Single-cell multimodal transcriptomics to study neuronal diversity in human stem cell-derived brain tissue and organoid models
Graphical abstract
Introduction
A thorough understanding of the cellular and molecular mechanisms underlying neurological and psychiatric disorders is necessary to discover new therapeutic targets and treatment strategies. The advent of induced pluripotent stem cell (iPSC) (Lowry et al., 2008; Park et al., 2008; Takahashi et al., 2007; Takahashi and Yamanaka, 2006; Yu et al., 2007) and direct cell fate conversion technologies (Caiazzo et al., 2011; Pfisterer et al., 2011; Son et al., 2011) has enabled the derivation of patient-specific live human neurons from easily accessible and renewable somatic cell types, such as skin fibroblasts. While in vitro tissue engineering with stem cells holds great potential for future scientific discoveries, the variability and heterogeneity of lab-engineered brain tissue may conceal important disease mechanisms that preclude therapeutic effects of novel drug candidates. Recent advances in single-cell genomics technologies and bioinformatics have enabled comprehensive and unbiased deconvolution of complex tissue into cellular subtypes based on molecular profiles (Stuart and Satija, 2019). Throughput, sensitivity and multimodal integration of these single-cell genomics methodologies are rapidly and continuously advancing, and it has become easier than ever to measure gene expression and other cellular data in thousands of single cells in an individual experiment. In this review, we discuss the current use, challenges and future perspectives of single-cell genomics methods for the characterization of cellular diversity in human stem cell-derived neuronal tissue. In addition, we detail how single-cell measurements can provide valuable insights into the quality and efficiency of the tissue engineering process, and guide the design of optimized protocols for neuronal differentiation.
Section snippets
Human neuronal tissue engineering: progress and challenges
The ability to derive, manipulate, and differentiate pluripotent stem cells (PSCs) has opened new avenues for generating neural tissue for disease modeling and drug candidate screening in vitro. In recent years, a panoply of protocols has been published for the generation of functional human neurons of specific brain region identities and neurotransmitter phenotypes [e.g., midbrain/dopaminergic (Boyer et al., 2012; Cho et al., 2008; Ma et al., 2011; Tofoli et al., 2019; Zhang et al., 2014),
Single-cell RNA-sequencing to characterize cell diversity and quality in neuronal tissue engineering
The quality of neuronal tissue generated in vitro has traditionally been assessed by morphological and functional (i.e., electrophysiological) characterization of generated neuronal subtypes, and by immunohistochemical detection of proteins that are known to be expressed in vivo. Analysis of transcriptome data from whole tissue can also reveal expression of cell type-relevant genes, but bulk RNA expression profiles represent average measures of gene expression in cells pooled en masse, thus
Multimodal single-cell analysis methods to study neuronal diversity
To acquire a more comprehensive understanding of the molecular-biological mechanisms underlying neuronal function in health, development and disease, recent research endeavors have been geared towards measuring multiple cellular aspects at the individual cell level. These “multimodal” single-cell analysis methods typically combine single-cell transcriptome profiling with the analysis of other cell-specific features, such as electrophysiological function, morphology, genome sequence, epigenetic
Current challenges in single-cell transcriptomics
While single-cell transcriptomics methods create unprecedented opportunities for profiling cellular heterogeneity, careful experimental considerations are necessary to address some important technical limitations, such as tradeoff between sequencing depth and number of cells profiled, detection of low expressed genes, presence of cell doublets, single-cell dissociation bias, and maintaining spatial context of single cells. We discuss these limitations below.
Conclusion and future perspectives
Advances in cell reprogramming and induced pluripotent stem cell technologies have opened new avenues for generating brain tissue for pre-clinical neuroscience studies. However, the large degree of cellular diversity within in vitro engineered neuronal tissue may confound disease phenotypes, hindering accurate detection of novel molecular targets. Single-cell genomics methods address this challenge by enabling a detailed, accurate and unbiased characterization of cellular types and states.
Acknowledgements
This work was supported by the Netherlands Organisation for Scientific Research Rubicon Fellowship (019.163LW.032) (to MvdH); the Brain Foundation, the Perpetual Impact Philanthropy (IPAP2017/0717), Rebecca L. Cooper Foundation and Flinders Foundation (to CB).
References (147)
- et al.
Direct reprogramming of adult human fibroblasts to functional neurons under defined conditions
Cell Stem Cell
(2011) - et al.
Patch-clamp recordings and calcium imaging followed by single-cell PCR reveal the developmental profile of 13 genes in iPSC-derived human neurons
Stem Cell Res.
(2014) - et al.
Single-cell, genome-wide sequencing identifies clonal somatic copy-number variation in the human brain
Cell Rep.
(2014) - et al.
The BRAIN initiative cell census consortium: lessons learned toward generating a comprehensive brain cell atlas
Neuron
(2017) - et al.
Single-neuron sequencing analysis of L1 retrotransposition and somatic mutation in the human brain
Cell
(2012) - et al.
Rapid and efficient generation of functional motor neurons from human pluripotent stem cells using gene delivered transcription factor codes
Mol. Ther.
(2011) - et al.
An organoid-based model of cortical development identifies non-cell-Autonomous defects in wnt signaling contributing to miller-dieker syndrome
Cell Rep.
(2017) - et al.
Midbrain-like organoids from human pluripotent stem cells contain functional dopaminergic and neuromelanin-producing neurons
Cell Stem Cell
(2016) - et al.
AMPA receptor subunits expressed by single Purkinje cells
Neuron
(1992) - et al.
Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets
Cell
(2015)
A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells
Cell
Neural differentiation of human embryonic stem cells induced by the transgene-mediated overexpression of single transcription factors
Biochem. Biophys. Res. Commun.
Lab-specific gene expression signatures in pluripotent stem cells
Cell Stem Cell
LRRK2 mutant iPSC-derived DA neurons demonstrate increased susceptibility to oxidative stress
Cell Stem Cell
Functional maturation of hPSC-derived forebrain interneurons requires an extended timeline and mimics human neural development
Cell Stem Cell
2D and 3D stem cell models of primate cortical development identify species-specific differences in progenitor behavior contributing to brain size
Cell Stem Cell
Cell fixation and preservation for droplet-based single-cell transcriptomics
BMC Biol.
Anatomical and physiological plasticity of dendritic spines
Annu. Rev. Neurosci.
Parallel single-cell sequencing links transcriptional and epigenetic heterogeneity
Nat. Methods
A practical solution for preserving single cells for RNA sequencing
Sci. Rep.
Single-nucleus and single-cell transcriptomes compared in matched cortical cell types
PLoS One
Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro
Proc. Natl. Acad. Sci. U. S. A.
Predicting the functional states of human iPSC-derived neurons with single-cell RNA-seq and electrophysiology
Mol. Psychiatry
Cell type purification by single-cell transcriptome-trained sorting
bioRxiv
Physiological properties of neurons derived from human embryonic stem cells using a dibutyryl cyclic AMP-based protocol
Stem Cells Dev.
Assembly of functionally integrated human forebrain spheroids
Nature
A functionally characterized test set of human induced pluripotent stem cells
Nat. Biotechnol.
Dopaminergic differentiation of human pluripotent cells
Curr. Protoc. Stem Cell Biol.
Modelling schizophrenia using human induced pluripotent stem cells
Nature
Electrophysiological, transcriptomic and morphologic profiling of single neurons using Patch-seq
Nat. Biotechnol.
Multimodal profiling of single-cell morphology, electrophysiology, and gene expression using Patch-seq
Nat. Protoc.
Direct generation of functional dopaminergic neurons from mouse and human fibroblasts
Nature
Sur la structure de l’ecorce cerebale de quelques mammiferes
Cellule
Textura de las circunvoluciones cerebrales de los mamíferos inferiores. Nota preventiva
Gac. Méd. Catalana
Human cerebral organoids recapitulate gene expression programs of fetal neocortex development
Proc. Natl. Acad. Sci. U. S. A.
Joint profiling of chromatin accessibility and gene expression in thousands of single cells
Science
Molecular and physiological diversity of cortical nonpyramidal cells
J. Neurosci.
Single-cell transcriptomics as a framework and roadmap for understanding the brain
J. Neurosci. Methods
Heterogeneity within classical cell types is the rule: lessons from hippocampal pyramidal neurons
Nat. Rev. Neurosci.
A human neurodevelopmental model for Williams syndrome
Nature
RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells
Science
Coupled electrophysiological recording and single cell transcriptome analyses revealed molecular mechanisms underlying neuronal maturation
Protein Cell
Efficient derivation of functional dopaminergic neurons from human embryonic stem cells on a large scale
Nat. Protoc.
Comprehensive qPCR profiling of gene expression in single neuronal cells
Nat. Protoc.
L1 retrotransposition in human neural progenitor cells
Nature
Multiplex single cell profiling of chromatin accessibility by combinatorial cellular indexing
Science
Integrated genome and transcriptome sequencing of the same cell
Nat. Biotechnol.
Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons
Science
Analysis of gene expression in single live neurons
Proc. Natl. Acad. Sci. U. S. A.
Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH
Nature
Cited by (24)
Neuronal Spike Shapes (NSS): A straightforward approach to investigate heterogeneity in neuronal excitability states
2024, Computers in Biology and MedicineLong-term adherence of human brain cells in vitro is enhanced by charged amine-based plasma polymer coatings
2022, Stem Cell ReportsCitation Excerpt :Therefore, the study focused on human brain cells, and further experimentation will be required to demonstrate the potential benefits of DAP coating for non-human cells. Patient-derived iPSCs are increasingly popular pre-clinical models of neurological and psychiatric disorders (Bardy et al., 2020; Chailangkarn et al., 2016; Israel et al., 2012; Mertens et al., 2016; Sarkar et al., 2018; Tran et al., 2020; van den Hurk and Bardy, 2019). Two-dimensional monolayer models offer reproducible and relatively high-throughput drug screening capabilities.
Midbrain organoids mimic early embryonic neurodevelopment and recapitulate LRRK2-p.Gly2019Ser-associated gene expression
2022, American Journal of Human GeneticsCitation Excerpt :We speculate that this NB cluster represents mfNPCs, which is the starting cell population for midbrain organoid generation. Although these cells are artificially patterned toward midbrain identity49 and show unspecific genetic identity,50 they can give rise to multiple physiologically relevant neuronal cell types and glia, similar to their in vivo neural progenitor counterpart. When comparing MUT to WT midbrain organoids, clear differences become visible regarding their cellular composition, revealing PD-associated phenotypic differences.
Reconstructing developmental landscapes and trajectories from single-cell data
2021, Current Opinion in Systems BiologyCitation Excerpt :This would be comparable with the transition from plate-based scRNA-seq to droplet-based methods, where one obtains far more cells at the expense of fewer reads per cell. Reconstruction of developmental landscapes and trajectories has numerous important applications, ranging from stem cell therapy and engineering organoids [43,54,4] to agriculture [37]. Although numerous methods have been proposed [36], there is relatively little work on rigorously understanding the performance of these methods.
Optical and genetic tools for in vivo single cell tracking
2021, Journal of Neuroscience MethodsInvestigating the pathophysiology of anorexia nervosa using induced pluripotent stem cells: Background, current trends, and perspectives
2021, iPSCs for Modeling Central Nervous System Disorders, Volume 6