Abstract
The development of brain organoids represents a major technological advance in the stem cell field, a novel bridge between traditional 2D cultures and in vivo animal models. In particular, the development of midbrain organoids containing functional dopaminergic neurons producing neuromelanin granules, a by-product of dopamine synthesis, represents a potential new model for Parkinson’s disease. To generate human midbrain organoids, we introduce specific inductive cues, at defined timepoints, during the 3D culture process to drive the stem cells towards a midbrain fate. In this method paper, we describe a standardized protocol to generate human midbrain organoids (hMOs) from induced pluripotent stem cells (iPSCs). This protocol was developed to demonstrate how human iPSCs can be successfully differentiated into numerous, high quality midbrain organoids in one batch. We also describe adaptations for cryosectioning of fixed organoids for subsequent histological analysis.
Highlights
Parkinson’s disease (PD) is a neurodegenerative disorder, affecting more than 1% of the population over 65 years of age
We describe a cryosectioning protocol that we adapted to produce high-quality histological sections from midbrain organoids, overcoming difficulties resulting from the particular texture of cultured tissue as well as their small size, relative to rodent brains
Complementary to these factors, heparin plays a role in enhancing the activity of Wnt signaling58. 2-mercaptoethanol regulates oxidative stress to maintain cell growth and avoid cell death due to Reagents mTeSRTM1 Basal medium/ mTeSRTM1 5x supplement Dulbecco’s Modified Eagle Medium/ Nutrient Mixture F-12 (DMEM/F12) Neurobasal B27 without vitamin A Glutamax Minimum Essential Medium- Non-Essential Amino Acids (MEM-NEAA) 2-mercaptoethanol Heparin SB431542 Noggin CHIR99021 ROCK inhibitor Sonic Hedgehog (SHH) Fibroblast Growth Factor 8 (FGF8) Insulin Laminin Penicillin-Streptomycin Brain-derived Neurotrophic Factor (BDNF) Glial cell-derived Neurotrophic Factor (GDNF) Ascorbic acid Dibutyryl- cyclic AMP Accutase Matrigel® reduced growth factor N2 Antibiotics-Antimycotic
Summary
Parkinson’s disease (PD) is a neurodegenerative disorder, affecting more than 1% of the population over 65 years of age. There is no treatment to halt the progression of the disease. Treatment of PD is limited to symptom management. Midbrain is of ectodermal origin, neuroectodermal differentiation towards a floor plate can be induced with the help of dual-SMAD inhibition factors, Noggin and SB431542 and a Wnt pathway activator, CHIR99021 (chemical name: 6- [[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino ]ethyl]amino]-3-pyridinecarbonitrile)[57]. Complementary to these factors, heparin plays a role in enhancing the activity of Wnt signaling. Supplier/ manufacturer Catalogue number STEMCELL Technologies 05851/05852 Gibco. Complementary to these factors, heparin plays a role in enhancing the activity of Wnt signaling58. 2-mercaptoethanol regulates oxidative stress to maintain cell growth and avoid cell death due to Reagents mTeSRTM1 Basal medium/ mTeSRTM1 5x supplement Dulbecco’s Modified Eagle Medium/ Nutrient Mixture F-12 (DMEM/F12) Neurobasal B27 without vitamin A Glutamax Minimum Essential Medium- Non-Essential Amino Acids (MEM-NEAA) 2-mercaptoethanol Heparin SB431542 Noggin CHIR99021 ROCK inhibitor Sonic Hedgehog (SHH) Fibroblast Growth Factor 8 (FGF8) Insulin Laminin Penicillin-Streptomycin Brain-derived Neurotrophic Factor (BDNF) Glial cell-derived Neurotrophic Factor (GDNF) Ascorbic acid Dibutyryl- cyclic AMP (db-cAMP) Accutase Matrigel® reduced growth factor N2 Antibiotics-Antimycotic
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