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Modeling SARS-CoV-2 infection in individuals with opioid use disorder with brain organoids

The COVID-19 pandemic has aggravated a preexisting epidemic: the opioid crisis. Much literature has shown that the circumstances imposed by COVID-19, such as social distancing regulations, medical and financial instability, and increased mental health issues, have been detrimental to those with opioid use disorder (OUD). In addition, unexpected neurological sequelae in COVID-19 patients suggest that COVID-19 compromises neuroimmunity, induces hypoxia, and causes respiratory depression, provoking similar effects as those caused by opioid exposure. Combined conditions of COVID-19 and OUD could lead to exacerbated complications. With limited human in vivo options to study these complications, they suggest that iPSC-derived brain organoid models may serve as a useful platform to investigate the physiological connection between COVID-19 and OUD. This mini-review highlights the advances of brain organoids in other neuropsychiatric and infectious diseases and suggests their potential utility for investigating OUD and COVID-19, respectively.


The worldwide COVID-19 pandemic, caused by the SARS-CoV-2 virus, arrived in the midst of another epidemic: the opioid crisis. Opioid use disorder (OUD), substance use and addictive disorder defined in part by the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (5th edition; DSM-5) as the desire to take opioids despite social and professional consequences, currently affects 40.5 million people worldwide, but the opioid crisis was actually beginning to decline as more people were gaining access to effective treatment options.


COVID-19 may reverse this trend, though, as people with OUD often experience significant challenges in healthcare that could be aggravated by the pandemic. For example, quarantine and social distancing measures could disrupt both vital social support groups and medication for addiction treatment, which is generally administered in person. Those with OUD could also be more likely to contract COVID-19 due to cognitive impairment, lower awareness of risk, and diminished efforts regarding personal protection in patients, further disrupting the treatment. Such premature treatment cessation dramatically increases overdose rates. In addition, the rise in fear, anxiety, and depression caused by COVID-19, induced by medical and financial instabilities, can be especially harmful to people with pre-existing mental health conditions and can cause relapses, worsening of these conditions, and increased substance usage. indeed, since the rise of COVID-19, the United States has seen an increase of 32% for non-prescribed fentanyl and an 11% increase in drug overdose deaths in the first 4 months of 2020 as compared with those of last year.



Human iPSCs self-assemble into embryoid bodies, and then sequentially undergo induction, differentiation, and maturation to form brain organoids. These organoids can mimic specific brain regions and model corresponding functional studies. Neurobiological mechanisms of various brain disorders can be investigated using patient-derived or genetically mutated iPSCs, as well as with the appropriate brain region.
Schematic representation of general brain organoid generation.

What are brain organoids?

Understanding the human brain, with both its vast number of specialized cell types and complex connectivity, has been historically challenging. Knowledge of the biological bases of neuropsychiatric disorders is unsatisfactory, too, and human health continues to suffer. This reality is primarily due to their limited access to the healthy human brain; most of their knowledge is built on human pathological or post-mortem specimens, animal studies, and in vitro 2D culture models. These models, although invaluable, are unable to capture the full scope of the living human brain, and they are limited by inherent species differences, concerns over tissue availability and manipulation, and lack of cell diversity and structural organization. Advancing neuroscience will require better human tissue-based models that can recapitulate the developmental and functional dynamics of the human brain.

Recent advances in cellular reprogramming and stem cell culture techniques have enabled the in vitro generation of 3D brain organoids from an individual’s unique genetic background.

Brain organoids are self-assembled 3D cellular aggregates that are generated from embryonic stem cells (ESCs) or iPSCs to mimic the brain. These brain organoids, which contain a 3D-organized heterogeneous cell population, can partially recapitulate some of the brain’s structure, developmental stages, and functionality, such as synapse formation and intercellular signal transmission. In addition, brain organoids can model specific regions of the brain; a variety of protocols demonstrate the generation of brain organoids to model the development of the cortex, hypothalamus, midbrain, and cerebellum. To generate brain organoids, iPSCs can be derived via the reprogramming of somatic cells. iPSCs can self-organize to form embryoid bodies (EBs). These EBs could then be cultured in the presence of neural induction molecules and region-specific patterning factors to give rise to brain organoids modeling specific brain regions. Each protocol details a distinct cocktail of patterning molecules (e.g. SMAD inhibitor and WNT3A) and generates brain organoids with cellular heterogeneity and spatial architecture. For instance, cerebral organoids consist of upper-layer and deep-layer neurons, radial glia cells, and neural progenitors that display a rosette-like structure mimicking the subventricular-like zone.1



Brain organoid models for OUD and neuropsychiatric diseases

OUD is part of the wide range of neuropsychiatric disorders in which neurodevelopmental features and neurological functions have been comprised.OUD and other substance use disorders are usually compounded with additional neuropsychiatric disorders, such as depression, anxiety, schizophrenia, bipolar disorder, attention-deficit hyperactivity disorder (ADHD), psychotic illness, borderline personality disorder, and antisocial personality disorder. Researchers have posed several reasons for such a high prevalence of comorbidity between OUD and other neuropsychiatric disorders: (1) they target similar brain regions (nucleus accumbens, ventral tegmental area, prefrontal cortex, hippocampus, amygdala) and neural circuitry (implicating the reward system, decision making, impulse control, stress response, and emotions); (2) they share common molecular mechanisms affected by various genetic, epigenetic, and environmental factors such as genetic mutations, stress, adversity, trauma, and drug exposure and/or access; (3) there are numerous clinical similarities and overlapping symptoms with each other, and one disease may unmask or exacerbate the symptoms of the other. However, the precise mechanisms for many neuropsychiatric diseases, including OUD, are unclear, and further investigation is crucial for a greater understanding of the nature of these diseases and their complex relationship.

Although OUD has yet to be investigated using brain organoids, many studies have been conducted on other neuropsychiatric diseases. These studies successfully investigated similar pathways and regions that would be relevant to OUD, suggesting that brain organoids can provide a viable framework for studying human brain disorders and explore potential therapeutics. To show the power of brain organoids for OUD, we will first highlight several significant advances that brain organoids have made in a few kinds of neuropsychiatric disorders: neurodevelopmental, psychotic, mood, and neurodegenerative.1



(a) Images of brain organoids revealing SARS-CoV-2 infection in both MAP2 mature neurons and SOX2 neural stem cells (top). Quantification shows an increase in SARS-CoV-2 positive cells in the organoids, suggesting productive replication (bottom). Adapted with permission. (b) Images of telencephalic organoids immunostained for HTR2C choroid plexus epithelial cells and HuCD neurons. Staining for SARS-CoV-2 viral spike protein expression reveals a much higher infection in choroid plexus cells than in other cortical cells and neurons. Adapted with permission. Copyright 2020, MRC Laboratory of Molecular Biology. (c) Images of TUNEL-positive cells in control (top) versus SARS-CoV-2-exposed (bottom) organoids reveal SARS-CoV-2 positive cells experienced significantly higher cell death. Adapted with permission. Copyright 2020, The Authors. (d) Immunofluorescence staining of ACE2 in brain organoids showed expression of ACE2 in MAP2-positive neurons (left). Immunofluorescence staining of organoids pre-incubated with anti-ACE2 antibodies (right) and infected with SARS-CoV-2 showed ACE2 antibody inhibited SARS-CoV-2 infection. Adapted with permission.(e) Selected enriched GO terms for SARS-CoV-2 versus control organoids, including biological process (red), molecular function (green), and cellular components (blue). Adapted with permission. Copyright 2020, Elsevier.


1. Willner MJ, Xiao Y, Kim HS, Chen X, Xu B, Leong KW. Modeling SARS-CoV-2 infection in individuals with opioid use disorder with brain organoids. Journal of Tissue Engineering. January 2021. doi:10.1177/2041731420985299

cover photo: https://www.the-scientist.com/news-opinion/human-cortical-organoids-make-brain-waves-66368

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