Single-cell transcriptomics reveals correct developmental dynamics and high-quality midbrain cell types by improved hESC differentiation

Nishimura, Kaneyasu; Yang, Shanzheng; Lee, Ka Wai; Ásgrímsdóttir, Emilía Sif; Nikouei, Kasra; Paslawski, Wojciech; Gnodde, Sabine; Lyu, Guochang; Hu, Lijuan; Saltó, Carmen; Svenningsson, Per; Hjerling-Leffler, Jens; Linnarsson, Sten; Arenas, Ernest

doi:10.1016/j.stemcr.2022.10.016

Cited by 16 publications

(14 citation statements)

References 59 publications

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“…Clusters were annotated based on the expression of known markers identified from several literatures. DA progenitors were annotated for their highly expressed markers such as NEUROG2, NHLH1 and SOX4 [29, 30]. DA-0 was annotated based on the high expression of ErbB4 known to be expressed in DA neurons and low level of SYT1 a post-synaptic marker[31].…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial Dysfunction and Neuronal Anomalies inPOLGMutant Midbrain Organoids

Chen,

Yangzom,

Sullivan

et al. 2023

Preprint

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Human pluripotent stem cell-derived midbrain organoids offer transformative potential for elucidating brain development, disease representation, and therapeutic innovations. We introduce a novel methodology to generate midbrain-specific organoids from both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). By employing tailored differentiation cues, notably dual-SMAD inhibition combined with FGF-8b and Sonic hedgehog agonist purmorphamine, we direct pluripotent stem cells towards a midbrain lineage. These organoids, growing beyond 2mm in diameter, house diverse neuroepithelial cell populations. Their midbrain character is affirmed by the pronounced expression of midbrain-specific markers and the absence of forebrain and hindbrain indicators. Critically, these organoids differentiate into dopaminergic neurons characteristic of the midbrain, displaying both morphological sophistication and electrophysiological vigor. Additionally, our experiments with POLG iPSC-derived midbrain organoids revealed a marked loss of dopaminergic neurons and diminished expression of genes governing mitochondrial pathways. This evidence underscores the model’s potential in simulating mitochondrial diseases and neurodegenerative conditions, notably Parkinson’s disease. Our protocol thus emerges as a pivotal instrument for crafting functionally adept, midbrain-centric organoids, paving avenues for advanced studies in midbrain evolution, disorders like Parkinson’s disease, and their interplay with mitochondrial dysfunction.

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Section: Methodsmentioning

confidence: 99%

Mitochondrial Dysfunction and Neuronal Anomalies inPOLGMutant Midbrain Organoids

Chen,

Yangzom,

Sullivan

et al. 2023

Preprint

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“…S7A) and then selected a few molecules from the Wnt, Bmp, and angiotensin II (AngII) pathways to investigate in human embryonic stem cells (hESCs) (Table 2). A human development-based protocol (Nishimura et al, 2023a) was used to differentiate WA09 cells into cells of the mDA lineage that are comparable to their endogenous counterparts, as assessed by scRNA-seq (Nishimura et al, 2023b). Ligands or small molecules were added to the cultures between days 22 and 35 of differentiation (Fig.…”

Section: Interactions Of Rgl3 Ligands With Cells Of the Mda Lineagementioning

confidence: 99%

“…Human embryonic stem cell differentiation. WA09 hESCs were obtained from WiCell, maintained, and differentiated as previously described (Nishimura et al, 2023b), with the addition of SHH-C24II (200 ng/ml, Miltenyi Biotec, 130-095) between days 7 and 10 of the differentiation protocol. On day 22, cells were prepared for ligand and ECM testing.…”

Section: Single-cell Transcription Factor (Tf) Pattern Mining Tfs Wit...mentioning

confidence: 99%

Midbrain Dopaminergic Neuron Development is Regulated by Two Molecularly Distinct Subtypes of Radial Glia Cells

Ásgrímsdóttir,

Bassini,

di Val Cervo

et al. 2024

Preprint

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Understanding midbrain dopaminergic (mDA) neuron development is key to advancing cell replacement therapies for Parkinson's disease (PD). Recent single-cell RNA-sequencing (scRNA-seq) studies have identified different subtypes of transient radial glia (Rgl) cell types in the developing mouse and human ventral midbrain. However, their individual functions and their impact on mDA neuron development are unclear. Here we analyze the transcriptome of endogenous mouse and human ventral midbrain Rgl and assess the function of key midbrain floor plate Rgl factors in human stem cells during mDA neuron differentiation. We find that Rgl1 is defined by a neurogenic network centered on Arntl, and that this transcription factor regulates human mDA neurogenesis. Conversely, the transcriptome of Rgl3 is dominated by signaling and extracellular matrix molecules that control different aspects of human mDA neuron development. Thus, our results suggest a function of Rgl1 as a mDA progenitor, and of Rgl3 as a signaling mDA niche cell. Moreover, using human stem cells we demonstrate that new knowledge of cell-type specific intrinsic and extrinsic developmental factors can readily be applied to improve the generation of cells with therapeutic interest, such as human mDA neurons for PD.

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“…This variability may be related to the donor's overall genetic background rather than the genotypes of research interest. Efforts have been focused on refining differentiation protocols with timely addition of signaling molecules mimicking in vivo embryonic development [14,15], but critical events for dopaminergic patterning, such as the level of Wnt activation [16][17][18], seem to be an inherent cell line characteristic [19]. Lastly, the final fraction of DA neurons is often limited in the culture, mixed with proliferating neural progenitor cells as well as other types of cells [20].…”

Section: Introductionmentioning

confidence: 99%

Comparative study of enriched dopaminergic neurons from siblings with Gaucher disease discordant for parkinsonism

Hertz,

Perez,

Hao

et al. 2024

Preprint

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Inducible pluripotent stem cells (iPSCs) derived from patient samples have significantly enhanced our ability to model neurological diseases. Comparative studies of dopaminergic (DA) neurons differentiated from iPSCs derived from siblings with Gaucher disease discordant for parkinsonism provides a valuable avenue to explore genetic modifiers contributing to GBA1-associated parkinsonism in disease-relevant cells. However, such studies are often complicated by the inherent heterogeneity in differentiation efficiency among iPSC lines derived from different individuals. To address this technical challenge, we devised a selection strategy to enrich dopaminergic (DA) neurons expressing tyrosine hydroxylase (TH). A neomycin resistance gene (neo) was inserted at the C-terminus of the TH gene following a T2A self-cleavage peptide, placing its expression under the control of the TH promoter. This allows for TH+ DA neuron enrichment through geneticin selection. This method enabled us to generate comparable, high-purity DA neuron cultures from iPSC lines derived from three sisters that we followed for over a decade: one sibling is a healthy individual, and the other two have Gaucher disease (GD) with GBA1 genotype N370S/c.203delC+R257X (p.N409S/c.203delC+p.R296X). Notably, the younger sister with GD later developed Parkinson disease (PD). A comprehensive analysis of these high-purity DA neurons revealed that although GD DA neurons exhibited decreased levels of glucocerebrosidase (GCase), there was no substantial difference in GCase protein levels or lipid substrate accumulation between DA neurons from the GD and GD/PD sisters, suggesting that the PD discordance is related to of other genetic modifiers.

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Single-cell transcriptomics reveals correct developmental dynamics and high-quality midbrain cell types by improved hESC differentiation

Cited by 16 publications

References 59 publications

Mitochondrial Dysfunction and Neuronal Anomalies inPOLGMutant Midbrain Organoids

Mitochondrial Dysfunction and Neuronal Anomalies inPOLGMutant Midbrain Organoids

Midbrain Dopaminergic Neuron Development is Regulated by Two Molecularly Distinct Subtypes of Radial Glia Cells

Comparative study of enriched dopaminergic neurons from siblings with Gaucher disease discordant for parkinsonism

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