Generation of 3D Midbrain Organoids from Human-Induced Pluripotent Stem Cells

This protocol describes a novel method for creating 3D midbrain organoids from human induced pluripotent stem cells, guiding their formation to mimic native midbrain tissue, thereby aiding in the study of development and disorders.

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain development, facilitating precise disease modeling, and advancing therapeutic research. This protocol outlines a method for generating midbrain-specific organoids using induced pluripotent stem cells (iPSCs), employing a strategic differentiation approach. Key techniques include dual-SMAD inhibition to suppress SMAD signaling, administration of fibroblast growth factor 8b (FGF-8b), and activation of the Sonic Hedgehog pathway using the agonist purmorphamine, guiding iPSCs towards a midbrain fate.

The organoids produced by this method achieve diameters up to 2 mm and incorporate a diverse array of neuroepithelial cell types, reflecting the midbrain's inherent cellular diversity. Validation of these organoids as authentic midbrain structures involves the expression of midbrain-specific markers, confirming their identity. A notable outcome of this methodology is the effective differentiation of iPSCs into dopaminergic neurons, which are characteristic of the midbrain.

The significance of this protocol lies in its ability to produce functionally mature, midbrain-specific organoids that closely replicate essential aspects of the midbrain, offering a valuable model for in-depth exploration of midbrain developmental processes and the pathophysiology of related disorders such as Parkinson's disease. Thus, this protocol serves as a crucial resource for researchers seeking to enhance our understanding of the human brain and develop new treatments for neurodegenerative diseases, making it an indispensable tool in the field of neurological research.

The human brain, with its intricate architecture and complex cellular and molecular composition, presents formidable challenges in neuroscience research, particularly in the context of disease modeling and cellular therapy development¹. These challenges are further complicated by the limited availability of sophisticated in vitro models that truly represent the complexity of the human brain. However, recent advancements in human induced pluripotent stem cell (iPSC) technology, enabling controlled differentiation into specific neuronal subtypes, have opened promising avenues for exploring human brain development, disease pathogenesis, and cell-b....

The iPSCs generated from human normal fibroblasts Detroit 551 and human embryonic stem cells (ESCs) hESC line 360 as previously described⁴. The iPSCs were obtained with the approval of the Western Norway Committee for Ethical Health Research REK nr. 2012/919. All cells were regularly monitored for Mycoplasma contamination using MycoAlert Mycoplasma Detection Kit. The Table of Materials contains information about all materials, reagents, and equipment used in this protocol. Table 1 describes the media and other stock used.

1. Thawing of iPSCs

1. Matrix-coated....

In this study, we introduce a pioneering protocol for the derivation of MOs from iPSCs. Central to our methodology is the innovative use of dual-SMAD inhibition, synergistically combined with FGF-8b and sonic hedgehog (SHH) pathway agonist purmorphamine (PM). This approach is depicted in Figure 1. The differentiation process begins by steering iPSCs towards neuroectoderm lineage, forming neuroepithelial or neural rosette colonies. This is achieved through dual SMAD signaling inhibition, empl.......

In this investigation, we have developed a methodology for the differentiation of MO from iPSCs. Our protocol employs a dual-SMAD inhibition strategy enhanced with morphogenic factors, including FGF-8b and the SHH agonist PM. This approach closely simulates the developmental cues crucial for midbrain ontogeny. The differentiation pathway we have instituted prompts the formation of neuroepithelial structures reminiscent of neural rosettes observed during natural brain development. This transformation from bidimensional cu.......

The authors have no conflicts of interest to disclose.

Figure 1 is created using BioRender.com. We thank University of Bergen Meltzers Høyskolefonds (project number: 103517133), Gerda Meyer Nyquist Guldbrandson, and Gerdt Meyer Nyquists legat (project number: 103816102) for funding.