Generation of Human Ventral Midbrain Organoids Derived from Pluripotent Stem Cells

Sozzi, Edoardo; Nilsson, Fredrik; Kajtez, Janko; Parmar, Malin; Fiorenzano, Alessandro

doi:10.1002/cpz1.555

Cited by 14 publications

(15 citation statements)

References 33 publications

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“…The correct developmental identity was confirmed by the presence of vMB floorplate progenitor markers LMX1A, FOXA2, and OTX2 (figure 3(c)). Coexpression of these markers at day 16 is in line with previous work on vMB organoids [18,30] and the reference 2D protocol for the generation of vMB dopamine (DA) neurons based on dual-SMAD inhibition and vMB patterning with SHH and GSK3i, followed by vMB progenitor caudalization with FGF8b [34,35]. Correct vMB patterning was further verified by RT-qPCR (figure S1a).…”

Section: Characterization Of Human Vmb Organoid Formationsupporting

confidence: 85%

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Modular 3D printed platform for fluidically connected human brain organoid culture

Rezaei,

Giacomoni,

Nilsson

et al. 2023

Biofabrication

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Brain organoid technology has transformed both basic and applied biomedical research and paved the way for novel insights into developmental processes and disease states of the human brain. While the use of brain organoids has been rapidly growing in the past decade, the accompanying bioengineering and biofabrication solutions have remained scarce. As a result, most brain organoid protocols still rely on commercially available tools and culturing platforms that had previously been established for different purposes, thus entailing suboptimal culturing conditions and excessive use of plasticware. To address these issues, we developed a 3D printing pipeline for the fabrication of tailor-made culturing platforms for fluidically connected but spatially separated brain organoid array culture. This all-in-one platform allows all culturing steps—from cellular aggregation, spheroid growth, hydrogel embedding, and organoid maturation—to be performed in a single well plate without the need for organoid manipulation or transfer. Importantly, the approach relies on accessible materials and widely available 3D printing equipment. Furthermore, the developed design principles are modular and highly customizable. As such, we believe that the presented technology can be easily adapted by other research groups and fuel further development of culturing tools and platforms for brain organoids and other 3D cellular systems.

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Section: Characterization Of Human Vmb Organoid Formationsupporting

confidence: 85%

“…Human vMB organoids were generated by following previously published protocol [18,30] in both conventional wells and the 3D printed platform. Single-cell suspension was generated by detaching hPSCs from the culturing dish using 0.5 mM Accutase (Thermo Fisher Scientific).…”

Section: Vmb Organoid Culturementioning

confidence: 99%

Modular 3D printed platform for fluidically connected human brain organoid culture

Rezaei,

Giacomoni,

Nilsson

et al. 2023

Biofabrication

Self Cite

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“…Previously, human embryonic stem cells were used as a source of pluripotent cells; however, in recent years, they have mostly been replaced by iPSCs [ 140 , 141 , 142 , 143 , 144 ]. As an example, a recent study analyzed single-cell RNA sequencing of the human fetal midbrain at 7.5 weeks of gestation and their subsequent differentiation into hMLOs [ 145 ]; however, the significance of using fetal midbrain remains uncertain.…”

Section: Exploring Transcriptional Modulation Downstream Of Epigeneti...mentioning

confidence: 99%

Unraveling the Complex Interplay between Alpha-Synuclein and Epigenetic Modification

Sugeno

Hasegawa

2023

IJMS

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Alpha-synuclein (αS) is a small, presynaptic neuronal protein encoded by the SNCA gene. Point mutations and gene multiplication of SNCA cause rare familial forms of Parkinson’s disease (PD). Misfolded αS is cytotoxic and is a component of Lewy bodies, which are a pathological hallmark of PD. Because SNCA multiplication is sufficient to cause full-blown PD, gene dosage likely has a strong impact on pathogenesis. In sporadic PD, increased SNCA expression resulting from a minor genetic background and various environmental factors may contribute to pathogenesis in a complementary manner. With respect to genetic background, several risk loci neighboring the SNCA gene have been identified, and epigenetic alterations, such as CpG methylation and regulatory histone marks, are considered important factors. These alterations synergistically upregulate αS expression and some post-translational modifications of αS facilitate its translocation to the nucleus. Nuclear αS interacts with DNA, histones, and their modifiers to alter epigenetic status; thereby, influencing the stability of neuronal function. Epigenetic changes do not affect the gene itself but can provide an appropriate transcriptional response for neuronal survival through DNA methylation or histone modifications. As a new approach, publicly available RNA sequencing datasets from human midbrain-like organoids may be used to compare transcriptional responses through epigenetic alterations. This informatic approach combined with the vast amount of transcriptomics data will lead to the discovery of novel pathways for the development of disease-modifying therapies for PD.

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“…In particular, the diversity of differentiation protocols for human neural organoids poses a unique challenge for organoid quality control that can be met by meta-analytic approaches. Neural organoids can either be undirected 25 (multiple brain region identities) or directed (specific brain region identity) with an increasing number of protocols striving to produce a wider variety of regionspecific organoids 11,[26][27][28][29][30][31][32][33][34][35][36][37] . Meta-analytic primary tissue/organoid comparisons across differentiation protocols stand to derive generalizable quality control metrics applicable to any differentiation protocol, fulfilling a currently unmet need for unified quality control metrics across heterogeneous neural organoids.…”

Section: Introductionmentioning

confidence: 99%

Preservation of co-expression defines the primary tissue fidelity of human neural organoids

Werner

Gillis

2023

Preprint

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Human neural organoid models offer an exciting opportunity for studying often inaccessible human-specific brain development; however, it remains unclear how precisely organoids recapitulate fetal/primary tissue biology. Here, we characterize field-wide replicability and biological fidelity through a meta-analysis of single-cell RNA-sequencing data for first and second trimester human primary brain (2.95 million cells, 51 datasets) and neural organoids (1.63 million cells, 130 datasets). We quantify the degree to which primary tissue cell-type marker expression and co-expression are recapitulated in organoids across 12 different protocol types. By quantifying gene-level preservation of primary tissue co-expression, we show neural organoids lie on a spectrum ranging from virtually no signal to co-expression near indistinguishable from primary tissue data, demonstrating high fidelity is within the scope of current methods. Additionally, we show neural organoids preserve the cell-type specific co-expression of developing rather than adult cells, confirming organoids are an appropriate model for primary tissue development. Overall, quantifying the preservation of primary tissue co-expression is a powerful tool for uncovering unifying axes of variation across heterogeneous neural organoid experiments.

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Generation of Human Ventral Midbrain Organoids Derived from Pluripotent Stem Cells

Cited by 14 publications

References 33 publications

Modular 3D printed platform for fluidically connected human brain organoid culture

Modular 3D printed platform for fluidically connected human brain organoid culture

Unraveling the Complex Interplay between Alpha-Synuclein and Epigenetic Modification

Preservation of co-expression defines the primary tissue fidelity of human neural organoids

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