denmark. 3Odense Center for Applied
observed during fetal brain development. The last aspect Neuroscience BRIDGE, University of Southern Denmark, Psychiatry in the
points to the importance of genomic/epigenomic methods Region of Southern Denmark Odense University Hospital Odense Denmark
in the evaluation of in vitro-derived neuronal tissues vs.
in vivo. The study came to the conclusion that the Competing interests
The authors declare that they have no competing financial interests.
remodeling of mCG and mCH during the organoid dif-
ferentiation resulted in an epigenomic state very similar to
the human fetal brain83. It is for the moment not clear Publisher's note
how epigenetic patterns of brain organoids could be Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
verified by comparison with a human ES line and this
should warrant further studies and be addressed. Received: 4 August 2017 Accepted: 26 October 2017
Studies concerned with iPSC epigenetic signatures
could aid in the investigation of alterations in epigenetic
regulation of gene expression in brain development.
There is also added hope that this technique will in the References
future aid in drug discovery research (see Fig. 2). 1. American Psychiatric Association. Diagnostic and Statistical Manual of Mental
Disorders 5th edn, (Washington, DC, 2013).
2. Steffenburg, S. et al. A twin study of autism in Denmark, Finland, Iceland,
Conclusions Norway and Sweden. J. Child Psychol. Psychiatry 30, 405 (1989).
Several findings suggest that epigenetic alterations play 3. Michel, T. M. et al. [Evaluation of diagnostic and therapeutic services in
German university hospitals for adults with autism spectrum disorder (ASD)].
a role in the development of ASD and further research is Fortschr. Neurol. Psychiatr. 78, 402 (2010).
therefore warranted. This review points out that there is a 4. Muhle, R., Trantacoste, S. V. & Rapin, I. The genetics of autism. Pediatrics 113,
necessity to carry out further EWAS studies. These should e472 (2004).
5. Abrahams, B. S. & Geschwind, D. H. Genetics: advances in autism genetics: on
include larger numbers of patients who should be clini- the threshold of a new neurobiology. Nat. Rev. 9, 341 (2008).
cally well characterized, as well as investigate other, dif- 6. Qureshi, I. A. & Mehler, M. F. Epigenetic mechanisms underlying the
ferent epigenetic mechanisms as the majority of research pathogenesis of neurogenetic diseases. Neurotherapeutics 11, 708 (2014).
7. van Vliet, J., Oates, N. A. & Whitelaw, E. CMLS: epigenetic mechanisms in the
has been focused on DNA methylation. The potential context of complex diseases. Cell. Mol. Life Sci. 64, 1531 (2007).
biomarkers, e.g., C11ORF21 and PRRT1, identified in 8. Meaney, M. J. & Ferguson-Smith, A. C. Epigenetic regulation of the neural
these studies should also be replicated. In addition, iPSCs transcriptome: the meaning of the marks. Nat. Neurosci. 13, 1313 (2010).
9. Gropman, A. L. & Batshaw, M. L. Epigenetics, copy number variation, and
and cerebral organoids could aid in bypassing the issue of other molecular mechanisms underlying neurodevelopmental disabilities:
obtaining sufficient postmortem brains, enabling a “dis- new insights and diagnostic approaches. J. Dev. Behav. Pediatrics 31, 582
order in a dish-setting”. Moreover, brain organoids are (2010).
10. Meaney, M. J. Maternal care, gene expression, and the transmission of
patient specific and can be utilized in the development of individual differences in stress reactivity across generations. Annu. Rev. Neu-
targeted personalized treatments. There are, however, rosci. 24, 1161 (2001).
some issues regarding this as well including, but not 11. Higley, J. D., Hasert, M. F., Suomi, S. J. & Linnoila, M. Nonhuman primate
model of alcohol abuse: effects of early experience, personality, and stress on
limited to, epigenetic memory of the tissue of origin in alcohol consumption. Proc. Natl. Acad. Sci. 88, 7261 (1991).
addition to issues regarding mutations and differing 12. McGowan, P. O. et al. Epigenetic regulation of the glucocorticoid receptor in
phenotypes from the same sample. For all the challenges human brain associates with childhood abuse. Nat. Neurosci. 12, 342 (2009).
13. Nickl-Jockschat, T. & Michel, T. M. The role of neurotrophic factors in autism.
posed, there are new findings that offer some valuable Mol. Psychiatry 16, 478 (2011).
clues and that might guide the field forward, the most 14. Thome, J. et al. Ciliary neurotrophic factor (CNTF) genotypes: influence on
recent advance being the first epigenomic sequencing of choline acetyltransferase (ChAT) and acetylcholine esterase (AChE) activities
and neurotrophin 3 (NT3) concentration in human post mortem brain tissue.
cerebral organoids and a newly acquired understanding of J. Hirnforsch. 38, 443 (1997).
how closely they could resemble the human brain. 15. Michel, T. M. et al. Altered glial cell line-derived neurotrophic factor (GDNF)
Studying organoids and the epigenetic alterations in the concentrations in the brain of patients with depressive disorder: a com-
parative post-mortem study. Eur. Psychiatry 23, 413 (2008).
different brain structures during embryonal development 16. Durany, N. et al. Brain-derived neurotrophic factor and neurotrophin 3 in
might open up new avenues for new treatments targeting schizophrenic psychoses. Schizophr. Res. 52, 79 (2001).
specific brain structures. 17. Durany, N. et al. Brain-derived neurotrophic factor and neurotrophin-3 levels
in Alzheimer’s disease brains. Int. J. Dev. Neurosci. 18, 807 (2000).
18. Nickl-Jockschat, T. & Michel, T. M. [Genetic and brain structure anomalies in
autism spectrum disorders. Towards an understanding of the aetiopatho-
Acknowledgements
genesis?]. Nervenarzt 82, 618 (2011).
We gratefully thank the Psychiatry Research Fund in Region of Southern
Denmark and BRIDGE Project for financial support, Morad Kamand and Niclas

Translational Psychiatry

Content courtesy of Springer Nature, terms of use apply. Rights reserved

Louise Forsberg et al. Translational Psychiatry (2018)8:14 Page 10 of 11

19. Chaste, P. & Leboyer, M. Autism risk factors: genes, environment, and gene- 48. Ladd-Acosta, C. et al. Common DNA methylation alterations in multiple brain
environment interactions. Dialogues Clin. Neurosci. 14, 281 (2012). regions in autism. Mol. Psychiatry 19, 862 (2014).
20. Dolinoy, D. C., Weidman, J. R. & Jirtle, R. L. Epigenetic gene regulation: linking 49. Nardone, S. et al. DNA methylation analysis of the autistic brain reveals
early developmental environment to adult disease. Reprod. Toxicol. 23, 297 multiple dysregulated biological pathways. Transl. Psychiatry 4, e433 (2014).
(2007). 50. Wong, C. C. Y. et al. Methylomic analysis of monozygotic twins discordant for
21. Jaenisch, R. & Bird, A. Epigenetic regulation of gene expression: how the autism spectrum disorder and related behavioural traits. Mol. Psychiatry 19,
genome integrates intrinsic and environmental signals. Nat. Genet. 03, 245 495 (2014). 04.
(2003). 51. Berko, E. R. et al. Mosaic epigenetic dysregulation of ectodermal cells in
22. Landrigan, P. J., Lambertini, L. & Birnbaum, L. S. A research strategy to dis- autism spectrum disorder. PLoS Genet. 10, e1004402 (2014).
cover the environmental causes of autism and neurodevelopmental dis- 52. Ardhanareeswaran, K., Coppola, G. & Vaccarino, F. The use of stem cells to
abilities. Environ. Health Perspect. 120, a258 (2012). study autism spectrum disorder. Yale J. Biol. Med. 88, 5 (2015).
23. Rasalam, A. D. et al. Characteristics of fetal anticonvulsant syndrome asso- 53. Zhubi, A. et al. Increased binding of MeCP2 to the GAD1 and RELN pro-
ciated autistic disorder. Dev. Med. Child Neurol. 47, 551 (2005). moters may be mediated by an enrichment of 5-hmC in autism spectrum
24. Akhtar, M. W. et al. Histone deacetylases 1 and 2 form a developmental disorder (ASD)cerebellum. Transl. Psychiatry 4, e349 (2014).
switch that controls excitatory synapse maturation and function. J. Neurosci. 54. James, S. J., Shpyleva, S., Melnyk, S., Pavliv, O. & Pogribny, I. P. Complex
29, 8288 (2009). epigenetic regulation of engrailed-2 (EN-2) homeobox gene in the autism
25. Atladóttir, H. O. et al. Maternal infection requiring hospitalization during cerebellum. Transl. Psychiatry 3, e232 (2013).
pregnancy and autism spectrum disorders. J. Autism Dev. Disord. 40, 1423 55. Nagarajan, R. P., Hogart, A. R., Gwye, Y., Martin, M. R. & LaSalle, J. M. Reduced
(2010). MeCP2 expression is frequent in autism frontal cortex and correlates with
26. Bækgaard Thorsen, M. et al. Oxidative stress – a promising candidate in aberrant MECP2 promoter methylation. Epigenetics 1, e1 (2006).
explaining the neurobiology of autism spectrum disorders. Eur. Psychiatry 33, 56. Nagarajan, R. P. et al. MECP2 promoter methylation and X chromosome
S182 (2016). inactivation in autism. Autism Res.1, 169 (2008).
27. Michel, T. M., Pülschen, D. & Thome, J. The role of oxidative stress in 57. Jack, A., Connelly, J. J. & Morris, J. P. DNA methylation of the oxytocin
depressive disorders. Curr. Pharm. Des. 18, 5890 (2012). receptor gene predicts neural response to ambiguous social stimuli. Front.
28. Michel, T. M. et al. Alteration of the pro-oxidant xanthine oxidase (XO) in the Hum. Neurosci.6, 280 (2012).
thalamus and occipital cortex of patients with schizophrenia. World J. Biol. 58. Yip, J., Soghomonian, J. J. & Blatt, G. J. Decreased GAD67 mRNA levels in
Psychiatry 12, 588 (2011). cerebellar Purkinje cells in autism: pathophysiological implications. Acta
29. Michel, T. M. et al. Increased xanthine oxidase in the thalamus and putamen Neuropathol. 113, 559 (2007).
in depression. World J. Biol. Psychiatry 11, 314 (2010). 59. Yip, J., Soghomonian, J. J. & Blatt, G. J. Increased GAD67 mRNA expression in
30. Michel, T. M. et al. Evidence for oxidative stress in the frontal cortex in cerebellar interneurons in autism: implications for Purkinje cell dysfunction. J.
patients with recurrent depressive disorder--a postmortem study. Psychiatry Neurosci. Res. 86, 525 (2008).
Res. 151, 145 (2007). 60. Takahashi, K. et al. Induction of pluripotent stem cells from adult human
31. Michel, T. M. et al. Cu, Zn- and Mn-superoxide dismutase levels in brains of fibroblasts by defined factors. Cell 131, 861 (2007).
patients with schizophrenic psychosis. J. Neural Transm. (Vienna) 111, 1191 61. Aasen, T. et al. Efficient and rapid generation of induced pluripotent stem
(2004). cells from human keratinocytes. Nat. Biotechnol. 26, 1276 (2008).
32. Kästner, A. et al. Autism beyond diagnostic categories: characterization of 62. Zhou, T. et al. Generation of human induced pluripotent stem cells from
autistic phenotypes in schizophrenia. BMC Psychiatry 15, 115 (2015). urine samples. Nat. Protoc. 7, 2080 (2012).
33. Melnyk, S. et al. Metabolic imbalance associated with methylation dysregu- 63. Wang, Y. et al. Induced pluripotent stem cells from human hair follicle
lation and oxidative damage in children with autism. J. Autism Dev. Disord. mesenchymal stem cells. Stem Cell Rev. 9, 451 (2013).
42, 367 (2012). 64. Marchetto, M. C. et al. Altered proliferation and networks in neural cells
34. Hu, V. W. The expanding genomic landscape of autism: discovering the derived from idiopathic autistic individuals. Mol. Psychiatry 22, 820 (2017).
‘forest’ beyond the ‘trees’. Future Neurol. 8, 29 (2013). 65. Torrent, R. et al. Using iPS cells toward the understanding of Parkinson’s
35. Leppig, K. A. & Disteche, C. M. Ring X and other structural X chromosome disease. J. Clin. Med. 4, 548 (2015).
abnormalities: X inactivation and phenotype. Semin. Reprod. Med. 19, 147 66. Gore, A. et al. Somatic coding mutations in human induced pluripotent stem
(2001). cells. Nature 471, 63 (2011).
36. Skuse, D. H. et al. Evidence from Turner’s syndrome of an imprinted X-linked 67. Kim, K. et al. Donor cell type can influence the epigenome and differentiation
locus affecting cognitive function. Nature 387, 705 (1997). potential of human induced pluripotent stem cells. Nat. Biotechnol. 29, 1117
37. Skuse, D. H. Imprinting, the X-chromosome, and the male brain: explaining (2011).
sex differences in the liability to autism. Pediatr. Res. 47, 9 (2000). 68. Laurent, L. C. et al. Dynamic changes in the copy number of pluripotency
38. Bulger, M. & Groudine, M. Looping versus linking: toward a model for long- and cell proliferation genes in human ESCs and iPSCs during reprogram-
distance gene activation. Genes Dev. 13, 2465 (1999). ming and time in culture. Cell Stem Cell 8, 106 (2011). 01.
39. Sykes, N. H. & Lamb, J. A. Autism: the quest for the genes. Expert Rev. Mol. 69. Kang, E. et al. Age-related accumulation of somatic mitochondrial DNA
Med. 9, 1 (2007). mutations in adult-derived human iPSCs. Cell Stem Cell 18, 625 (2016).
40. Steffenburg, S., Gillberg, C. L., Steffenburg, U. & Kyllerman, M. Autism in 70. Beagan, J. A. et al. Local genome topology can exhibit an incompletely
Angelman syndrome: a population-based study. Pediatr. Neurol. 14, 131 rewired 3D-folding state during somatic cell reprogramming. Cell Stem Cell
(1996). 18, 611 (2016).
41. Bonati, M. T. et al. Evaluation of autism traits in Angelman syndrome: a 71. Meyer, S., Wörsdörfer, P., Günther, K., Thier, M. & Edenhofer, F. Derivation of
resource to unfold autism genes. Neurogenetics 8, 169 (2007). adult human fibroblasts and their direct conversion into expandable neural
42. Crespi, B. Genomic imprinting in the development and evolution of psy- progenitor cells. J. Vis. Exp.07, e52831 (2015).
chotic spectrum conditions. Biol. Rev. Camb. Philos. Soc. 83, 441 (2008). 72. Xue, Y. et al. Direct conversion of fibroblasts to neurons by reprogramming
43. Vogels, A. et al. Psychotic disorders in Prader-Willi syndrome. Am. J. Med. PTB-regulated microRNA circuits. Cell152, 82 (2013).
Genet. A 127A, 238 (2004). 73. Thier, M. et al. Direct conversion of fibroblasts into stably expandable neural
44. Dykens, E. M., Lee, E. & Roof, E. Prader-Willi syndrome and autism spectrum stem cells. Cell Stem Cell 10, 473 (2012).
disorders: an evolving story. J. Neurodev. Disord. 3, 225 (2011). 74. Liu, M. L. et al. Small molecules enable neurogenin 2 to efficiently convert
45. Cook, E. H. et al. Autism or atypical autism in maternally but not paternally human fibroblasts into cholinergic neurons. Nat Commun. 4, 2183 (2013).
derived proximal 15q duplication. Am. J. Hum. Genet. 60, 928 (1997). 75. Ladewig, J. et al. Small molecules enable highly efficient neuronal conversion
46. Kim, Y. et al. Targeting the histone methyltransferase G9a activates imprinted of human fibroblasts. Nat. Methods 9, 575–578 (2012).
genes and improves survival of a mouse model of Prader-Willi syndrome. 76. Antoni, D., Burckel, H., Josset, E. & Noel, G. Three-dimensional cell culture: a
Nat. Med. 23, 213 (2017). breakthrough in vivo. Int. J. Mol. Sci. 16, 5517 (2015).
47. Gregory, S. G. et al. Genomic and epigenetic evidence for oxytocin receptor
deficiency in autism. BMC Med. 7, 62 (2009).

Translational Psychiatry

Content courtesy of Springer Nature, terms of use apply. Rights reserved

Louise Forsberg et al. Translational Psychiatry (2018)8:14 Page 11 of 11

77. LaPlaca, M. C., Vernekar, V. N., Shoemaker, J. T. & Cullen, D. K. in Methods in spectrum disorders and a novel autism candidate gene, RORA, whose pro-
Bioengineering. 3D Tissue Engineering. (eds Berthiaume, F. & Morgan, J.) tein product is reduced in autistic brain. FASEB J. 24, 3036 (2010).
(Artech House, London, 2010). 105. Wang, Y. et al. Hypermethylation of the enolase gene (ENO2) in autism. Eur. J.
78. Mariani, J. et al. FOXG1-dependent dysregulation of GABA/glutamate neuron Pediatrics 173, 1233 (2014).
differentiation in autism spectrum disorders. Cell 162, 375 (2015). 106. Sun, W. et al. Histone acetylome-wide association study of autism spectrum
79. Eiraku, M. et al. Self-organized formation of polarized cortical tissues from disorder. Cell 167, 1385 (2016).
ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 3, 519 107. Delahanty, R. J. et al. Maternal transmission of a rare GABRB3 signal peptide
(2008). variant is associated with autism. Mol. Psychiatry 16, 86 (2011).
80. Paşca, A. M. et al. Functional cortical neurons and astrocytes from human 108. Hogart, A., Nagarajan, R. P., Patzel, K. A., Yasui, D. H. & Lasalle, J. M. 15q11-13
pluripotent stem cells in 3D culture. Nat. Methods 12, 671 (2015). GABAA receptor genes are normally biallelically expressed in brain yet are
81. Jo, J. et al. Midbrain-like organoids from human pluripotent stem cells subject to epigenetic dysregulation in autism-spectrum disorders. Hum. Mol.
contain functional dopaminergic and neuromelanin-producing neurons. Cell Genet. 16, 691 (2007).
Stem Cell 19, 248 (2016). 109. Coghlan, S. et al. GABA system dysfunction in autism and related disorders:
82. Qian, X. et al. Brain-region-specific organoids using mini-bioreactors for from synapse to symptoms. Neurosci. Biobehav. Rev. 36, 2044 (2012).
modeling ZIKV exposure. Cell 165, 1238 (2016). 110. Hogart, A. et al. Chromosome 15q11-13 duplication syndrome brain reveals
83. Luo, C. et al. Cerebral organoids recapitulate epigenomic signatures of the epigenetic alterations in gene expression not predicted from copy number.
human fetal brain. Cell Rep. 17, 3369 (2016). J. Med. Genet. 46, 86 (2009).
84. Camp, J. G. et al. Human cerebral organoids recapitulate gene expression 111. Meguro-Horike, M. et al. Neuron-specific impairment of inter-chromosomal
programs of fetal neocortex development. PNAS 112, 15672–15677 (2015). pairing and transcription in a novel model of human 15q-duplication syn-
85. Marx, V. Cell culture: a better brew. Nature 496, 253 (2013). drome. Hum. Mol. Genet. 20, 3798 (2011).
86. Quadrato, G. et al. Cell diversity and network dynamics in photosensitive 112. Makedonski, K., Abuhatzira, L., Kaufman, Y., Razin, A. & Shemer, R. MeCP2
human brain organoids. Nature 05, 545 (2017). deficiency in Rett syndrome causes epigenetic aberrations at the PWS/AS
87. Pampaloni, F., Reynaud, E. G. & Stelzer, E. H. Molecular cell biology: the third imprinting center that affects UBE3A expression. Hum. Mol. Genet. 14, 1049
dimension bridges the gap between cell culture and live tissue. Nat. Rev. 8, (2005).
839 (2007). 113. Yashiro, K. et al. Ube3a is required for experience-dependent maturation of
88. Renner, M. et al. Self-organized developmental patterning and differentiation the neocortex. Nat. Neurosci. 12, 777 (2009).
in cerebral organoids. EMBO J. 36, 1316–1329 (2017). 114. Zec, N., Rowitch, D. H., Bitgood, M. J. & Kinney, H. C. Expression of the
89. Xiang, Y. et al. Fusion of regionally specified hPSC-derived organoids models homeobox-containing genes EN1 and EN2 in human fetal midgestational
human brain development and interneuron migration. Cell Stem Cell 21, medulla and cerebellum. J. Neuropathol. Exp. Neurol. 56, 236 (1997).
1–16 (2017). 115. James, S. J., Shpyleva, S., Melnyk, S., Pavliv, O. & Pogribny, I. P. Elevated 5-
90. Lancaster, M. A. et al. Guided self-organization and cortical plate formation in hydroxymethylcytosine in the Engrailed-2 (EN-2) promoter is associated with
human brain organoids. Nat. Biotechnol. 35, 659–666 (2017). increased gene expression and decreased MeCP2 binding in autism cere-
91. Birey, F. et al. Assembly of functionally integrated human forebrain spheroids. bellum. Transl. Psychiatry 4, e460 (2014).
Nature 545, 54–59 (2017). 116. Fatemi, S. H. Reelin glycoprotein: structure, biology and roles in health and
92. Haycock, J. W. 3D cell culture: a review of current approaches and techni- disease. Mol. Psychiatry 10, 251 (2005).
ques. Methods Mol. Biol. 695, 1 (2011). 117. Fatemi, S. H., Kroll, J. L. & Stary, J. M. Altered levels of Reelin and its isoforms in
93. Prestwich, G. D. Simplifying the extracellular matrix for 3-D cell culture and schizophrenia and mood disorders. Neuroreport 12, 3209 (2001).
tissue engineering: a pragmatic approach. J. Cell. Biochem.101, 1370–1383 118. Kavalali, E. T., Nelson, E. D. & Monteggia, L. M. Role of MeCP2, DNA methy-
(2007). lation, and HDACs in regulating synapse function. J. Neurodev. Disord. 3, 250
94. Ranga, A., Gjorevski, N. & Lutolf, M. P. Drug discovery trough stem-cell based (2011).
organoid models. Adv. Drug. Deliv. Rev. 69-70, 19–28 (2014). 119. Xi, C. Y. et al. Analysis of MECP2 gene copy number in boys with autism. J.
95. Fang, Y. & Eglen, R. M. Three-dimensional cell cultures in drug discovery and Child Neurol. 26, 570 (2011).
development. SLAS Discov. 22, 456–477 (2017). 120. Shahbazian, M. D. & Zoghbi, H. Y. Rett syndrome and MeCP2: linking epi-
96. Griffith, L. G., Swartz, M. A. Capturing complex 3D tissue physiology in vitro. genetics and neuronal function. Am. J. Hum. Genet. 71, 1259 (2002).
Nat. Rev. Mol. Cell Biol. 7, 211–224 (2006). 121. Balmer, D., Goldstine, J., Rao, Y. M. & LaSalle, J. M. Elevated methyl-CpG-
97. Ilieva, M. & Dufva, M. SOX2 and OCT4 mRNA-expressing cells, detected by binding protein 2 expression is acquired during postnatal human brain
molecular beacons, localize to the center of neurospheres during differ- development and is correlated with alternative polyadenylation. J. Mol. Med.
entiation. PLoS ONE 8, e73669 (2013). 81, 61 (2003).
98. Colacino, J. A. Epigenomics: 3D human tissue culture: modeling environ- 122. LaSalle, J. M., Goldstine, J., Balmer, D. & Greco, C. M. Quantitative localization
mental effects on the stem cell epigenome. Epigenomics 8, 1453 (2016). of heterogeneous methyl-CpG-binding protein 2 (MeCP2) expression phe-
99. Stevens, H. E., Mariani, J., Coppola, G. and Vaccarino, F. M. Neurobiology notypes in normal and Rett syndrome brain by laser scanning cytometry.
meets genomic science: the promise of human-induced pluripotent stem Hum. Mol. Genet. 10, 1729 (2001).
cells. Dev. Psychopathol. 24, 1443–1451 (2012). 123. Samaco, R. C., Nagarajan, R. P., Braunschweig, D. & LaSalle, J. M. Multiple
100. Vaccarino, F. M. et al. Induced pluripotent stem cells: a new tool to confront pathways regulate MeCP2 expression in normal brain development and
the challenge of neuropsychiatric disorders. Neuropharmacology 60, exhibit defects in autism-spectrum disorders. Hum. Mol. Genet. 13, 629 (2004).
1355–1363 (2011). 124. Gong, X. et al. Analysis of X chromosome inactivation in autism spectrum
101. Vaccarino, F. M. et al. Annual research review: the promise of stem cell disorders. Am. J. Med. Genet. B Neuropsychiatr. Genet. 147B, 830 (2008).
research for neuropsychiatric disorders. J. Child Psychol. Psychiatry52, 504 125. Talebizadeh, Z., Bittel, D. C., Veatch, O. J., Kibiryeva, N. & Butler, M. G. Brief
(2011). report: non-random X chromosome inactivation in females with autism. J.
102. Kelava, I. & Lancaster, M. A. Dishing out mini-brains: current progress and Autism Dev. Disord. 35, 675 (2005).
future prospects in brain organoid research. Dev. Biol. 420, 199 (2016). 126. Chadwick, L. H. & Wade, P. A. MeCP2 in Rett syndrome: transcriptional
103. Ginsberg, M. R., Rubin, R. A., Falcone, T., Ting, A. H. & Natowicz, M. R. Brain repressor or chromatin architectural protein? Curr. Opin. Genet. Dev. 17, 121
transcriptional and epigenetic associations with autism. PLoS ONE 7, e44736 (2007).
(2012). 127. Woods, R. et al. Long-lived epigenetic interactions between perinatal PBDE
104. Nguyen, A., Rauch, T. A., Pfeifer, G. P. & Hu, V. W. Global methylation profiling exposure and Mecp2308 mutation. Hum. Mol. Genet. 21, 2399 (2012).
of lymphoblastoid cell lines reveals epigenetic contributions to autism

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