Differentiation of Enteric Nervous System Lineages from Human Pluripotent Stem Cells

作者： Homa Majd1,2, Mikayla N. Richter1,2, Ryan M. Samuel1,2, Ali Kalantari1,2, Jonathan T. Ramirez1,2, Faranak Fattahi1,2,3 1Department of Cellular and Molecular Pharmacology,University of California, San Francisco, 2Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research,University of California, San Francisco, 3Program in Craniofacial Biology,University of California, San Francisco

简介：

Overview

This study presents a detailed in vitro protocol for deriving human enteric neurons from human pluripotent stem cells (hPSCs) under chemically defined conditions. The platform enables scalable culture of enteric nervous system components, opening avenues for studying ENS development, disease modeling, and regenerative medicine.

Key Study Components

Area of Science

• Neuroscience
• Cell Biology
• Regenerative Medicine

Background

• Understanding human enteric nervous system (ENS) development and function is challenging due to the rarity of ENS cells.
• Access to tissues and isolation of these cells has been difficult.
• Existing methods for generating enteric neurons are often limited and not fully defined.
• There is a need for scalable solutions for ENS components to facilitate research.

Purpose of Study

• To provide a reliable method for generating functional human enteric neurons from hPSCs.
• To overcome challenges associated with tissue access and rarity of cells.
• To use generated neurons for disease modeling and drug discovery.

Methods Used

• The main platform used is cell culture of human pluripotent stem cells.
• The biological model involves differentiating hPSCs into enteric neurons through neural crest induction.
• The method includes incubating cells in defined media (Medium A, B, C) over 30 to 40 days with specific feeding schedules.
• Key steps include aspiration of media, cell detachment, centrifugation, and resuspension in NCC and ENC media.
• Critical timelines include daily feeding and cell counting to ensure successful neuron differentiation.

Main Results

• The protocol yielded authentic and functional enteric neurons, with the observation of neurites between days 25 to 30.
• High-quality free-floating spheroids expressing the neural crest marker SOX10 were generated.
• Cell viability and morphology indicated successful differentiation and growth in culture.
• Overall, the results validated the method as robust for generating ENS components.

Conclusions

• This study demonstrates an efficient approach to generating human enteric neurons from hPSCs, facilitating ENS research.
• The method enables scalable cultures for future studies on ENS development and diseases.
• These advancements have significant implications for understanding ENS mechanisms and developing regenerative therapies.

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