An assembloid is an ''

in vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology an ...

'' model that combines two or more

organoid An organoid is a miniaturized and simplified version of an Organ (anatomy), organ produced in vitro in three dimensions that shows realistic micro-anatomy. They are derived from one or a few Cell (biology), cells from a Tissue (biology), tissue, ...

spheroids A spheroid, also known as an ellipsoid of revolution or rotational ellipsoid, is a quadric surface (mathematics), surface obtained by Surface of revolution, rotating an ellipse about one of its principal axes; in other words, an ellipsoid with ...

, or cultured cell types to recapitulate structural and functional properties of an organ. They are typically derived from

induced pluripotent stem cell Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from a somatic cell. The iPSC technology was pioneered by Shinya Yamanaka's lab in Kyoto, Japan, who showed in ...

s. Assembloids have been used to study

cell migration Cell migration is a central process in the development and maintenance of multicellular organisms. Tissue formation during embryonic development, wound healing and immune responses all require the orchestrated movement of cells in particular dire ...

neural circuit A neural circuit is a population of neurons interconnected by synapses to carry out a specific function when activated. Neural circuits interconnect to one another to form large scale brain networks. Biological neural networks have inspired the ...

assembly, neuro-immune interactions,

metastasis Metastasis is a pathogenic agent's spread from an initial or primary site to a different or secondary site within the host's body; the term is typically used when referring to metastasis by a cancerous tumor. The newly pathological sites, then, ...

, and other complex tissue processes. The term "assembloid" was coined by

Sergiu P. Pașca Sergiu P. Pașca (born January 30, 1982) is a Romanian-American scientist and physician at Stanford University in California. He is renowned for his groundbreaking work creating and developing stem cell-based models of the human brain to gain insi ...

's lab in 2017.

Generation of assembloids

Assembloids were described in 2017 in a study from a laboratory at

Stanford Stanford University, officially Leland Stanford Junior University, is a private research university in Stanford, California. The campus occupies , among the largest in the United States, and enrolls over 17,000 students. Stanford is considere ...

to model forebrain development. Assembloids joining ventral and dorsal forebrain neural organoids demonstrated that cortical interneurons migrate and integrate into synaptically connected cortical microcircuits. This was confirmed by multiple research groups applying similar approaches to model regionalized organoid interactions and study interneuron migration. Assembloids have subsequently been generated to model projections between brain regions, such as cortico-striatal, cortico-spinal, or retino-thalamic. Methods such as Cre recombination combined with G-deleted rabies tracing can be used to identify cells projecting within assembloids; additionally,

optogenetic Optogenetics is a biological technique to control the activity of neurons or other cell types with light. This is achieved by expression of light-sensitive ion channels, pumps or enzymes specifically in the target cells. On the level of individ ...

stimulation can demonstrate the assembly of functional neural circuits in vitro. Assembloid formation starts with the generation of organoids. Initially, human induced pluripotent stem (hiPS) cells are aggregated to generate regionalized organoids through

directed differentiation Directed differentiation is a bioengineering methodology at the interface of stem cell biology, developmental biology and tissue engineering. It is essentially harnessing the potential of stem cells by constraining their differentiation in vitro t ...

. There are multiple ways in which organoids can be assembled. Regionalized organoids can be put in close proximity resulting in their fusion to generate multi-region assembloids. Alternatively, organoids can be assembled by co-culture with other cell lineages, such as microglia or endothelial cells, or with tissue samples from animal dissection, leading to multi-lineage assembloids. Lastly, organoids can be assembled with morphogenic or organizer-like cells, thus generating polarized assembloids. The assembloid type depends on the scientific question and the accessibility of cell types required. Major biological fields utilizing the assembloid technique include cancer, gastroenterology, cardiology, and neuroscience. For instance, there are liver assembloids, kidney assembloids, pericytes assembloids to study SARS-COVID2, endometrium assembloids, and bladder assembloids.

Types

Assembloids are composed of at least two organoids and/or cells derived from stem cells or primary tissue. They can be assembled to form multi-region or multi-lineage assembloids, as described above. A. Multi-region assembloids of the nervous system There are techniques to guide organoid differentiation into specific regions of the nervous system. For example, fusion of thalamic and cortical neural organoids models thalamo-cortical projections of ascending sensory input while cortico-striatal assembloids generate the initial projections of motor planning circuits.

Forebrain In the anatomy of the brain of vertebrates, the forebrain or prosencephalon is the Anatomical terms of location#Directional terms, rostral (forward-most) portion of the brain. The forebrain (prosencephalon), the midbrain (mesencephalon), and hin ...

assembloids model interneuron migration into the cerebral cortex. Cortico-motor assembloids can reconstitute aspects of the cortico-spinal-muscle circuit in vitro. Finally, retinal organoids can be combined with thalamic and cortical organoids to model aspects of the ascending visual pathway. B. Multi-lineage assembloids of the nervous system Some cell types of interest are challenging to differentiate within organoids but can be isolated from tissue explants or derived in monolayer culture. These tissue samples or enriched cell populations can then be integrated with organoid(s) of interest to study their interaction. For example, one current limitation of organoids and assembloids is their lack of functional vasculature, which hinders the supply of nutrients and trophic factors. In a technical advancement, researchers have been able to achieve vascularization by combining neural organoids with

endothelial The endothelium is a single layer of squamous endothelial cells that line the interior surface of blood vessels and lymphatic vessels. The endothelium forms an interface between circulating blood or lymph in the lumen and the rest of the vessel ...

organoids and mesenchymal cells or human embryonic stem cell-derived vascular organoids. Next,

microglia Microglia are a type of neuroglia (glial cell) located throughout the brain and spinal cord. Microglia account for about 7% of cells found within the brain. As the resident macrophage cells, they act as the first and main form of active immune de ...

-like cells derived from hiPS cells can be introduced into midbrain neural organoids to model neuro-immune interactions. Similarly,

oligodendrocyte Oligodendrocytes (), or oligodendroglia, are a type of neuroglia whose main functions are to provide support and insulation to axons in the central nervous system of jawed vertebrates, equivalent to the function performed by Schwann cells in the ...

s can be generated in neural organoids and then migrate from the ventral forebrain to the dorsal forebrain. Lastly, combining hiPS cell-derived intestinal organoids with

neural crest Neural crest cells are a temporary group of cells unique to vertebrates that arise from the embryonic ectoderm germ layer, and in turn give rise to a diverse cell lineage—including melanocytes, craniofacial cartilage and bone, smooth muscle, per ...

cells can derive assembloids of the enteric nervous system. Additionally, assembloids can be categorized as inter-individual or inter-species, depending on whether the organoids are combined from different stem cell lines (e.g., control with disease-associated lines) or different species, respectively. These combinations help determine what aspects of development are cell-autonomous.

Disease models and applications

Assembloids help determine the complex pathophysiology of developmental disorders. For example, Timothy syndrome, which affects

L-type calcium channel The L-type calcium channel (also known as the dihydropyridine channel, or DHP channel) is part of the high-voltage activated family of voltage-dependent calcium channel. "L" stands for long-lasting referring to the length of activation. This c ...

s, was modeled in neural assembloid experiments. When dorsal and ventral forebrain organoids were integrated into an assembloid, interneurons migrated into the dorsal cortical neurons. Timothy syndrome-derived interneurons showed impaired migration. The resulting assembloids developed hypersynchronous neuronal activity, hypothesized to be due to abnormal interneuron integration into circuits. Next,

Phelan-McDermid syndrome 22q13 deletion syndrome, also known as Phelan–McDermid syndrome (PMS), is a genetic disorder caused by deletions or rearrangements on the q terminal end (long arm) of chromosome 22. Any abnormal genetic variation in the q13 region that present ...

, also known as 22q13.3 deletion syndrome, is a neurodevelopmental disorder with a high risk of autism spectrum disorder that was modeled in assembloids containing cortical and striatal organoids. This research demonstrated increased striatal medium spiny neuron activity in Phelan-McDermid-derived assembloids after fusion of striatal and cortical organoids but not in isolated striatal organoids.

Rett syndrome Rett syndrome (RTT) is a genetic disorder that typically becomes apparent after 6–18 months of age and almost exclusively in females. Symptoms include impairments in language and coordination, and repetitive movements. Those affected often h ...

-derived assembloids displayed hypersynchronous activity perhaps due to an increase in calretinin interneurons.

Alzheimer's disease Alzheimer's disease (AD) is a neurodegeneration, neurodegenerative disease that usually starts slowly and progressively worsens. It is the cause of 60–70% of cases of dementia. The most common early symptom is difficulty in short-term me ...

risk allele

APOE4 Apolipoprotein E (APOE) is a protein involved in the metabolism of fats in the body of mammals. A subtype is implicated in Alzheimer's disease and cardiovascular disease. APOE belongs to a family of fat-binding proteins called apolipoproteins. ...

, which increases the risk of dementia, has been modeled in assembloids. APOE4-derived assembloids of neural organoids combined with microglia demonstrated increased amyloid-beta-42 secretion, a known Alzheimer biomarker. APOE4 microglia in assembloids had a more complex morphology than in two-dimensional culture and had limited amyloid-beta-42 clearance.

Limitations

Despite the research benefits of assembloids, as for any model system, they have limitations. First, assembloids, like organoids, lack vascularisation, which impairs nutrient diffusion to the surface and eventually leads to necrosis in the core, thus limiting their growth. One way to address this limitation is through transplantation. Grafting cortical organoids into the brains of laboratory rats leads to improved growth and neural development.Another critique of both assembloids and organoids is the lack of sensory input, which is important for the maturation and shaping of circuits during embryonic development. Assembloids and organoids do not currently have a blood brain barrier or immune cells, limiting the biological validity for drug screening or disease modeling.There is a temporal limitation on the investigation of clinically relevant pathophysiology; organoids most closely model initial developmental stages corresponding to fetal and infant neurodevelopment and thus may not accurately model later-onset psychiatric disorders or degenerative conditions. Future directions to address this limitation include studies to understand and accelerate developmental clocks.Next, organoids and assembloids have batch-to-batch variability. Guided differentiation methods reduce variability significantly, yet reproducibility still requires optimization. Finally, the derivation and maintenance of organoids and assembloids require expertise and can be time-intensive and expensive.

References

{{reflist , refs= {{cite journal , author=Pașca SP, Arlotta P, Bateup HS, Camp JG, Cappello S, Gage FH , title=A nomenclature consensus for nervous system organoids and assembloids , journal=Nature , year=2022 , volume=609 , issue=7929 , pages=907–910 , pmid=36171373 , doi=10.1038/s41586-022-05219-6 , pmc=10571504, bibcode=2022Natur.609..907P {{cite journal , author=Pașca SP , title=The rise of three-dimensional human brain cultures , journal=Nature , year=2018 , volume=553 , issue=7689 , pages=437–445 , doi=10.1038/nature25032 , pmid=29364288, bibcode=2018Natur.553..437P , s2cid=205262820 {{cite journal , author=Paşca SP , title=Assembling human brain organoids , journal=Science , year=2019 , volume=363 , issue=6423 , pages=126–127 , doi=10.1126/science.aau5729 , pmid=30630918, bibcode=2019Sci...363..126P , s2cid=57825925 {{cite journal , author=Schmidt C , title=The rise of the assembloid , journal=Nature , year=2021 , volume=597 , issue=7878 , pages=S22–S23 , doi=10.1038/d41586-021-02628-x, bibcode=2021Natur.597S..22S , s2cid=238229376 {{cite journal , author=Birey F, Andersen J, Makinson CD, Islam S, Wei W, Huber N , title=Assembly of functionally integrated human forebrain spheroids , journal=Nature , year=2017 , volume=545 , issue=7652 , pages=54–59 , doi=10.1038/nature22330 , pmid=28445465 , pmc=5805137, bibcode=2017Natur.545...54B {{Cite AV media , people=Pașca, Sergiu , title=How we're reverse engineering the human brain in the lab , date=August 24, 2022 , language=en , location=Vancouver, Canada , publisher=TED , url=https://www.ted.com/talks/sergiu_p_pasca_how_we_re_reverse_engineering_the_human_brain_in_the_lab , access-date=2023-12-10 {{cite journal , author=Bagley JA, Reumann D, Bian S, Lévi-Strauss J, Knoblich JA , title=Fused cerebral organoids model interactions between brain regions , journal=Nat Methods , year=2017 , volume=14 , issue=7 , pages=743–751 , doi=10.1038/nmeth.4304 , pmid=28504681 , pmc=5540177 {{cite journal , author=Xiang Y, Tanaka Y, Patterson B, Kang YJ, Govindaiah G, Roselaar N , display-authors=etal , title=Fusion of regionally specified hPSC-derived organoids models human brain development and interneuron migration , journal=Cell Stem Cell , year=2017 , volume=21 , issue=3 , pages=383–398.e7 , doi=10.1016/j.stem.2017.07.007 , pmid=28757360 , pmc=5720381 {{cite journal , author=Miura Y, Li MY, Birey F, Ikeda K, Revah O, Thete MV , display-authors=etal , title=Generation of human striatal organoids and cortico-striatal assembloids from human pluripotent stem cells , journal=Nat Biotechnol , year=2020 , volume=38 , issue=12 , pages=1421–1430 , doi=10.1038/s41587-020-00763-w , pmid=33273741 , pmc=9042317 {{cite journal , author=Andersen J, Revah O, Miura Y, Thom N, Amin ND, Kelley KW , display-authors=etal , title=Generation of functional human 3d cortico-motor assembloids , journal=Cell , year=2020 , volume=183 , issue=7 , pages=1913–1929.e26 , doi=10.1016/j.cell.2020.11.017 , pmid=33333020 , pmc=8711252 {{cite journal , author=Fligor CM, Lavekar SS, Harkin J, Shields PK, VanderWall KB, Huang KC, Gomes C, Meyer JS , title=Extension of retinofugal projections in an assembled model of human pluripotent stem cell-derived organoids , journal=Stem Cell Rep , year=2021 , volume=16 , issue=9 , pages=2228–2241 , doi=10.1016/j.stemcr.2021.05.009 , pmid=34115986 , pmc=8452489 {{cite journal , author=Miura Y, Li MY, Revah O, Yoon SJ, Narazaki G, Pașca SP , title=Engineering brain assembloids to interrogate human neural circuits , journal=Nat Protoc , year=2022 , volume=17 , issue=1 , pages=15–35 , doi=10.1038/s41596-021-00632-z , pmid=34992269, s2cid=245773595 {{cite journal , author=Sloan SA, Andersen J, Pașca AM, Birey F, Pașca SP , title=Generation and assembly of human brain region–specific three-dimensional cultures , journal=Nat Protoc , year=2018 , volume=13 , issue=9 , pages=2062–2085 , doi=10.1038/s41596-018-0032-7 , pmid=30202107 , pmc=6597009 {{cite journal , author=Lin YT, Seo J, Gao F, Feldman HM, Wen HL, Penney J , display-authors=etal , title=APOE4 causes widespread molecular and cellular alterations associated with Alzheimer's disease phenotypes in human ipsc-derived brain cell types , journal=Neuron , year=2018 , volume=98 , issue=6 , pages=1141–1154.e7 , doi=10.1016/j.neuron.2018.05.008 , pmid=29861287 , pmc=6023751 {{cite journal , author=Cederquist GY, Asciolla JJ, Tchieu J, Walsh RM, Cornacchia D, Resh MD, Studer L , title=Specification of positional identity in forebrain organoids , journal=Nat Biotechnol , year=2019 , volume=37 , issue=4 , pages=436–444 , doi=10.1038/s41587-019-0085-3 , pmid=30936566 , pmc=6447454 {{cite journal , author=Takeishi K, Collin de l'Hortet A, Wang Y, Handa K, Guzman-Lepe J, Matsubara K , display-authors=etal , title=Assembly and function of a bioengineered human liver for transplantation generated solely from induced pluripotent stem cells , journal=Cell Rep , year=2020 , volume=31 , issue=9 , pages=107711 , doi=10.1016/j.celrep.2020.107711 , pmid=32492423 , pmc=7734598 {{cite journal , author=Tsujimoto H, Kasahara T, Sueta SI, Araoka T, Sakamoto S, Okada C , display-authors=etal , title=A modular differentiation system maps multiple human kidney lineages from pluripotent stem cells , journal=Cell Rep , year=2020 , volume=31 , issue=1 , pages=107476 , doi=10.1016/j.celrep.2020.03.040 , pmid=32268094, s2cid=215610752 , doi-access=free , hdl=2433/250216 , hdl-access=free {{cite journal , author=Wang L, Sievert D, Clark AE, Lee S, Federman H, Gastfriend BD , display-authors=etal , title=A human three-dimensional neural-perivascular "assembloid" promotes astrocytic development and enables modeling of SARS-CoV-2 neuropathology , journal=Nat Med , year=2021 , volume=27 , issue=9 , pages=1600–1606 , doi=10.1038/s41591-021-01443-1 , pmid=34244682 , pmc=8601037 {{cite journal , author=Birey F, Li MY, Gordon A, Thete MV, Valencia AM, Revah O , display-authors=etal , title=Dissecting the molecular basis of human interneuron migration in forebrain assembloids from Timothy syndrome , journal=Cell Stem Cell , year=2022 , volume=29 , issue=2 , pages=248–264.e7 , doi=10.1016/j.stem.2021.11.011 , pmid=34990580, doi-access=free {{cite journal , author=Rawlings TM, Makwana K, Taylor DM, Molè MA, Fishwick KJ, Tryfonos M , display-authors=etal , title=Modelling the impact of decidual senescence on embryo implantation in human endometrial assembloids , journal=eLife , year=2021 , volume=10 , doi=10.7554/eLife.69603 , pmid=34487490 , pmc=8523170 , doi-access=free {{cite journal , author=Kim E, Choi S, Kang B, Kong J, Kim Y, Yoon WH , display-authors=etal , title=Creation of bladder assembloids mimicking tissue regeneration and cancer , journal=Nature , year=2020 , volume=588 , issue=7839 , pages=664–669 , doi=10.1038/s41586-020-3034-x , pmid=33328632, bibcode=2020Natur.588..664K , s2cid=229293144 {{cite journal , author=Song L, Yuan X, Jones Z, Griffin K, Zhou Y, Ma T, Li Y , title=Assembly of human stem cell-derived cortical spheroids and vascular spheroids to model 3-D brain-like tissues , journal=Sci Rep , year=2019 , volume=9 , issue=1 , pages=5977 , doi=10.1038/s41598-019-42439-9 , pmid=30979929 , pmc=6461701, bibcode=2019NatSR...9.5977S {{cite journal , author=Cakir B, Xiang Y, Tanaka Y, Kural MH, Parent M, Kang YJ , display-authors=etal , title=Engineering of human brain organoids with a functional vascular-like system , journal=Nat Methods , year=2019 , volume=16 , issue=11 , pages=1169–1175 , doi=10.1038/s41592-019-0586-5 , pmid=31591580 , pmc=6918722 {{cite journal , author=Sun XY, Ju XC, Li Y, Zeng PM, Wu J, Zhou YY , display-authors=etal , title=Generation of vascularized brain organoids to study neurovascular interactions , journal=eLife , year=2022 , volume=11 , doi=10.7554/eLife.76707 , pmid=35506651 , pmc=9246368 , doi-access=free {{cite journal , author=Sabate-Soler S, Nickels SL, Saraiva C, Berger E, Dubonyte U, Barmpa K , display-authors=etal , title=Microglia integration into human midbrain organoids leads to increased neuronal maturation and functionality , journal=Glia , year=2022 , volume=70 , issue=7 , pages=1267–1288 , doi=10.1002/glia.24167 , pmid=35262217 , pmc=9314680 {{cite journal , author=Madhavan M, Nevin ZS, Shick HE, Garrison E, Clarkson-Paredes C, Karl M , display-authors=etal , title=Induction of myelinating oligodendrocytes in human cortical spheroids , journal=Nat Methods , year=2018 , volume=15 , issue=9 , pages=700–706 , doi=10.1038/s41592-018-0081-4 , pmid=30046099 , pmc=6508550 {{cite journal , author=Marton RM, Miura Y, Sloan SA, Li Q, Revah O, Levy RJ , display-authors=etal , title=Differentiation and maturation of oligodendrocytes in human three-dimensional neural cultures , journal=Nat Neurosci , year=2019 , volume=22 , issue=3 , pages=484–491 , doi=10.1038/s41593-018-0316-9 , pmid=30692691 , pmc=6788758 {{cite journal , author=Shaker MR, Pietrogrande G, Martin S, Lee JH, Sun W, Wolvetang EJ , title=Rapid and efficient generation of myelinating human oligodendrocytes in organoids , journal=Front Cell Neurosci , year=2021 , volume=15 , doi=10.3389/fncel.2021.631548 , pmid=33815061 , pmc=8010307 , doi-access=free {{cite journal , author=Workman MJ, Mahe MM, Trisno S, Poling HM, Watson CL, Sundaram N , display-authors=etal , title=Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system , journal=Nat Med , year=2017 , volume=23 , issue=1 , pages=49–59 , doi=10.1038/nm.4233 , pmid=27869805 , pmc=5562951 {{cite journal , author=Kanton S, Paşca SP , title=Human assembloids , journal=Development , year=2022 , volume=149 , issue=20 , doi=10.1242/dev.201120 , pmid=36317797, s2cid=253246025 , doi-access=free {{cite journal , author=Samarasinghe RA, Miranda OA, Buth JE, Mitchell S, Ferando I, Watanabe M , display-authors=etal , title=Identification of neural oscillations and epileptiform changes in human brain organoids , journal=Nat Neurosci , year=2021 , volume=24 , issue=10 , pages=1488–1500 , doi=10.1038/s41593-021-00906-5 , pmid=34426698 , pmc=9070733 {{cite journal , author=Qian X, Song H, Ming GL , title=Brain organoids: advances, applications and challenges , journal=Development , year=2019 , volume=146 , issue=8) , doi=10.1242/dev.166074 , pmid=30992274 , pmc=6503989 {{cite journal , author=Revah O, Gore F, Kelley KW, Andersen J, Sakai N, Chen X , display-authors=etal , title=Maturation and circuit integration of transplanted human cortical organoids , journal=Nature , year=2022 , volume=610 , issue=7931 , pages=319–326 , doi=10.1038/s41586-022-05277-w , pmid=36224417 , pmc=9556304, bibcode=2022Natur.610..319R {{cite journal , author= Makrygianni EA, Chrousos GP , title=From brain organoids to networking assembloids: implications for neuroendocrinology and stress medicine , journal=Frontiers in Physiology , year=2021 , volume=12 , doi=10.3389/fphys.2021.621970 , pmid=34177605 , pmc=8222922 , doi-access=free {{cite journal , author=Andrews MG, Kriegstein AR , title=Challenges of organoid research , journal=Annu Rev Neurosci , year=2022 , volume=45 , pages=23–39 , doi=10.1146/annurev-neuro-111020-090812 , pmid=34985918 , pmc=10559943 {{cite journal , author=Levy RJ, Paşca SP , title=What have organoids and assembloids taught us about the pathophysiology of neuropsychiatric disorders? , journal=Biol Psychiatry , year=2023 , volume=93 , issue=7 , pages=632–641 , doi=10.1016/j.biopsych.2022.11.017 , pmid=36739210, s2cid=254167227 Biological models Stem cells Cell culture techniques

Generation of assembloids

Types

Disease models and applications

Limitations

See also

References