Cerebellar Modelling Using Human Induced Pluripotent Stem Cells

Essen, Max J. van; Nayler, Samuel; Apsley, Elizabeth J.; Jacob, John R.; Becker, Esther B. E.

doi:10.1007/978-1-0716-2026-7_1

Cited by 3 publications

(2 citation statements)

References 34 publications

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“…Cerebellar organoid differentiation was performed as described previously. 9 In brief, human iPSC line AH017-3 cells 10 were detached by incubation with pre-warmed TrypLE for 5 minutes. Cells were collected by centrifugation and resuspended in induction medium (50% IMDM, 50% F12, 7ug/mL Insulin, 5mg/mL BSA, 1X Chemically defined Lipid concentrate, 450uM Monothioglycerol, 15ug/mL Apo-transferrin, 1X Penicillin/Streptomycin) at a final concentration of 100,000 cells/ml.…”

Section: Methodsmentioning

confidence: 99%

Sonic hedgehog medulloblastoma cells in co-culture with cerebellar organoids converge towardsin vivomalignant cell states

van Essen,

Nicheperovich,

Schuster-Böckler

et al. 2024

Preprint

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Background In the malignant brain tumour sonic hedgehog medulloblastoma (SHH-MB) the properties of cancer cells are influenced by their microenvironment, but the nature of those effects and the phenotypic consequences for the tumour are poorly understood. The aim of this study was to identify phenotypic properties of SHH-MB cells that were driven by the non-malignant tumour microenvironment. Methods Human induced pluripotent cells (iPSC) were differentiated to cerebellar organoids to simulate the non-malignant tumour microenvironment. Tumour spheroids were generated from two distinct, long-established SHH-MB cell lines which were co-cultured with cerebellar organoids. We profiled the cellular transcriptomes of malignant and non-malignant cells by performing droplet-based single-cell RNA-sequencing (scRNA-seq). The transcriptional profiles of tumour cells in co-culture were compared with those of malignant cells cultured in isolation and with public SHH-MB datasets of patient tumours and patient- derived xenograft (PDX) models. Results SHH-MB cell lines in organoid co-culture adopted patient tumour-associated phenotypes and showed increased heterogeneity compared to monocultures. Sub-populations of co-cultured SHH-MB cells activated a key marker of differentiating granule cells, NEUROD1 that was not observed in tumour monocultures. Other sub-populations expressed transcriptional determinants consistent with a cancer stem cell (CSC)-like state that resembled cell states identified in vivo. Conclusion For SHH-MB cell lines in co-culture, there was a convergence of malignant cell states towards patterns of heterogeneity in patient tumours and PDX models implying these states were non-cell autonomously induced by the microenvironment. Therefore, we have generated an advanced, novel in vitro model of SHH-MB with potential translational applications.

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Section: Methodsmentioning

confidence: 99%

Sonic hedgehog medulloblastoma cells in co-culture with cerebellar organoids converge towardsin vivomalignant cell states

van Essen,

Nicheperovich,

Schuster-Böckler

et al. 2024

Preprint

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“…Cerebellar organoids were generated as described (van Essen et al, 2022;Nayler et al, 2021;Watson et al, 2018). In brief, iPSC were detached using TrypLE™ Express Enzyme (1X) (Gibco™, Cat# 12604013) and resuspended in Induction Medium (50% Iscove's Modified Dulbecco's Medium (Gibco™, Cat# 31980022), 50% Ham's F-12 Nutrient Mix (Gibco™, Cat# 31765027), 7𝜇g/mL insulin (Sigma, Cat# I1882), 5mg/mL BSA (|Sigma, Cat# A3156-5G), 1% Chemically Defined Lipid Concentrate (Gibco™, Cat# 11905031), 450𝜇M 1-thioglycerol (Sigma, Cat# M-6145), 15𝜇g/mL apo-transferrin (Sigma, Cat# T1147), 1% Penicillin/Streptomycin (Gibco™, Cat#15140122)) containing 50µM Rho-kinase inhibitor(Abcam, Cat#ab120129) and 10µM SB431542 (Tocris, Cat#1614/10).…”

Section: Cerebellar Organoid Differentiationmentioning

confidence: 99%

PTCH1mutant human cerebellar organoids are associated with altered neural development and early pathways of medulloblastoma oncogenesis

Essen

Riepsaame

Cowley

et al. 2023

Preprint

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SummaryPatched 1 (PTCH1) is the primary receptor for Sonic Hedgehog (SHH) ligand and negatively regulates SHH signalling, an essential pathway in human embryogenesis. Loss-of-function mutations inPTCH1are associated with altered neuronal development and the malignant brain tumour medulloblastoma (MB). As a result of differences between murine and human development, molecular and cellular perturbations that arise from humanPTCH1mutations remain poorly understood. Here, we employ cerebellar organoids differentiated from human induced pluripotent stem cells (iPSC) combined with CRISPR/Cas9 gene editing to investigate the earliest molecular and cellular consequences ofPTCH1mutations on human cerebellar development. Our findings support the occurrence of developmental mechanisms in cerebellar organoids that mirrorin vivoprocesses of regionalisation and SHH signalling, and offer new insight into early pathophysiological events of MB tumorigenesis.HiglightsDifferentiation of human iPSC into cerebellar organoidsHomozygous LOF ofPTCH1prevents cerebellar organoid differentiationPTCH1+/-cerebellar organoids display tissue-specific effects of SHH signallingEarly altered gene expression relevant for MB inPTCH1+/-cerebellar organoids

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PTCH1-mutant human cerebellar organoids exhibit altered neural development and recapitulate early medulloblastoma tumorigenesis

van Essen,

Apsley,

Riepsaame

et al. 2024

Disease Models &Amp; Mechanisms

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Patched 1 (PTCH1) is the primary receptor for the sonic hedgehog (SHH) ligand and negatively regulates SHH signalling, an essential pathway in human embryogenesis. Loss-of-function mutations in PTCH1 are associated with altered neuronal development and the malignant brain tumour medulloblastoma. As a result of differences between murine and human development, molecular and cellular perturbations that arise from human PTCH1 mutations remain poorly understood. Here, we used cerebellar organoids differentiated from human induced pluripotent stem cells combined with CRISPR/Cas9 gene editing to investigate the earliest molecular and cellular consequences of PTCH1 mutations on human cerebellar development. Our findings demonstrate that developmental mechanisms in cerebellar organoids reflect in vivo processes of regionalisation and SHH signalling, and offer new insights into early pathophysiological events of medulloblastoma tumorigenesis without the use of animal models.

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Cerebellar Modelling Using Human Induced Pluripotent Stem Cells

Cited by 3 publications

References 34 publications

Sonic hedgehog medulloblastoma cells in co-culture with cerebellar organoids converge towardsin vivomalignant cell states

Sonic hedgehog medulloblastoma cells in co-culture with cerebellar organoids converge towardsin vivomalignant cell states

PTCH1mutant human cerebellar organoids are associated with altered neural development and early pathways of medulloblastoma oncogenesis

PTCH1-mutant human cerebellar organoids exhibit altered neural development and recapitulate early medulloblastoma tumorigenesis

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