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Padlet made by Sabrin Cabdi, Wai Yan Oo, Fathima Farha Mohamed Farzan, Isaiah Grimmond-Thompson, Isabella O'Kane , Jasmee Navaratnarajah, Blanka Bajka-Reynier

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Studies where neural organoids have been developed from stem cells with disease causing mutations has allowed researchers to understand the pathology of neurological diseases. This can help us better understand the disease mechanisms such as changes in signalling pathways which could allow researchers to develop potential drug treatments.

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This aids in making the treatment ‘safe’, and through recent development, it will be used to curate medicine in humans against disease(1).

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More specifically in the digestive system with the potential to treat short bowel syndrome, or human inflammatory bowel disease, by using organoids to develop functional tissue to be reintroduced into the patient, for effective function(1)

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(Google Document)
https://docs.google.com/document/d/1DuZjb9Ockyvi3vfgECa1lOYKNnw4UMZYcugr2_Zzac8/edit

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Cerebral organoids can also be genetically modified to study gene expression. It was found that the stage that the modifications occurred, as well as the medium in which the modifications were made, affected the development of the organoid. Those modified earlier resulted in a larger impact on the development of the organoid.

'Long-lasting genetic modification' can be achieved through the Crispr/Cas9 method [9] as using 'genetic cargo' to insert mutations resulted in reduced modification ever round of cell division.

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This is a brain scan from a study where researchers implanted lab grown human neurons into the cortex of newborn rats (7) .Their aim was to see whether the human neurons could engage with the rat's, and it was successful. This study shows that growing more complex organoids in the future may be possible, which will let us study brain development in more detail than ever before.

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Cajal–Retzius cells drawn by Santiago Ramón y Cajal in 1891

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During development, cerebral cortical neurons begin to develop on the preplate, which then develops into the cortical plate. The cells are also reelin positive, a glycoprotein, secreted from Cajal-Retzius cells [8], involved in the correct organisation of the brain as it develops.

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For example, the tiny 1cm size of brain organoids, and only 100,000 neurones, means they lack the vast complexity of human brains, and can never therefore accurately reflect interactions with the outside world.

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In this 5 minute-long video, the development of mini brain-organoids is explained, along with various limitations.

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What are mini brains? - Madeline Lancaster

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Check out our Patreon page: https://www.patreon.com/teded View full lesson: https://ed.ted.com/lessons/can-we-grow-human-brains-outside-our-bodies-madeline-lancaster Shielded by our thick skulls and swaddled in layers of protective tissue, the human brain is extremely difficult to observe in action. Luckily, scientists can use brain organoids - pencil eraser-sized masses of cells that function like human brains but aren't part of an organism - to look closer.

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Limitations include:

High-fidelity cell types

Limited maturation

Atypical physiology

Lack of arealization

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On the other hand, in a study conducted by Sloane et al. 2017 where astrocytes taken from the brain, showed an accurate transformation from fetal to early postnatal development. After this period the rate of phagocytosis steadily declined, however.

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The current lack of organisation in the physical distribution of relevant genes, especially within neural organoids, limits the use of organoids in a clinical setting.
More accurately patterned brain organisation is needed for use in accurate disease diagnoses. (1)

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How to we determine the limitations of the technology to avoid the biggest ethical conundrum of them all: creating something too human.

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Left, a whole brain organoid imaged in bright field showing a lobe of cortical tissue developing. Right, a section through a lobe of cortical tissue, with neurons in green, neural stem cells in pink/red and nuclei stained blue.

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Axons can be formed by cerebral organoids, shown to have complex branching and growth cone formation seen in Green Fluorescence Protein (GFP) staining.

Spontaneous calcium surges and calcium oscillations have been shown to occur in organoids as well when calcium dyed

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as proposed by philosopher Hilary Putnam, in the far future, the development of brains could lead to the highly unlikely but potentially possible event where a brain is put into a nutrient broth and connected to a supercomputer as a new form of machinery (2)

also, a brain could be connected to other organs and create a complex, or be implanted into animals to form chimeric animal models, thereby raising various additional moral and ethical questions (2)

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Additionally, neural organoids could be used to model various neurological diseases such as schizophrenia (6). Normally, the molecular mechanisms of schizophrenia were studied using animal models or by post mortem brain studies (6). However, these studies often lack reliability and validity as  tissues in post mortem studies could have structural abnormalities due to decay and animal models aren’t representative to human brains.

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1. Start in the middle with the "History and development of organoids" section (white)

2. Next, understanding "How organoids are generated" (purple)

3. Then, "Applications of neural organoids" (blue)

4. Then, "Limitations of organoid development" (red)

5. Next, "Spinal cord organoids" (green)

6. Finally, "Ethical considerations of organoids" (purple)

7. Reflection & Sources Section (yellow)

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REFLECTING ON OUR PROCESS

FIRST IDEAS: During our first meeting, we discussed ideas for our anatomy Padlet. Our initial ideas included: ideal anatomy/physiology for living in space, ideal anatomy/physiology for a predator, organoids, regenerative medicine, anatomy in art and society

NARROWING IT DOWN: Organoids were our main interest since they combined most of the concepts we wanted to investigate, and therefore we chose this topic.

WHY BRAIN ORGANOIDS: We chose to research brain organoids as we thought it would be best to focus on one organ to ensure that our research was specific and easy for the reader to understand. If we had looked at other types of organoids (e.g. liver organoids), our Padlet would have contained too much detail and would therefore be less appealing to the reader. We chose to include Spinal cord organoids since they directly related to the topic at hand, but abstained from anything else.

CHALLENGES: We had difficulty understanding the software since we had never faced it before (Padlet), but we persevered as a team to figure out the interface.

OUR TEAMWORK:  We prioritized our more immediate coursework, but consistently met to discuss new ideas and offer suggestions, meeting goals set as a team. We each had some specific interests in the topic and allocated topics accordingly, helping each other along the way. To successfully finish this project, we set internal deadlines and met more often as the due date approached to ensure our work would demonstrate our best effort and be completed on time.

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If this process could be improved (ie. the applications made more plentiful and the limitations decreased, the options for developing and researching neural organoids would expand greatly and have the chance to change many lives. (4)

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potentially produce inaccurate results due to a shortcoming and provide inadequate information, which could be dangerous if the research is implemented

as the technology develops, could lead to an actual issue whereby a fabricated brain develops something akin to consciousness, which would have great ethical ramifications

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Now, there is a far reaching and relatively accepted theory of consciousness, called the Integrated Information Theory of Consciousness (IIT). (3)

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however, this falls short when considering machine learning and AI, whereby software and machinery can reach higher intellect than humans without having a consciousness (2)

of course, this is purely presumed, though this may change in the future and is a main point of contention amongst scientists and philosophers, and essentially all of mankind (4)

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The moral significance of consciousness is highly relevant, since the meaning of consciousness itself is debated.

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This aims to make it possible to transplant organs while overcoming problems of scarcity, compatibility, and rejection.

Though it is an incredible goal and could change many lives for the better, it is also a central debate topic for cerebral organoids specifically, since there are many moral and ethical issues with the manipulation of neural tissue. (3)

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living beings aren’t destroyed/impaired

scientific reliability may be higher than the model organism's

may be a more acceptable way to test hypotheses and carry out brain research, when compared to human fetuses, animals, or even voluntary adults

may very well be cheaper than certain large-scale trials

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Some schools of thought argue that intelligence is the key aspect of ‘humanity’, ie. the individual’s functional capacity of the subject in performing tasks. (2)

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epistemologically, some experts argue that the logic used for comatose patients should be applied to organoids. (2)

however, this is highly diminutive, and many argue that there needs to be a developed, objective index of brain-based consciousness that doesn't rely on motor outputs, sensory processing, or executive functions.

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Potential Benefits, Dangers & Additional Dilemmas (7) 

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Potential Benefits, Dangers & Additional Dilemmas (7) 

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Ethical Considerations of Brain Organoids (1)

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Ethical Considerations of Brain Organoids (1)

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Human cerebral organoids and the various brain regions marked by genetic markers. They are present in different stages of development such as initially where PAX6 shows neural induction or AUTS2 showing the cerebral cortex.

Genetic markers for the hippocampus, ventral forebrain, and choroid plexus are also present in cerebral organoids, however, the overall structures of these regions have not yet been formed at this stage of development.

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Anatomy of the inside of the brain, showing the pineal and pituitary glands, optic nerve, ventricles (with cerebrospinal fluid shown in blue), and other parts of the brain.

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Organoids self organise during development through 'cell sorting' and 'spatially restricted lineage commitment'.

At different periods of time, different types and volumes of cells develop e.g primarily neurons are generated during early development, versus glial cells during later stages [3].

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https://www.youtube.com/watch?v=9ksLuRoEq6A&ab_channel=NewScientist

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Human brain cells in a dish learn to play Pong

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Summary for cellular composition and tissue type in brain organoid

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Would you beat a brain organoid at pong?

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Would you beat a brain organoid at pong?

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Consisting of around 800,000 neurons, a group of researchers managed to make a cerebral organoid capable of playing Pong! [1]

Using electrodes, the electrical signals and subsequent cellular changes were converted into a visual representation, shown in the video linked.

It's a long way away from the 80-100 billion neurons of a human, but it is a promising step towards developing more complex brains, or even developing more advanced AI!

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Another aspect of such is in regenerative medicine, where organoids can be used to heal or replace, damaged or scarred tissue, within the body.

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Cerebral organoids were used because the cerebral cortex and spinal cord have similar physiological properties. [6]

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Matured cerebral organoids were used to repair small sections of the spinal cord.

Cerebral organoids were transplanted to the spinal cord sections. The cerebral organoids fused with the spinal cord tissue.

Research showed that successful fusion between tissues is selective. The cortical organoids were more likely to fuse with the spinal cord organoids compared to the skeletal muscle organoids. [6]

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Spinal cord injury treatment can be trialled via organoids, which can be used to visualise the effects of used biomaterials.

Spinal cord spheroids can be differentiated into dorsal, intermediate and ventral spinal cord units by restricting the direction of differentiation. [5]

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Research has been conducted on mice but there is limited correlation between mice and human mutations. [7]

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During development, the spinal cord is formed via neurulation. Errors during early neurulation can lead to neural tube defects (NTDs). Use of organoids can lead to better understanding of human development. [7]

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They can’t undergo full patterning, have high variability in formed organoids and high tissue heterogeneity. [8]

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It can partly recapitulate DV and AP patterning of the neural tube. [8]

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Throughout development, various cell types begin to differentiate. These include astrocytes, oligodendrocytes, glutamatergic neurons and various others.

In addition, synapses and dendritic spines, structural traits of mature neurons, were also found around 8 months into development, suggesting that the mature brain organoid can be used to investigate more advanced nervous functions [7].

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Research was carried out on organoids with functional neuromuscular junctions, which were then exposed to different ALS subgroups. This allowed identification of different structures such as motor neurons and skeletal muscle. [4]

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Cerebral organoids show brain regionalisation

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Cerebral organoids show brain regionalisation

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During development, extracellular signalling at different stages can dictate development of specific regions [4].

These regions include the choroid plexus, hippocampus and frontal brain. Photosensitive cells can be developed, mimicking a structure similar to the retina.

Genetic markers can be added to outline certain regions [6] to see how the organoid develops.

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To generate a cerebral organoid, embryonic stem cells are used. In normal embryonic development, the nervous system develops from ectoderm.

Hence, the stem cells are placed in neuroectoderm encouraging broth, grown in a gel scaffold, spun in a bioreactor to distribute O2 and nutrients to maintain viability.

Rich populations of radial glial cells are present during growth, helping increase the number of developing neurons [2].

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The cellular composition of the spinal cord organoids were investigated through immunostaining at different stages. As the organoids matured, an apical-to-basal patterning was seen. [2]

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Usually, neurotoxicity is dependent on animal models, which has several disadvantages. This includes differences in anatomy, ethical concerns and costs.

Advancements of neural organoids allow testing of chemicals that can affect human CNS development. Using spinal cord organoids has helped visualise the potential risk of neural tube defects. [8]

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Spinal cord organoids can be used for toxicology screening.

The effects of antiepileptic drugs (AEDs) were trialled on spinal cord organoid models. The models were treated with 3 concentrations of 6 selected AEDs. Results showed that two types of AEDs resulted in neural tube closure failure after 8 days of treatment. These results were consistent with clinical risks of AEDs. [7]

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Neural organoids could be developed further in the future by incorporating vascularization, as this would mimic the in vivo environment of a real life brain (4) .Recently, researchers have developed microglia- containing human brain organoids, (MC-HBOs) as microglia play a key role as the immune defence, in the central nervous system (5) .This allows researchers to study diseases using a more reproducible and accurate model, which encapsulates the main features of the human brain.

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Microglia play crucial roles in the brain

EMPs arise at embryonic day (E) 7.5 during the first wave of hematopoiesis in the yolk sac. EMPs-derived PMPs colonize the developing brain at E9.5 and further differentiate into microglia.

Microglia are well known for their critical functions in immune surveillance. In the brain, microglia serve as resident phagocytes that dynamically monitor the environment. In normal physiological conditions, resting microglia are highly thin and ramified. While in disease status or other conditions that disrupt the brain homeostasis or immune milieu, microglia become activated with short and thick morphologies [ 13]. Activated microglia rapidly respond to endogenous or exogenous pathological insults, and eliminate those pathogenic species through pinocytosis, phagocytosis, or receptor-mediated endocytosis, thus protecting the brain from damages caused by the pathological insults [ 14].

In addition to their immune functions, microglia also play critical roles in neural development [ 15], synaptic formation and plasticity [ 16, 17], and neural network maturation [ 18]. Microglia selectively colonize the cortical proliferative zones and phagocytose neural precursor cells (NPCs) to regulate neural development [ 19]. During postnatal brain development, microglia actively engulf synaptic material and eliminate weak synapses to shape neuronal circuits through synaptic pruning [ 18].

Models to study microglia

Studies conducted over the past decades have implicated microglia dysfunction in multiple brain disorders [ 20], ranging from psychiatric disorders (e.g., schizophrenia, bipolar disorder, and autism) [ 21, 22] to neurodegenerative diseases including Alzheimer's disease (AD) and Parkinson's disease (PD) [ 23, 24]. Developing therapeutics by targeting microglia has emerged as a promising approach to the therapy of microglia-related brain disorders. To illuminate how microglia contribute to brain development and pathology, multiple microglial models including in-vivo animal models, two-dimensional (2D) cell cultures, human microglia-mouse chimeric models, and the emerging three-dimensional (3D) cultured microglia-containing human brain organoids (MC-HBOs) have been constructed. The major pros and cons of these microglial models are shown in Table 1.

Table 1 Major pros and cons of microglial models.

In-vivo animal models

In-vivo animal models including leech, zebrafish, rodent, and non-human primates have been intensively used for microglia study [ 25]. By the use of technologies including in-vivo labeling, living imaging, gene editing, and high-throughput sequencing, etc., animal models have provided us with much understanding of the basic characteristics, developmental paths, and functions of microglia [ 26]. Animal models are undoubtedly useful in uncovering important new findings of microglia biology. However, key features such as transcriptional and pharmacological differences between animal and human microglia limited the application of animal models in human microglia studies [ 27, 28, 29, 30]. For example, human and mouse microglia have opposite responses to valproic acid, a drug for epilepsy and bipolar disorder. When exposed to valproic acid, mouse microglia were selectively killed but human microglia were not [ 29]. Another key limitation of animal models is the genetic differences between animals and humans. For example, the schizophrenia risk gene complement component 4 (C4) [ 31] has C4A and C4B isotypes in human, but solely C4b in mouse. Considering that greater C4A expression in the brain is associated with increased schizophrenia risk [ 31], the C4b knockout mouse may not be a good model for schizophrenia and other microglia pruning-related disorders. In addition, animal models usually need a long experimental period and motivate the development of in-vitro cell culture models for human microglia study.

2D microglial cultures

Primary microglial cultures

Since primarily isolated microglia have the same genetic background and similar phenotypes as in-vivo cells, microglia isolated from brains of rodents, non-human primates, and humans have been used for ex-vivo cultures [ 26, 32]. Especially, human primary microglia could be isolated from postmortem brain tissues, epilepsy surgery, or brain tumors [ 26, 32], allowing investigations to be conducted under human genetic background. However, the transfer of human or animal microglia to the in-vitro environment resulted in transcriptome-wide gene expression changes, including the downregulation of microglia-specific genes [ 28, 33]. The acutely isolated primary microglia are likely ex-vivo activated and may present characteristics different from the in-vivo microglia. The isolated primary microglia may also show inconsistent phenotypes since they could be isolated from different brain regions or individuals with different healthy conditions [ 26]. Moreover, high-quality human postmortem brains are limited to obtain, and extensive preparations are needed for each surgical operation. These limitations hinder the application of primary microglia cultures [ 34].

Microglial cell lines

Microglial cell lines are generally homogeneous cell populations with high cell proliferation rates. The use of microglial cell lines could shorten the experimental period and enable low-cost cell culture, making them suitable for basic research and high-throughput screening assays. With these advantages, a number of immortalized microglial cell lines with mouse, rat, rhesus macaque, and human origin, including the commonly used BV2, N9, and HMO6 cell lines, have been used for microglia studies [ 35]. However, complex cellular interactions and in-vivo like environment are absent from microglial cell lines. Studies underlined the limitations of microglial cell lines in recapitulating transcriptional signatures, morphologies, and functions of in-vivo microglia [ 32, 35]. Moreover, the phenotypes and transcriptional patterns of microglia may be altered after viral infection or immortalization.

Induced microglia-like cells (iMGs)

Another strategy to generate iMGs is to transdifferentiate human peripheral blood mononuclear cells [ 48, 49] or hiPSCs-derived monocytes [ 50] into microglia by adding IL34 and GM-CSF or M-CSF into the differentiation medium. After about two weeks of culture, cells would differentiate into iMGs that transcriptionally resembled human primary microglia and showed typical characteristics of in-vivo microglia. These transdifferentiation approaches could be rapidly established and are easy to operate as an alternative iMGs model.

Though iMGs are readily available sources for microglia study, shortcomings of iMGs also exist. iMGs showed transcriptional and morphological heterogeneity across differentiation protocols. For example, iMGs generated from studies of Pandya et al. [ 40] and Muffat et al. [ 38] did not separate well from blood dendritic cells and macrophages according to the correlation-based hierarchical clustering analysis. iMGs generated in some studies [ 40, 45, 50] did not show highly ramified morphologies as reported in other iMGs studies [ 38, 39, 41, 42, 43, 44]. Moreover, how much iMGs could mimic the in-vivo microglia are unclear since microglia-neuron interactions and in-vivo like brain microenvironment are usually absent from the iMGs culture systems.

Human microglia-mouse chimeric model

3D cultured MC-HBOs

Introduction of HBOs

Microglia function through interacting with other brain cell types. Intercellular signaling pathways enable microglia to communicate with one another and their surrounding microenvironment [ 56, 57]. Since complex cellular architectures similar to the in-vivo human brain are usually not available in 2D cell cultures, studies underscored the shortcomings of 2D microglial cultures in recapitulating in-vivo signatures [ 32, 55, 58, 59]. Though animal models or chimeric models could provide an in-vivo microenvironment for microglia, the lack of human genetic background or interactions between microglia and other human brain cell types is another concern for microglia study. A model containing multiple human cell types that facilitates microglia-neuron/macroglia interactions is desirable.

Generation of MC-HBOs

The neuronal lineage cells are of ectoderm origin. Differentiation towards endoderm and mesoderm lineages is generally suppressed during the formation of HBOs. Therefore, microglia are generally assumed to be absent from HBOs due to their non-neuroectodermal origin. Several strategies now have been developed to generate MC-HBOs to enable a better understanding of microglial functions and their connections to brain disorders. The major pros and cons of these strategies are listed in Table 2 to guide the choice for microglia study.

Table 2 Summary of strategies used to generate MC-HBOs.

Co-culture of microglia with HBOs

Since microglia are thought to be absent from HBOs, we would naturally propose to co-culture HBOs with exogenous microglia to build MC-HBOs (Fig. 2). To our knowledge, Abud et al. [ 39] were the first to co-culture hiPSCs-derived iMGs with HBOs that contained neurons, astrocytes, and oligodendrocytes. They found that iMGs were able to integrate into HBOs, mature, ramify, and respond to injury similar as in-vivo microglia. Both iMGs in 2D culture and xenotransplanted in immune-deficient AD mice could internalize and phagocytose Aβ plaques or tau neurofibrillary tangles, two hallmark AD pathologies [ 39]. However, whether iMGs integrated within HBOs could phagocytose the pathological species was not investigated.

After the study by Abud et al., several other studies also incorporated iMGs into HBOs to build MC-HBOs. By co-culturing HBOs with isogenic iMGs, Song et al. [ 74] generated dorsal or ventral forebrain spheroids containing iMGs (D-iMGs and V-iMGs, respectively). In their study, D-iMGs and V-iMGs showed differential microglia migration ability, intracellular Ca ²⁺ signaling, and the response to pro-inflammatory stimuli. Increased cell proliferation (higher BrdU⁺ cells) and reduced reactive oxygen species in D-iMGs and V-iMGs were observed when compared with those in iMGs only. By comparing bulk transcriptome from D-iMGs and from iMGs only, they identified sets of differentially expressed genes, including the neuron and glial cell-specific genes. However, it is questionable to make such a comparison since the D-iMGs contain diverse cell types in addition to microglia, while the iMGs contain microglia only. The sources of the phenotypic and transcriptional differences cannot be defined. These differences may just reflect the intrinsic cell composition differences between cortical spheroids and iMGs. Similarly, the study by Bejoy et al. [ 75] investigating the impact of 3D culturing microenvironment on the metabolic phenotypes of iMGs came across the same problem. They re-analyzed the transcriptome data of Song et al. [ 74] to identify transcriptional differences in metabolic pathways between D-iMGs and iMGs only. Genes related to glycolysis, hypoxia signaling, and several metabolic pathways were found to be upregulated in D-iMGs. However, it might be premature to conclude that 3D microenvironment may reshape the immunity of in-vitro cortical spheroids [ 75] by comparing transcriptome from D-iMGs and from iMGs only.

Ao et al. [ 76] used a stereolithography 3D printer to develop a tubular device for brain organoid culture. They stated that the tubular device reduced organoid necrosis and hypoxia. Isogenic microglia could incorporate into the tubular organoids and show a stronger cytokine response compared to that in 2D iMGs. Specifically, the level of tumor necrosis factor α (TNFα) was significantly elevated in microglia-incorporated tubular organoids but not in 2D iMGs post lipopolysaccharide (LPS) exposure, suggesting the advantage of MC-HBOs in modeling neuroinflammation.

The above studies established the MC-HBOs models, but how microglia within HBOs resemble human primary microglia and how they affect functions of HBOs were not investigated. Recently, Popova et al. [ 55] comprehensively compared the transcriptomic signatures of microglia across different models, including iMGs, iMGs transplanted in mouse brain, primary microglia cultured ex-vivo or transplanted in HBOs, to create a microglia report card. They found that human primary microglia transplanted into HBOs had the closest resemblance to their in-vivo counterparts, suggesting that brain organoid microenvironment preserves the homeostatic microglia state [ 55]. They further found that radial glia and dividing cells in HBOs were two top cell types responding to microglia transplantation. Microglia transplanted into HBOs induced transcriptional changes, reduced cell stress, and attenuated expression of interferon response genes. Microglia also increased the synchronization and frequency of oscillatory bursts in the HBOs that facilitated maturation of neural networks [ 55], highlighting the microglial contribution to neural development. The study by Popova et al. [ 55] presented better evidence to illustrate how microglia and brain organoids affect each other.

Using MC-HBOs, studies investigated how microglia acted in brain pathology. Lin et al. [ 77] co-cultured APOE3- or APOE4-carrying iMGs with APP gene duplication HBOs that displayed Aβ aggregates, to investigate how microglia impacted AD pathology. They found that APP duplication organoids co-cultured with APOE4 iMGs exhibited more extracellular Aβ aggregates than those co-cultured with APOE3 iMGs. Moreover, APOE4 iMGs within APP duplication organoids showed longer processes than those in APOE3 iMGs, suggesting that iMGs with APOE4 variant were less able to sense and respond to extracellular Aβ. These results supported that APOE4 variant impacted AD pathology mainly through affecting the ability of iMGs to clear extracellular Aβ.

Several other studies applied MC-HBOs to model ZIKA [ 78, 79], Dengue [ 78, 79], and HIV-1 viral infection [ 80]. For example, Muffat et al. [ 78] showed that ZIKA virus pre-infected iMGs penetrated the HBOs and spread ZIKA virus by initiating infection of adjacent cells, resulting in a stalled growth of HBOs. Abreu et al. [ 79] also used MC-HBOs to model ZIKA and Dengue viral infection by incorporating virus-infected human SV40 microglial cells into HBOs. MC-HBOs supported replication of the ZIKA and Dengue viruses. Increased expression of several cytokine/chemokine genes was observed in MC-HBOs after infection of ZIKA ( IL6, IL1β, TNFα, and CCL2) and Dengue virus ( IL1β and CCL2). However, due to the rapid and unlimited proliferation of immortalized SV40 microglial cells, the MC-HBOs could only maintain for 7 days after microglia incorporation. In another study, Reis et al. [ 80] incorporated HIV-1 infected human HMC3 microglial cells into HBOs, finding that HIV-1 infection induced inflammatory responses and the release of pro-inflammatory factors (TNFα and IL1β). Similar to Abreu et al. [ 79] study, the MC-HBOs could only maintain for 15 days after microglia incorporation. To enable a long-term culture of MC-HBOs, Reis et al. incorporated HIV-1 pre-infected primary human adult microglia into HBOs. They found that HIV-1 infection resulted into significant cytotoxicity, neuronal loss, and astrocyte activation in MC-HBOs, which are major hallmark features seen in the brains of HIV-1 infected individuals [ 80]. These studies indicated that MC-HBOs could support viral replication and trigger strong immune responses to viral infection.

Taken together, the above studies showed that microglia could incorporate into HBOs and reshape the immunity and functions of HBOs. This co-culture model could be a useful tool for modeling brain development and pathology. However, microglia from different studies could vary in their maturity and the abilities in response to immune stimuli, which may result into inconsistent results after microglia incorporation into HBOs. For example, iMGs in Abud et al. study [ 39] secreted higher levels of cytokines IL6, IL8, IL10, and TNFα after exposure to LPS. However, no change of TNFα was detected in iMGs after exposure to LPS in AO et al. study [ 76]. In Abreu et al. study [ 79], gene expression of IL6, IL8, and IL10 was also not increased in SV40 microglial cells after LPS treatment. Interestingly, LPS treatment upregulated gene expression of IL6, IL8, and IL10 in HBOs containing SV40 microglia when compared with HBOs without microglia [ 79]. These results suggest the cross-talk between microglia and other cell types including astrocytes and oligodendrocytes within brain organoids. Such cross-talk may play important roles in immune response [ 81, 82] and is required for a specific inflammatory response.

Co-culture of MPCs with HBOs

Microglia are cells of mesodermal origin. Incorporation of MPCs (EMPs or PMPs) into HBOs may also generate microglia under the microenvironment of HBOs (Fig. 3). By co-culture of hiPSCs-derived mesodermal progenitors (Brachyury⁺) with neural spheroids, Wörsdörfer et al. [ 83] reproducibly generated vascularized neural organoids with vessel-like structures (CD31⁺) and microglia-like cells included. This study provided a model for studying angiogenesis and neural development, but how microglia functioned in the organoids was not investigated. In another study, Fagerlund et al. [ 84] reported that hiPSCs-derived EMPs (CD41⁺) migrated into HBOs, differentiated into microglia-like cells, and interacted with synaptic materials. Whole-cell patch-clamp and multi-electrode array recordings showed that microglia within the organoids promoted neural network maturation. Specifically, spontaneous and NMDA-induced neuronal bursting activity was stronger and more prevalent in organoids with microglia than that in organoids without microglia. Excitatory post-synaptic currents (EPSCs) were present only in neurons from microglia- containing organoids after day 107 [ 84]. A recent study [ 85] that co-cultured human midbrain organoids with hiPSCs-derived macrophage progenitors also reported that microglia integration led to increased neural maturation and functionality. Whole-cell patch-clamp and multi-electrode array recordings showed that a lower threshold for action potential generation and a shorter interspike interval were observed in microglia-integrated midbrain organoids, suggesting that microglia integration promoted neural maturation. The presence of microglia in midbrain organoids also increased cytokine release (including IL6, IL10, IL1β, and TNFα), and affected expression of genes related to immune response, inflammation, phagocytosis, and synaptic remodeling and maturation [ 85].

The incorporation of MPCs into HBOs provides a promising model for microglia study, since this strategy could generate MC-HBOs with vessel-like structures included. The emergence of vessel-like structures has the possibility to improve survival and maturity of brain organoids, though the current vascular structures are far from functional. Importantly, microglia developed in this strategy mimic the developmental trajectories of in-vivo microglia, resembling the process that microglial progenitors migrate into early developing brain for further maturation. However, the amount of microglia and other potential mesodermal lineage cells developed in HBOs is uncontrolled after incorporation of MPCs. For example, the number of microglia developed in MC-HBOs using this strategy only accounted for 1.18% of total cells as revealed by single-cell RNA sequencing (scRNA-seq) [ 85]. This number is much lower than that in the healthy human brain.

Co-culture of MPCs with NPCs

Studies have demonstrated the capacity of NPCs to self-assemble into 3D brain organoids [ 86]. Co-culture of human MPCs with NPCs could be an alternative to develop MC-HBOs (Fig. 4). This strategy allows researchers to study the interactions between microglia and NPCs or developing neurons during the organoid formation, and to generate uniformed and cell-type ratio-controlled MC-HBOs. By co-clustering of hiPSCs-derived PMPs (CD43⁺ and CD235⁺) and NPCs, Xu et al. [ 87] developed brain-region specific MC-HBOs with a controllable proportion of microglia (8% in total cells). In the MC-HBOs, microglia exhibited the capacity to phagocytose NPCs, remove apoptotic cells, prune synapses, and respond to immune stimuli. The microglia could also respond to ZIKA virus and exhibit increased synaptic elimination in MC-HBOs. Importantly, neurons in the MC-HBOs (~90 days) exhibited EPSCs, suggesting the establishment of functional neural network in the organoids.

By using Xu's co-culture methods [ 87], Jin et al. [ 88] generated MC-HBOs to explore microglial functions in Down syndrome (DS). In their study, DS microglia in HBOs exhibited enhanced synaptic pruning without significantly morphological changes when compared with control microglia. After transplantation into mouse brain, both DS and control microglia showed increasingly complex morphologies. However, the xenografted DS microglia displayed less intricate morphologies than control microglia, as indicated by enlarged cell volume, shortened process length, fewer endpoints, and branch numbers. These results suggested that MC-HBOs recapitulated the enhanced synaptic pruning function of DS microglia but were not optimal for modeling the morphological changes of DS microglia. This might be due to that microglia in HBOs did not exhibit highly branched and ramified morphologies as they did in-vivo. In addition, the MC-HBOs were limited to examine how DS microglia affected synaptic transmission in HBOs, as neurons didn't exhibit robust EPSCs until after a long-term culture (~90 days) [ 88]. The authors transplanted microglia into mouse brain and found that DS chimeric mice had miniature EPSCs with significantly reduced frequency and amplitude. DS microglia showed dystrophic and senescent phenotypes in response to pathological tau. Reducing expression of Hsa21 encoded type I interferon receptor genes (IFNARs) rescued the accelerated senescence and dystrophic phenotypes of DS microglia [ 88], implying therapeutic targets for treating AD in DS.

Spontaneous formation of microglia in HBOs

Pluripotent stem cells including hiPSCs and hESCs have the potential to differentiate into all cell types of three germ layers (endoderm, mesoderm, and ectoderm). Due to the ectoderm origin of neuronal cells, dual-SMAD signaling inhibition was commonly used for neuroectoderm induction in order to suppress the differentiation towards endoderm and mesoderm lineages [ 89]. Since SMAD signaling inhibitors were not used in the unguided protocol for cerebral organoid formation [ 60], it is possible that non-neuronal lineage cells exit in the cerebral organoids. Indeed, scRNA-seq analysis identified mesodermal progenitors [ 62], myeloid cells [ 90], and even microglia clusters [ 68] in HBOs generated from unguided protocols. With the hypothesis that mesodermal progenitors within HBOs are able to differentiate into mature microglia under the organoid microenvironment, Ormel et al. firstly developed a model with microglia innately developed within cerebral organoids [ 91] (Fig. 5). Interestingly, the authors only simply modified the unguided protocol by reducing the concentration of heparin (from 1 ug/mL to 0.1 ug/mL) and delaying matrigel embedment of the organoids, resulting in the production of mesodermal progenitors and following microglia generation in organoids (as early as day 24). The organoid-grown microglia displayed ramified morphologies, expressed microglia-specific markers, functionally resembled adult human microglia, and elicited immune responses in organoids. Interestingly, EPSCs could be detected in MC-HBOs as early as day 52 in Ormel et al. study [ 91], while it was not until organoid day 90 and day 107 that EPSCs could be detected in the studies of Xu et al. [ 87] and Fagerlund et al. [ 84].

Based on the protocol of Ormel et al. [ 91], other studies [ 92, 93, 94] also generated HBOs containing innately-developed microglia. The MC-HBOs were used to model SARS-CoV-2 infection [ 93] or Western Pacific Amyotrophic Lateral Sclerosis and Parkinsonism-Dementia Complex (ALS-PDC, a neurodegenerative disease) [ 94]. Upon SARS-CoV-2 infection, the innately-developed microglia in organoids responded to viral infection through upregulating phagocytosis- and interferon-related pathways. Several AD and multiple sclerosis susceptibility genes were also found to be upregulated in microglia upon SARS-CoV-2 infection, suggesting potential contribution of SARS-CoV-2 to neurological symptoms [ 93]. By using MC-HBOs, the ALS-PDC study [ 94] revealed that the shift of microglia to a more pro-inflammatory state, which exacerbated inflammation and reduced extracellular matrix strength, may account for the etiology of ALS-PDC. Unlike previous studies [ 91, 92, 93], this study didn't reduce the concentration of heparin to 0.1 ug/mL but also resulted into microglia generation in cerebral organoids.

Though microglia could innately develop in HBOs, the proportion and distribution of microglia in organoids are highly variable due to the spontaneous and stochastic features of unguided differentiation [ 91]. For example, the proportion of CD11B⁺ microglia in MC-HBOs was about 7% in Bodnar et al. study [ 92], while it was up to 1.5% in the study of Ormel et al. [ 91]. To develop HBOs with a tunable proportion of microglia included, Cakir et al. [ 95] mixed myeloid-specific transcription factor PU.1-overexpressed hESCs (10%) with parental hESCs (90%) to generate MC-HBOs. scRNA-seq analysis revealed that transcriptional profiles of PU.1-induced microglia resembled those in the middle to late stages of fetal microglia development. Notably, the authors stated that the PU.1-induced microglia demonstrated a more similar transcriptome profile to human primary microglia than microglia generated in Omel's study [ 91]. The presence of microglia within HBOs significantly attenuated transcriptional dysregulation of dendritic and synaptic development and apoptotic genes caused by Aβ exposure. Using the MC-HBOs, the authors further found that suppression of AD risk genes ( TREM2 and SORL1) in PU.1⁺ cells did not affect microglia generation but their responses to Aβ exposure. This study provided a strategy to develop a tunable proportion (~10%) of microglia within organoids for microglia study. However, not all PU.1 overexpressed cells in organoids were converted into microglia-like cells, leading to these myeloid cells with a distinct identity and maturity in organoids [ 95].

Perspectives and future directions

The recently developed MC-HBOs have become a promising model to study how microglia modulate brain development and pathology. Several strategies now have been developed to generate MC-HBOs to study brain development and disorders and viral infection as reviewed in this paper. Those studies highlighted MC-HBOs in resembling in-vivo microglia signatures, such as human-specific expression patterns of cytokine and chemokine genes [ 55] and unique inflammatory responses to viral infection [ 79]. In such an emerging field, most of the current studies focused on establishing robust MC-HBOs models. Regarding future work, the incorporation of microglia into HBOs could be used to study how microglia-specific genes or genetic variations affect brain phenotypes, and how microglia per se modulate brain functions. The co-culture of MPCs with HBOs, which may better mimic the developmental trajectories of in-vivo microglia, could be utilized to study how HBOs environment affects microglia development. The co-culture of MPCs with NPCs could be used to investigate co-development of microglia and neurons. More importantly, co-culture of MPCs with NPCs [ 87, 88] could generate MC-HBOs with a controllable proportion of microglia (~8%) similar to the physiologic conditions. In addition to co-culture methods, the strategy that innately develops microglia within HBOs is a simple method to generate MC-HBOs for the study of genetic variations existing in all cell types. This simplified method is suitable for large-sample studies such as case-control transcriptomic studies based on MC-HBOs. Considering that microglia affect transcriptional patterns and phenotypes of brain organoids, applying MC-HBOs to case-control transcriptomic studies may yield novel insights that could be missed by studies based on brain organoids without microglia.

We have to keep in mind that a robust brain organoid model is essential to yield reproducible results for microglia studies. It is urgent to put forward gold standards to define what "true" brain organoids are, as diverse protocols will likely generate HBOs with distinct features that lead to inconsistent results. A human brain organoid may have to show similar cell composition (progenitors, neurons and glia cells, etc.), cellular structures (the emergence of ventricular zone, cortical layers, etc), gene expression patterns, and developmental trajectories as the in-vivo human brain. Thus, it is challenging to call 3D cell cultures by mixing neurons with other cell types (e.g., microglia and astrocyte) as "organoids", since cellular organization and composition, and cell developmental paths in the cell mixture system could not mimic those in the human brain. For the reasons, those studies [ 103, 104] that developed 3D assembloids [ 105] by co-culture of neurons, microglia, and astrocytes were not included in this review. Assembloids, as defined by Pasca [ 105], are 3D structures formed from the fusion and functional integration of multiple cell types. MC-HBOs formed by the co-culture strategies could also be defined as assembloids [ 105]. In the future, generating assembloids by fusing MC-HBOs with different brain region identities will be beneficial to study regional heterogeneity of microglia.

In addition to the standards for brain organoids, the fidelity of microglia within the MC-HBOs is another key concern in microglia study. Microglia is a highly complex and dynamic cell type. The morphologies and transcriptional patterns of microglia could be changed in response to environment. How much the induced microglia can mimic in-vivo cells to produce consistent responses remains to be learned. Microglia produced by different protocols could vary in their origin and the abilities to recapitulate in-vivo signatures. For example, hiPSCs-derived microglia from different methods showed transcriptional and morphological heterogeneity, resulting in potential heterogeneous phenotypes after incorporation into HBOs. Assays used to examine microglia identity and state, phagocytotic function, response to immune stimuli, etc., are required for microglia characterization. A systematic evaluation is also needed to assess which strategy is better to generate MC-HBOs that recapitulate the true ontogeny and in-vivo signatures of human microglia.

This review highlights the merits and applications of MC-HBOs among the several microglial models, some apparent shortcomings of current MC-HBOs should be noted. First, MC-HBOs are in-vitro models in nature and unlikely fully recapitulate features of in-vivo human brain. Current MC-HBOs lack the vascular systems and lead to increased cellular stress that hinders cell type specification and maturation of brain organoids [ 106]. Moreover, the lack of complex neural circuits and intact immune system also limit the further maturation of HBOs to study adult brain and late-onset brain disorders using current MC-HBOs. In the future, approaches including air-liquid-interphase culture [ 107], vascularization system [ 108], and microfluidic system [ 109] could be applied to brain organoid culture to attenuate cellular stress, and to promote organoid maturation.

Every microglial model has its advantages and disadvantages (Table 1), using the appropriate model in a specific situation or combining the use of different models could yield robust results for microglia study. For example, researchers combined the use of HBOs and mouse model and found that HBOs established long-distance subcortical projections in the mouse brain after transplantation. Neurons differentiated from the transplanted HBOs functionally integrated into mouse neural circuits [ 110, 111]. The HBOs-mouse chimeric models enable investigations to be conducted under in-vivo like conditions. Since mouse model could provide an in-vivo microenvironment to enhance fidelity of native microglia state [ 55], and to promote maturation of brain organoids [ 106, 111, 112, 113], MC-HBOs transplanted into mouse brain could be a promising model to better mimic the real human brain.

In summary, MC-HBOs have become a promising model to illustrate what roles microglia play in brain functions and disease etiologies. The MC-HBOs are not only valuable for microglia research, but also beneficial to the construction of organoids that better mimic the real human brain. With the MC-HBOs, we have the unique opportunity to advance our understanding of how the human brain develops and how it works. In the future studies, developing a robust and reproducible MC-HBOs model by combining with gene editing and multi-omics analyses will largely promote the fields of disease modeling and functional studies.

Funding

This work was supported by grants from the National Natural Science Foundation of China (Nos. 82171504, 82022024, 31970572, and 81901359), the National Key R&D Project of China (No. 2017YFC0908701), the Natural Science Foundation of Hunan Province (No. 2022JJ20035), the Science and Technology Innovation Program of Hunan Province (2022RC1214), the Natural Science Foundation of Guangxi (No. 2020AC20026), and the Innovation-Driven Project of Central South University (No. 2020CX003), and the National Institute of Health (No. 1U01MH116489).

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Author notes

These authors contributed equally: Wendiao Zhang, Jiamei Jiang.

Authors and Affiliations

1. The First Affiliated Hospital, Multi-Omics Research Center for Brain Disorders, Hengyang Medical School, University of South China, 421001, Hengyang, Hunan, China

Wendiao Zhang, Jiamei Jiang, Zhenhong Xu, Hongye Yan, Beisha Tang & Qingtuan Meng

2. Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, and Department of Psychiatry, The Second Xiangya Hospital, Central South University, 410008, Changsha, Hunan, China

Wendiao Zhang & Chao Chen

3. The First Affiliated Hospital, Clinical Research Center for Immune-Related Encephalopathy of Hunan Province, Hengyang Medical School, University of South China, 421001, Hengyang, Hunan, China

Wendiao Zhang, Jiamei Jiang, Zhenhong Xu, Hongye Yan & Qingtuan Meng

4. The First Affiliated Hospital, Department of Neurology, Hengyang Medical School, University of South China, 421001, Hengyang, Hunan, China

Zhenhong Xu, Hongye Yan & Qingtuan Meng

5. Department of Neurology, Xiangya Hospital, Central South University, 410008, Changsha, Hunan, China

Beisha Tang

6. Department of Psychiatry, SUNY Upstate Medical University, Syracuse, NY, 13210, USA

Chunyu Liu

7. Hunan Key Laboratory of Animal Models for Human Diseases, Central South University, 410008, Changsha, Hunan, China

Chao Chen

8. Hunan Key Laboratory of Molecular Precision Medicine, Central South University, 410008, Changsha, Hunan, China

Chao Chen

Contributions

QM, CC, CL, WZ, JJ, and BT wrote and revised the manuscript. ZX and HY draw the figures.

Corresponding authors

Correspondence to Chunyu Liu, Chao Chen or Qingtuan Meng.

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Zhang, W., Jiang, J., Xu, Z. et al. Microglia-containing human brain organoids for the study of brain development and pathology. Mol Psychiatry 28, 96-107 (2023). https://doi.org/10.1038/s41380-022-01892-1
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DOI: https://doi.org/10.1038/s41380-022-01892-1

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Microglia-containing human brain organoids for the study of brain development and pathology - Molecular Psychiatry

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Microglia are resident immune cells in the central nervous system, playing critical roles in brain development and homeostasis. Increasing evidence has implicated microglia dysfunction in the pathogenesis of various brain disorders ranging from psychiatric disorders to neurodegenerative diseases. Using a human cell-based model to illuminate the functional mechanisms of microglia will promote pathological studies and drug development.

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Application of organoids on the pharmaceutical industry

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Application of organoids on the pharmaceutical industry

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Drug Discovery:

Neural organoids or organoids, have been used and tested in mice against cancer with personalized drug treatment. As the organoid will closely mimic the genetics of the mouse, it can be used to see what drug or treatment, would be most effective, to deal with different diseases, whilst mitigating as many side effects as possible(1).

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Fused in sarcoma (FUS) is a protein that is involved in regulating transcription and processing RNA.

ALS patients usually have mutated FUS in the cytoplasm of affected neurons.

Reducing levels of FUS in a spinal cord organoid decreased neural proliferation and differentiation, suggesting that FUS is important for those roles. [9]

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Studying Brain Development:
The development of neural organoids has allowed researchers to gain a deeper understanding of the functioning of the human brain during development (4). Since neural organoids are similar to the human brain at a molecular and cellular level, researchers can study the mechanisms underlying brain development, which will then allow them to understand, how certain neurological disorders arise (4).

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ALS affects motor neurons, resulting in progressive muscle weakness. The condition has an average survival rate of about 3 years after symptoms onset. [3]

Organoids derived from patients with ALS have shown irregular neuromuscular junctions. [4]

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This disorder is caused by mutations affecting levels of spinal motor neurons. This can result in paralysis or death in extreme cases.

Spinal organoid models can be derived to observe development of the patient’s motor neurons and for testing what small molecules can help increase motor neuron levels. [2]

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specifically the distribution of cells in cortical layers being in multiple rosettes, instead of continuous layers as typical of the human brain. (1)

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it does not accurately reflect the developmental events of typical human growth, occurring more rapidly than normal, especially relating to neural organoids. (1)

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1. History and development of organoids - a timeline

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1. History and development of organoids - a timeline

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A lot of progress has been made with the development of organoids, beginning with Henry Van Peters Wilson showing sponge cells self organising into a whole organism [1]. Organoid research then advanced to the development of different human organoids [1] and is now at a stage where they are being transplanted into humans [3]. Despite all this, there is still a long way to go with organoid research before they can be widely used in clinical settings.

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Specificity of gene expression are not yet perfectly replicated in organoid-derived cells (1)

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Defining Consciousness & its Importance (6)

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Defining Consciousness & its Importance (6)

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Challenges of Organoid Research

Madeline G. Andrews and Arnold R. Kriegstein

Annual Review of Neuroscience 2022 45:1, 23-39
(1)

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Examining the morphogenesis of an organ can provide useful insight into its development, and how they function at a cellular level.

Therefore this understanding of disease mechanisms can be understood, and treatments can be curated.

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Spinal cord organoids can be formed by combining induced pluripotent stem cells (iPSCs), derived neural and non-neural cells. This can form a replica of a mature human spinal cord. [1]