Science Tuberous sclerosis human brain organoids: expansion of interneuronal progenitors drives tumor and cortical nodule development

Written | Edited by Qi | Malformations of cortical development (MCD) are responsible for more than 40% of medically intractable childhood seizures
.

Among them, tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder caused by mutations in TSC1 or TSC, which encode proteins that form complexes and inhibit mTOR kinase
.

Eighty percent of patients present with subependymal nodules (SEN), benign tumors that proliferate along the lateral ventricle and develop into subependymal giant cell astrocytoma (SEGA) [1]
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In mice, TSC requires simultaneous biallelic inactivation of TSC1 or TSC2, and analysis of patients showed that although this biallelic defect is present in most SEN/SEGAs, it is almost absent in cortical nodules Medium【2-4】
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This challenges the previously proposed second-strike models [5, 6]
.

Recently, Jürgen A.
Knoblich's team from the Vienna Biocenter published an article in the journal Science entitled Amplification of human interneuron progenitors promotes brain tumors and neurological defects.
They completed the TSC human brain organoid model of tuberous sclerosis complex , by scRNA-seq and histological validation, identified a specific interneuron progenitor population, caudal late interneuron progenitors (caudal late interneuron progenitors) originating from the caudal ganglionic eminence (CGE) in mid-gestation cells, CLIP), which are hyperproliferative in TSC and can cause brain tumors and cortical nodules
.

Tumor burden can be reduced by inhibition of epidermal growth factor receptor (EGFR), suggesting a potential therapeutic option for TSC and related diseases
.

To mimic the brain pathology of TSC, we generated induced pluripotent stem cells (iPSCs) from patients with drug-resistant epilepsy who harbored cortical nodules and subependymal tumors with known TSC2 mutations, respectively.
Organoids that specifically predispose to subependymal tumor or cortical nodule formation are grown in high-nutrient (H) and low-nutrient (L) media
.

The genotypes of the two cultures were not significantly different during the first 90 days of culture corresponding to the early stages of neural development, but TSC2+/- organoids cultured in H 110 days after embryoidbody (EB) formation (TSC2+/-H-organoids) formed cell nodular aggregates expressing the proliferation marker Ki67 and the mTOR activation marker pS6, morphologically similar to SEN, and positive labeling for Nestin, ASCL1, and SOX2 demonstrated the reported SEN activity.
The origin of neural stem cells (NSC) [7]
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Organoids cultured for 220 days in H were almost entirely composed of tumor tissue.
To characterize the cellular composition at this stage, we performed single-cell transcriptome analysis and identified 4 major clusters: interneurons, interneuron progenitors , dividing interneuron progenitors and excitatory neurons
.

Based on the reported inconsistency in the number of allelic inactivation in mouse models and patients, is biallelic inactivation of TSC required for tumor initiation at all? We tested early-stage TSC2+/-H-organoids for mutational status, and genotyping after FACS sorting indicated that many tumors were still heterozygous and only partially displayed loss of heterozygosity (LOH), Analysis of allele frequencies in scRNA-seq data also further confirmed that tumors in TSC organoids originate from heterozygous interneuron progenitors, and that copyneutral LOH (cnLOH) is only acquired during tumor progression
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Similarly, the formation of cortical nodule-like structures does not depend on cnLOH, denying the previous conclusion based on mouse models that LOH of Tsc1 or Tsc2 is necessary for a TSC-like phenotype
.

So do these two phenotypes in TSC originate from the same cell type? The researchers analyzed the gene expression characteristics in TSC organoids.
The expression of markers such as DLX2, DLX5, and SP8 suggested that the lineage originated from the caudal ganglion eminence.
Further RNA velocity and pseudo-event analysis found that the two phenotypes shared from CGE progenitors.
Developmental trajectories of cells to CGE interneurons
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Furthermore, comparing these data with published scRNA-seq data from different fetal ages, it was observed that 98% of the fetal cells co-clustered with the expanded CGE progenitors originated at week 22 of gestation, based on these cells The origin and emergence period of CGE, the authors named such cells as late tail interneuron progenitors
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These data may also explain why SEN/SEGA is usually present in the caudate thalamic sulcus, the region where the CGE is located during fetal development
.

Figure 1.
Schematic illustration of hyperproliferation of TSC2 heterozygous mutation-derived CLIP cells derived from mid-trimester CGE driving brain tumor and cortical nodule formation
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mTOR inhibitors have been clinically used to treat SEN/SEGA in TSC patients, but there are tumor recurrence and other side effects after drug discontinuation, so alternative therapies need to be sought
.

CLIP cells from TSC tumors are known to express EGFR, and organoids were treated with the EGFR receptor tyrosine kinase inhibitors afatinib and everolimus for 30 days to define efficacy as a reduction in the area co-expressing pS6 and EGFR
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Among them, everolimus treatment almost completely eliminated tumors in 140-day organoids, and after afatinib treatment, both tumor burden and mean tumor size were significantly reduced compared with untreated organoids
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Thus, targeting the EGFR pathway may be an alternative strategy for the treatment of TSC brain injury
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This study identifies CLIP cells as the origin of brain injury in TSC using organoids derived from iPSCs of TSC patients and confirms that cnLOH occurs during TSC tumor progression and is dispensable for the initiation of the lesion phenotype, suggesting a mutant disease Correlation should always be assessed in the context of a specific cell of origin
.

In conclusion, this work demonstrates an example of a human progenitor cell type that contributes to neurodevelopmental disease in humans and demonstrates the need to use human organoid models to identify disease mechanisms that are not conserved in mammals
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In response to this study, Professor Rebecca A.
Ihrie of Vanderbilt University School of Medicine and Professor Elizabeth P.
Henske of Harvard Medical School published an opinion article in the same journal titled Modeling tuberous sclerosis with organoids, who pointed out that hindering the formation of A key issue with progress in ganglionic sclerosis research has been the failure to develop models that fully mimic the brain malformations and neuropsychiatric manifestations of TSC, such as animal models that often require the loss of additional tumor suppressor genes to promote tumor development, or exhibit seizures despite But not accompanied by obvious cortical nodules and so on
.

Eichmüller et al.
used iPSCs from TSC patients to generate organoids that recapitulate the two different phenotypes of TSC and identify cells of common origin, which may be a breakthrough in understanding the nervous system performance of TSC
.

It is also important to note that while EGFR is expressed in CLIP cells and targeted inhibition has been shown to alleviate tumor progression in Eichmüller et al.
, other populations of progenitor cells in the brain also dynamically express EGFR, and whether these progenitor cells are susceptible Affected by TSC2 mutation? In addition, it is still unknown how TSC is affected by gender, immune infiltration, genetic modification, the dependence of phenotype and severity on genotype, and individuals with TSC in the same family have widely different clinical manifestations.
It may be solved by optimization of the model
.

Original link: https://doi.
org/10.
1126/science.
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x; pmid: 18564101 Instructions for reprinting【Original article】BioArt original article, welcome to repost and share it.
Reprinting is prohibited without permission.
The copyright of all published works is owned by BioArt10.
1007/s00401-013-1085-x; pmid: 233863246.
AG Knudson Jr.
, Mutation and cancer: Statistical study of retinoblastoma.
Proc.
Natl.
Acad.
Sci.
USA 68, 820–823 (1971).
doi: 10.
1073/ pnas.
68.
4.
820; pmid: 52795237.
AM Buccoliero et al.
, Subependymal giant cell astrocytoma (SEGA): Is it an astrocytoma? Morphological, immunohistochemical and ultrastructural study.
Neuropathology 29, 25–30 (2009).
doi: 10.
1111/ j.
1440-1789.
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00934.
x; pmid: 18564101 Instructions for reprinting【Original article】BioArt original article, welcome to repost and share it.
Reprinting is prohibited without permission.
The copyright of all published works is owned by BioArtimmunohistochemical and ultrastructural study.
Neuropathology 29, 25–30 (2009).
doi: 10.
1111/j.
1440-1789.
2008.
00934.
x; pmid: 18564101 Notes for reprinting [Original article] BioArt original article, welcome to forward and share without permission Reprinting is prohibited, the copyright of all works published is owned by BioArtimmunohistochemical and ultrastructural study.
Neuropathology 29, 25–30 (2009).
doi: 10.
1111/j.
1440-1789.
2008.
00934.
x; pmid: 18564101 Notes for reprinting [Original article] BioArt original article, welcome to forward and share without permission Reprinting is prohibited, the copyright of all works published is owned by BioArt
.

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