Curated Papers > Recent high-impact research on "organoids" (IF≥30, last 5 years)

Specification of positional identity in forebrain organoids.

Literature Information

DOI	10.1038/s41587-019-0085-3
PMID	30936566
Journal	Nature biotechnology
Impact Factor	41.7
JCR Quartile	Q1
Publication Year	2019
Times Cited	176
Keywords	forebrain organoids, signaling center, Sonic Hedgehog, spatial topography, cholesterol metabolism
Literature Type	Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't
ISSN	1087-0156
Pages	436-444
Issue	37(4)
Authors	Gustav Y Cederquist, James J Asciolla, Jason Tchieu, Ryan M Walsh, Daniela Cornacchia, Marilyn D Resh, Lorenz Studer

TL;DR

This study introduces a method to create spatially organized human forebrain organoids by incorporating a Sonic Hedgehog (SHH) protein gradient, which enables the establishment of in vivo-like topographic features and major forebrain subdivisions. The findings highlight the potential of inductive signaling to improve the development of brain organoids, offering valuable tools for studying brain development and associated metabolic processes.

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forebrain organoids · signaling center · Sonic Hedgehog · spatial topography · cholesterol metabolism

Abstract

Human brain organoids generated with current technologies recapitulate histological features of the human brain, but they lack a reproducible topographic organization. During development, spatial topography is determined by gradients of signaling molecules released from discrete signaling centers. We hypothesized that introduction of a signaling center into forebrain organoids would specify the positional identity of neural tissue in a distance-dependent manner. Here, we present a system to trigger a Sonic Hedgehog (SHH) protein gradient in developing forebrain organoids that enables ordered self-organization along dorso-ventral and antero-posterior positional axes. SHH-patterned forebrain organoids establish major forebrain subdivisions that are positioned with in vivo-like topography. Consistent with its behavior in vivo, SHH exhibits long-range signaling activity in organoids. Finally, we use SHH-patterned cerebral organoids as a tool to study the role of cholesterol metabolism in SHH signaling. Together, this work identifies inductive signaling as an effective organizing strategy to recapitulate in vivo-like topography in human brain organoids.

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Primary Questions Addressed

1. How do different signaling molecules interact with Sonic Hedgehog to influence positional identity in forebrain organoids?
2. What are the implications of establishing in vivo-like topography in forebrain organoids for understanding human brain development?
3. How might variations in cholesterol metabolism affect SHH signaling and the development of forebrain organoids?
4. What are the limitations of current technologies in replicating the topographic organization of the human brain in organoid models?
5. In what ways could the findings about SHH-patterned forebrain organoids be applied to regenerative medicine or brain injury repair?

Key Findings

Research Background and Objectives

The study focuses on enhancing the reproducibility and topographic organization of human brain organoids derived from pluripotent stem cells (hPSCs). Current organoid technologies often produce structures that lack the defined spatial organization seen in vivo, which is crucial for modeling human brain development and disease. The authors hypothesize that introducing a signaling center, specifically a Sonic Hedgehog (SHH) protein gradient, can effectively specify the positional identity of neural tissue in forebrain organoids.

Main Methods/Materials/Experimental Design

The research employs a novel approach to create a SHH gradient in forebrain organoids using an inducible SHH-expressing hPSC line (iSHH). The methodology involves the following key steps:

1. Generation of iSHH Cells: The iSHH line is created using TALEN-mediated gene targeting to introduce a tetracycline-inducible SHH cassette into the AAVS1 locus of an hPSC line.
2. Spheroid Formation: iSHH cells are aggregated in low-attachment microwells, followed by the addition of wild-type hPSCs to form chimeric spheroids (SHH-spheroids).
3. Differentiation Protocol: The SHH-spheroids are cultured in a medium containing TGFβ, BMP, and WNT inhibitors for 6-8 days, then embedded in matrigel to promote neuroepithelial organization.
4. Induction of SHH Gradient: Doxycycline is added to the culture medium to induce SHH expression, creating a gradient that mimics in vivo conditions.
5. Assessment of SHH Activity: The spatial expression of various markers (e.g., PAX6, NKX2.1, FOXG1, OTX2) is analyzed to confirm the specification of dorso-ventral and antero-posterior axes.

Key Results and Findings

• The introduction of the SHH gradient successfully induced distinct forebrain regions in organoids, mimicking the spatial organization of the human brain.
• SHH signaling led to the establishment of major forebrain subdivisions that are positioned in a manner consistent with in vivo anatomy.
• The study revealed that SHH exhibits long-range signaling capabilities in 3D cultures, significantly exceeding the signaling range observed in 2D cultures.
• Specific gene expression patterns (e.g., PAX6 for dorsal identity and NKX2.1 for ventral identity) were confirmed in the resulting organoids, demonstrating effective patterning along both dorso-ventral and antero-posterior axes.

Main Conclusions/Significance/Innovation

This research identifies a novel method to enhance the topographic organization of brain organoids through the application of an SHH signaling gradient. The findings indicate that SHH is a potent organizing factor that can induce positional identity in neural tissues, offering a valuable tool for studying human brain development and diseases. The successful establishment of anatomically relevant brain structures in vitro has significant implications for neurodevelopmental disease modeling and regenerative medicine.

Research Limitations and Future Directions

• Limitations: The variability in organoid size and regional domain specification may affect reproducibility. Additionally, the long-term maintenance of organoid topography remains a challenge.
• Future Directions: Future studies should focus on improving the consistency of organoid topography over extended culture periods and exploring the potential for applying this methodology to other regions of the central nervous system. Further investigation into the role of cholesterol metabolism in SHH signaling may also provide insights into the underlying mechanisms of neurodevelopmental disorders.

References

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Literatures Citing This Work

1. Polarizing brain organoids. - Yuki Miura;Sergiu P Pașca - Nature biotechnology (2019)
2. Pluripotent Stem Cell-Derived Cerebral Organoids Reveal Human Oligodendrogenesis with Dorsal and Ventral Origins. - Hyosung Kim;Ranjie Xu;Ragunathan Padmashri;Anna Dunaevsky;Ying Liu;Cheryl F Dreyfus;Peng Jiang - Stem cell reports (2019)
3. Brain Organoids as Tools for Modeling Human Neurodevelopmental Disorders. - Jason W Adams;Fernanda R Cugola;Alysson R Muotri - Physiology (Bethesda, Md.) (2019)
4. Complex Oscillatory Waves Emerging from Cortical Organoids Model Early Human Brain Network Development. - Cleber A Trujillo;Richard Gao;Priscilla D Negraes;Jing Gu;Justin Buchanan;Sebastian Preissl;Allen Wang;Wei Wu;Gabriel G Haddad;Isaac A Chaim;Alain Domissy;Matthieu Vandenberghe;Anna Devor;Gene W Yeo;Bradley Voytek;Alysson R Muotri - Cell stem cell (2019)
5. Inner ear organoids: new tools to understand neurosensory cell development, degeneration and regeneration. - Marta Roccio;Albert S B Edge - Development (Cambridge, England) (2019)
6. Human brain development through the lens of cerebral organoid models. - Madeline G Andrews;Tomasz J Nowakowski - Brain research (2019)
7. Building Bridges Between the Clinic and the Laboratory: A Meeting Review - Brain Malformations: A Roadmap for Future Research. - Tamar Sapir;Tahsin Stefan Barakat;Mercedes F Paredes;Tally Lerman-Sagie;Eleonora Aronica;Wlodzimierz Klonowski;Laurent Nguyen;Bruria Ben Zeev;Nadia Bahi-Buisson;Richard Leventer;Noa Rachmian;Orly Reiner - Frontiers in cellular neuroscience (2019)
8. Wnt-Notch Signaling Interactions During Neural and Astroglial Patterning of Human Stem Cells. - Julie Bejoy;Brent Bijonowski;Mark Marzano;Richard Jeske;Teng Ma;Yan Li - Tissue engineering. Part A (2020)
9. Brain Organoids: Human Neurodevelopment in a Dish. - Silvia Benito-Kwiecinski;Madeline A Lancaster - Cold Spring Harbor perspectives in biology (2020)
10. Reverse engineering human brain evolution using organoid models. - Mohammed A Mostajo-Radji;Matthew T Schmitz;Sebastian Torres Montoya;Alex A Pollen - Brain research (2020)

... (166 more literatures)

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