Human Spinal Cord Organoids Recapitulate Developmental and Disease-associated Oligodendrocyte Lineage Signatures Open Access

Abstract

Oligodendrocytes play essential roles in central nervous system development and homeostasis, and their dysfunction is a hallmark of numerous neurological disorders. However, human in vitro systems that support oligodendrocyte lineage progression while enabling the study of disease-relevant states remain limited. Through this work, we establish human spinal cord organoids (hSpO) and cortico-motor assembloids as platforms to model oligodendrocyte development, neuron-glia interactions, and cytokine-induced dysfunction. We show that hSpO generate oligodendrocyte lineage populations that transcriptionally resemble those found in the developing human spinal cord, and oligodendrocyte progenitor cells that exhibit physiologically-relevant functional properties, including migration and monosynaptic input from neurons. Exposure of assembloids to pro-inflammatory cytokines induces transcriptional changes across the oligodendrocyte lineage, characterized by altered lineage progression and acquisition of disease-associated gene expression programs that mirror signatures observed in multiple sclerosis patient tissue. Together, this work establishes hSpO and assembloids as in vitro systems for studying oligodendrocyte lineage development and disease-associated states in a human multi-cellular context.

Table of Contents

LIST OF FIGURES

Main

Supplemental

LIST OF TABLES

ABBREVIATIONS

CHAPTER 1: GENERAL INTRODUCTION

1.1 Oligodendrocyte lineage dynamics and function

1.2 Oligodendrocyte lineage in disease

1.3 Spinal cord organoids as a model of human oligodendrocytes

1.4 Acknowledgement of reproduction

CHAPTER 2: HISTORICAL BACKGROUND

2.1 The development and application of induced pluripotency

2.2 Three-dimensional modeling of human neural development

2.3 The case for in vitro oligodendrocyte generation

CHAPTER 3: HSPO OLIGODENDROCYTE LINEAGE CHARACTERIZATION

3.1 Introduction

3.2 Results

3.2.1 Timeline of gene expression in hSpO

3.2.2 Oligodendrocyte lineage at the single cell level

3.2.3 Identifying mGPCs in organoid culture

3.3 Discussion

CHAPTER 4: FUNCTIONAL CHARACTERIZATION OF HSPO OLIGODENDROCYTES

4.1 Introduction

4.2 Results

4.2.1 Oligodendrocyte migration

4.2.2 Neuron-OPC synapses

4.2.3 Oligodendrocyte engraftment

4.3 Discussion

CHAPTER 5: MODELING DISEASE IN HSPO OLIGODENDROCYTE LINEAGE CELLS

5.1 Introduction

5.2 Results

5.2.1 Modeling neuroinflammation-induced oligodendrocyte lineage disruption

5.2.2 Acquisition of disease-associated oligodendrocyte signatures

5.3 Discussion

CHAPTER 6: GENERAL DISCUSSION

6.1 Oligodendrocyte lineage cell dynamics in the adult CNS

6.2 hSpO-derived oligodendrocyte lineage cells

6.3 Limitations

6.4 Conclusion

METHODS

Data, code, and protocol availability

hiPSC-derived cultures

Culture of hiPSCs

Generation of hSpO from hiPSCs

Generation of hCO from hiPSCs

Generation of hCO-hSpO assembloids

Generation of 3D hSkM

Generation of hCO-hSpO-hSkM assembloids

Vehicle and TIC treatment of assembloids

Dissociation and primary culture

Human tissue

Single cell dissociation

Immunopanning

Engraftment

Viral transfection experiments

Organoid viral labeling and monosynaptic rabies tracing

Virus generation

Immunohistochemistry and imaging

Organoid cryopreservation and immunohistochemistry

Whole organoid immunohistochemistry

Imaging

Human primary fetal 2D immunocytochemistry

Imaging quantification

Migration quantification

Immunohistochemistry and immunocytochemistry quantification

RNA isolation and qPCR

RNA collection, isolation, and Real-time quantitative PCR

RNA sequencing

Single cell RNA sequencing analysis and quality control

Bulk RNAseq library preparation and analysis

Cytokine-release luminex immunoassay

Schematics and statistics

Statistical analysis

Reagents

Table 1: Media bases

Table 2: Media reagents

Table 3: Antibodies

Table 4: Viruses

Table 5: Plasmids

Table 6: Primers

Table 7: Top 15 marker genes per oligodendrocyte lineage cluster

Table 8: Top 15 differentially expressed genes with TIC treatment

REFERENCES

About this Dissertation

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience
Keyword	neuron-glia interactions oligodendrocytes spinal cord organoids oligodendrocyte progenitor cells disease-associated oligodendrocytes
Committee Chair / Thesis Advisor	Andersen, Jimena, Emory University
Committee Members	Raj, Nisha, Emory University Alvarez, Francisco, Emory University Sloan, Steven, Emory University Birey, Fikri, Emory University

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