Generation of human midbrain organoids from induced pluripotent stem cells

Mohamed, Nguyen‐Vi; Mathur, Meghna; Silva, Ronan V. da; Thomas, Rhalena A.; Lépine, P; Beitel, Lenore K.; Fon, Edward A.; Durcan, Thomas M.

doi:10.12688/mniopenres.12816.2

Cited by 13 publications

(43 citation statements)

References 68 publications

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“…Details on iPSC passaging is found in our QC workflow for iPSC maintenance 29 . hMOs were generated according to the published method 30 . Briefly, 10,000 cells from a single cell suspension of iPSCs were seeded in each well of a 96 well ultra-low attachment plate, containing neuronal induction media.…”

Section: Maintenance Of Ipscs and Hmo Differentiationmentioning

confidence: 99%

“…Finally, the following day, embedded hMOs were transferred into a 6-well ultra-low attachment plate filled with final differentiation medium and cultured on an orbital shaker until required for experiments 30 . For quality control purposes, mycoplasma tests were run monthly on iPSC cells and hMOs to ensure the absence of contamination.…”

Section: Maintenance Of Ipscs and Hmo Differentiationmentioning

confidence: 99%

“…Fixation and immunofluorescence staining of hMO cryosections hMOs were fixed and cryosectioned according to our published methods 30 . For staining, 14 μm cryosections were rehydrated in PBS for 15 min and surrounded with a hydrophobic barrier drawn with a barrier pen.…”

Section: Genomic Stability Testingmentioning

confidence: 99%

“…Following our previously published methods 30 , we derived hMOs from the SNCA iPSC lines using classical midbrain patterning factors 26,[39][40][41][42][43][44][45] (Fig. 1A).…”

Section: Generation Of Midbrain Organoids From Patient-derived Ipscs With An Snca Triplicationmentioning

confidence: 99%

“…1B). It has been reported that, upon exogenous treatment with dopamine, hMOs accumulate neuromelanin granules, a by-product of dopamine synthesis 26,30,46 . Thus, we treated 35-day old hMOs for 10 days with 100 μm of dopamine followed by Fontana Masson staining of hMOs sections.…”

Section: Generation Of Midbrain Organoids From Patient-derived Ipscs With An Snca Triplicationmentioning

confidence: 99%

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Midbrain organoids with anSNCAgene triplication model key features of synucleinopathy

Mohamed

Sirois

Ramamurthy

et al. 2021

Preprint

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SNCA, the first gene associated with Parkinson′s disease, encodes the α-synuclein (α-syn) protein, the predominant component within pathological inclusions termed Lewy bodies (LBs). The presence of LBs is one of the classical hallmarks found in the brain of patients with Parkinson′s disease, and LBs have also been observed in patients with other synucleinopathies. However, the study of α-syn pathology in cells has relied largely on two-dimensional culture models, which typically lack the cellular diversity and complex spatial environment found in the brain. Here, to address this gap, we use 3D midbrain organoids (hMOs), differentiated from human induced pluripotent stem cells derived from patients carrying a triplication of the SNCA gene and from CRISPR/Cas9 corrected isogenic control iPSCs. These hMOs recapitulate key features of α-syn pathology observed in the brains of patients with synucleinopathies. In particular, we find that SNCA triplication hMOs express elevated levels of α-syn and exhibit an age-dependent increase in α-syn aggregation, manifested by the presence of both oligomeric and phosphorylated forms of α-syn. These phosphorylated α-syn aggregates were found in both neurons and glial cells and their time-dependent accumulation correlated with a selective reduction in dopaminergic neuron numbers. Thus, hMOs from patients carrying SNCA gene multiplication can reliably model key pathological features of Parkinson′s disease and provide a powerful system to study the pathogenesis of synucleinopathies.

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Section: Maintenance Of Ipscs and Hmo Differentiationmentioning

confidence: 99%

Section: Maintenance Of Ipscs and Hmo Differentiationmentioning

confidence: 99%

Section: Genomic Stability Testingmentioning

confidence: 99%

“…Following our previously published methods 30 , we derived hMOs from the SNCA iPSC lines using classical midbrain patterning factors 26,[39][40][41][42][43][44][45] (Fig. 1A).…”

Section: Generation Of Midbrain Organoids From Patient-derived Ipscs With An Snca Triplicationmentioning

confidence: 99%

Section: Generation Of Midbrain Organoids From Patient-derived Ipscs With An Snca Triplicationmentioning

confidence: 99%

See 3 more Smart Citations

Midbrain organoids with anSNCAgene triplication model key features of synucleinopathy

Mohamed

Sirois

Ramamurthy

et al. 2021

Preprint

Self Cite

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Microfabricated dynamic brain organoid cocultures to assess the effects of surface geometry on assembloid formation

Cassel de Camps,

Rostami,

et al. 2024

Biotechnology Journal

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Organoids have emerged as valuable tools for the study of development and disease. Assembloids are formed by integrating multiple organoid types to create more complex models. However, the process by which organoids integrate to form assembloids remains unclear and may play an important role in the resulting organoid structure. Here, a microfluidic platform is developed that allows separate culture of distinct organoid types and provides the capacity to partially control the geometry of the resulting organoid surfaces. Removal of a microfabricated barrier then allows the shaped and positioned organoids to interact and form an assembloid. When midbrain and unguided brain organoids were allowed to assemble with a defined spacing between them, axonal projections from midbrain organoids and cell migration out of unguided organoids were observed and quantitatively measured as the two types of organoids fused together. Axonal projection directions were statistically biased toward other midbrain organoids, and unguided organoid surface geometry was found to affect cell invasion. This platform provides a tool to observe cellular interactions between organoid surfaces that are spaced apart in a controlled manner, and may ultimately have value in exploring neuronal migration, axon targeting, and assembloid formation mechanisms.

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CelltypeR: A flow cytometry pipeline to characterize single cells from brain organoids

Thomas,

Sirois,

et al. 2024

iScience

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Generation of human midbrain organoids from induced pluripotent stem cells

Cited by 13 publications

References 68 publications

Midbrain organoids with anSNCAgene triplication model key features of synucleinopathy

Midbrain organoids with anSNCAgene triplication model key features of synucleinopathy

Microfabricated dynamic brain organoid cocultures to assess the effects of surface geometry on assembloid formation

CelltypeR: A flow cytometry pipeline to characterize single cells from brain organoids

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