Generation of midbrain-specific organoids from human pluripotent stem cells

An organoid and cell technology, applied in the field of organoid generation, which can solve problems such as unclear 3D organoid models

Pending Publication Date: 2019-07-09
AGENCY FOR SCI TECH & RES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While these studies will certainly advance the field, it is unclear whether 3D organoid models of the midbrain can be developed

Method used

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  • Generation of midbrain-specific organoids from human pluripotent stem cells
  • Generation of midbrain-specific organoids from human pluripotent stem cells
  • Generation of midbrain-specific organoids from human pluripotent stem cells

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Embodiment Construction

[0037] Organoids are cellular structures that mimic the organization and function of organs. In contrast to single-cell cultures, organoids consist of multiple cell types that are spatially organized. The present disclosure demonstrates that 3D culture conditions allow cells of organoids to unravel the self-organizing capacity of multiple cell types, which enhances better survival and maturation of specific cell types by creating an appropriate niche environment. Accordingly, the present disclosure describes methods for generating organoids capable of recapitulating midbrain characteristics by differentiating human embryonic stem cells into midbrain-like organoids.

[0038] Efficient differentiation of pluripotent stem cells (PSCs) into most homogeneous cell types, such as neurons, can be directed. These in vitro generated neurons enable downstream research and potential therapeutic applications. Recent advances in three-dimensional (3D) culture systems have led to the gener...

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Abstract

The present invention provides a method of deriving and maintaining a midbrain-like organoid in culture, comprising (a) culturing pluripotent stem cells to obtain neuronal lineage embryoid bodies; (b)culturing the neuronal lineage embryoid bodies from (a) to obtain midbrain regionalized tissues; (c) embedding and culturing the midbrain regionalized tissues from (b) in an extracellular matrix to obtain neuroepithelial tissues; and (c) culturing the neuroepithelial tissues from (c) to obtain a midbrain-like organoid. Also disclosed herein are culture media suitable for deriving and maintainingneuronal lineage embryoid bodies comprising (a) TGF-beta inhibitor and / or SMAD2 / 3 inhibitors; and (b) WNT-signaling activator; culture media suitable for deriving and maintaining midbrain regionalizedtissues comprising (a) TGF-beta inhibitor and / or SMAD2 / 3 inhibitors; (b) WNT-signaling activator; (c) hedgehog signaling protein; and (d) a fibroblast growth factor; culture media suitable for deriving and maintaining neuroepithelial tissues comprising (a) hedgehog signaling protein; and (b) a fibroblast growth factor and culture media suitable for deriving and maintaining a midbrain-like organoid comprising (a) neurotrophin factor; (b) ascorbic acid; and (c) an activator of cAMP-dependent pathway.

Description

[0001] Cross References to Related Applications [0002] This application claims priority from Singapore Provisional Application No. 10201601965P filed on 14 March 2016, the contents of which are hereby incorporated by reference in their entirety for all purposes. [0003] field of invention [0004] The present invention generally relates to the field of biotechnology. In particular, the invention relates to methods of producing organoids. Background technique [0005] Limited access to functional human brain tissue is the greatest challenge to understanding the development of the human nervous system and its dysfunction in brain diseases. This challenge is being overcome recently with rapid research progress in human pluripotent stem cells (hPSCs), including for example embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), which can be generated from any individual and can be directed to differentiate in vitro into derivatives representing all germ laye...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0793
CPCC12N5/0619C12N2501/119C12N2501/13C12N2501/15C12N2501/33C12N2501/41C12N2501/415C12N2501/727C12N2501/999C12N2506/02C12N2513/00C12N2533/90C12N5/0018C12N5/0062C12N5/0696C12N2533/54C12N2533/56C12N2533/72C12N2533/74C12N2533/76C12N2533/78
Inventor 曹重铉黄学晖诸炫秀
Owner AGENCY FOR SCI TECH & RES
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