Organotypic Cultures of Adult Human Cortex as an <em>Ex vivo</em> Model for Human Stem Cell Transplantation and Validation

Sara Palma-Tortosa; Raquel Mart&#237;nez-Curiel; Constanza Aretio-Medina; Natalia Avaliani; Zaal Kokaia

doi:10.3791/64234

인간 줄기 세포 이식 및 검증을 위한 생체 외 모델로서의 성인 인간 피질의 기관형 배양

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

인간 줄기 세포 이식 및 검증을 위한 생체 외 모델로서의 성인 인간 피질의 기관형 배양

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07:16 min

December 9, 2022

DOI: 10.3791/64234-v

Sara Palma-Tortosa¹, Raquel Martínez-Curiel¹, Constanza Aretio-Medina¹, Natalia Avaliani², Zaal Kokaia¹

¹Laboratory of Stem Cells and Restorative Neurology, Lund Stem Cell Center,Lund University, ²Lund Stem Cell Center,Lund University

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Overview

This study details a novel methodology for long-term organotypic cultures of adult human cortex and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. The approach allows for the investigation of stem cell therapies in a human-to-human context, addressing challenges faced in prior animal model studies for neurodegenerative disorders.

Key Study Components

Area of Science

Neuroscience
Stem Cell Biology
Neurodegeneration

Background

Current therapies often fail in clinical translation due to species differences.
Organotypic cultures offer a platform for testing cellular interactions within a human system.
There is a critical need to enhance understanding of human neural circuitry.
Observations of functional recovery are necessary for therapeutic advancements.

Purpose of Study

To establish a methodology for studying grafted and host cell interactions.
To assess the viability and functionality of transplanted cells in a human model.
To ultimately improve functional recovery post-brain damage through enhanced therapy testing.

Methods Used

Organotypic cultures of adult human cortex were utilized.
Induced pluripotent stem cell-derived cortical progenitors were transplanted ex vivo.
Key procedures included tissue preparation, culture maintenance, and assessment of cellular function after transplantation.
Critical timelines involved immediate processing of tissue post-collection for optimal viability.
Detailed procedures for culturing and transplanting cells were followed to ensure successful integration.

Main Results

Successful transplantation resulted in the survival and maturation of grafted cells.
Grafted cells exhibited complex neuronal activity and integration into host tissue.
Post-transplantation, cells displayed membrane potentials and synaptic activity indicating functional integration.
Microglial response showed a transition from resting to activated states, influencing overall tissue health.

Conclusions

This study demonstrates the feasibility of using human organotypic cultures to investigate stem cell therapies.
The findings contribute to understanding neuronal mechanisms relevant to human brain damage and recovery.
Insights gained may inform future therapeutic strategies for neurodegenerative conditions.

Frequently Asked Questions

What are the advantages of using organotypic cultures?

Organotypic cultures allow for the assessment of human-to-human cellular interactions, providing insights that are more relevant than those obtained from rodent models.

How is the human adult cortex prepared for culture?

Tissue is collected from patients, equilibrated, and then sliced into coronal or sagittal sections for culture.

What outcomes can be measured from this model?

Outcomes include neuronal survival, differentiation, synaptic activity, and microglial response, reflecting the health of the tissue.

How can this method be applied to other studies?

The methodology can be adapted for various types of neuronal or glial cells and can be used to evaluate the efficacy of different therapeutic approaches.

What are the key limitations of this study?

Key limitations may include the variable preservation of tissue and the challenges associated with maintaining long-term culture viability.

What can be learned about human neuronal circuitry through this study?

The study enables a deeper understanding of human neural circuitry interactions and functionality, essential for advancing treatment approaches for neurodegenerative diseases.

이 프로토콜은 유도 만능 줄기 세포 유래 피질 전구체의 생체 외 피질 내 이식과 결합된 성인 인간 피질의 장기 기관형 배양을 설명하며, 이는 인간 신경퇴행성 장애에 대한 줄기 세포 기반 치료법을 추가로 테스트하기 위한 새로운 방법론을 제시합니다.

인간 성인 피질의 기관형 배양은 지금까지 인간 이식 된 세포와 인간 숙주 세포 사이의 상호 작용을 연구 할 수있는 손상된 피질에 대한 줄기 세포 치료법을 테스트하는 유일한 시스템입니다. 동물 모델에서 테스트된 치료법은 일반적으로 설치류와 인간 세포의 차이로 인해 임상 환경으로 번역될 때 실패합니다. 여기에서 우리는 인간 대 인간 환경에서 이식된 세포, 생존, 분화 및 기능을 연구할 수 있습니다.

이 방법론은 인간의 뇌에서 신경 회로가 어떻게 작동하는지 이해하는 데 도움이 될 것이며, 또한 뇌 손상 후 기능 회복을 개선하기 위해 이 지식을 임상 환경으로 번역하는 것을 자극할 것입니다. 이 절차를 시연하는 것은 박사 과정 학생 인 Raquel Martinez-Curiel과 석사 과정 학생 인 Costanza Aretio-Medina가 내 연구실에서 나온 것입니다. 시작하려면 통풍이 잘되는 후드 아래 세포 배양 실험실에서 집게를 사용하여 6웰 플레이트에 배양 삽입물을 놓습니다.

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