In vitro-derived medium spiny neurons recapitulate human striatal development and complexity at single-cell resolution

Conforti, Paola; Bocchi, Vittoria Dickinson; Campus, Ilaria; Scaramuzza, Linda; Galimberti, Maura; Lischetti, Tiziana; Talpo, Francesca; Pedrazzoli, Matteo; Murgia, Alessio; Ferrari, Ivan; Cordiglieri, Chiara; Fasciani, Alessandra; Arenas, Ernest; Felsenfeld, Dan P.; Biella, Gerardo; Besusso, Dario; Cattaneo, Elena

doi:10.1016/j.crmeth.2022.100367

Cited by 9 publications

(10 citation statements)

References 32 publications

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“…In this study, we utilized isogenic HD72 and corrected iPSCs-derived MSNs to investigate the effects of HD on the development of human MSNs as they differentiate from NSCs to mature MSNs. Consistent with previous studies [26], our results show that iPSCs-derived developing MSNs recapitulate multiple aspects of fetal striatum development. Specifically, we observed similar gene-expression patterns and cell-type compositions between the two systems, including early and intermediate progenitors and mature MSNs.…”

Section: Discussionsupporting

confidence: 92%

“…Our transcriptomic analysis of isogenic HD72 NSCs suggested that HD is linked to developmental impairments that prevent proper generation of MSNs and subsequent loss of MSNs identity [6,8,[23][24][25]. Similarly, HD iPSC derived neurons feature dysregulation of multiple pathways related to development [21,26,27]. We also found that deletion of transcription factors required for striatum development leads to HDlike phenotypes [28].…”

Section: Introductionmentioning

confidence: 90%

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Transcriptomic Characterization Reveals Disrupted Medium Spiny Neuron Trajectories in Huntington’s Disease and Possible Therapeutic Avenues

Aquirre

Tshilenge

Battistoni

et al. 2023

Preprint

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Huntington's disease (HD) is a neurodegenerative disorder caused by an expansion of CAG repeats in exon 1 of the HTT gene, ultimately resulting in the generation of a mutant HTT (mHTT) protein. Although mHTT is expressed in various tissues, it significantly affects medium spiny neurons (MSNs) in the striatum, resulting in their loss and subsequent motor function impairment in HD. While HD symptoms typically emerge in midlife, disrupted MSN neurodevelopment has an important role. To explore the effects of mHTT on MSN development, we differentiated HD induced pluripotent stem cells (iPSC) and isogenic controls into neuronal stem cells, and then generated a developing MSN population encompassing early, intermediate progenitors, and mature MSNs. Single-cell RNA sequencing revealed that the developmental trajectory of MSNs in our model closely emulated the trajectory of fetal striatal neurons. However, in the HD MSN cultures, the differentiation process downregulated several crucial genes required for proper MSN maturation, including Achaete-scute homolog 1 and members of the DLX family of transcription factors. Our analysis also uncovered a progressive dysregulation of multiple HD-related pathways as the MSNs matured, including the NRF2-mediated oxidative stress response and mitogen-activated protein kinase signaling. Using the transcriptional profile of developing HD MSNs, we searched the L1000 dataset for small molecules that induce the opposite gene expression pattern. Our analysis pinpointed numerous small molecules with known benefits in HD models, as well as previously untested novel molecules. A top novel candidate, Cerulenin, partially restored the DARPP-32 levels and electrical activity in HD MSNs, and also modulated genes involved in multiple HD-related pathways.

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Section: Discussionsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 90%

Transcriptomic Characterization Reveals Disrupted Medium Spiny Neuron Trajectories in Huntington’s Disease and Possible Therapeutic Avenues

Aquirre

Tshilenge

Battistoni

et al. 2023

Preprint

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“…Both 2D neuronal and 3D cerebral organoid protocols have been regionally specified toward forebrain and midbrain lineages that are expected to include MSNs and DNs, respectively. However, while several 2D neuronal [20][21][22]31,40,43,44,46,47,[54][55][56][57][58] and 3D organoid [26][27][28]32,38,49,50] protocols have been shown to generate MSNs and DNs, 3D organoid models lack mutually compatible media conditions for early coculture or fusion. Our first goal was to develop regionally specified organoid models for both the forebrain and midbrain using media conditions mutually compatible with early fusion (at day 25).…”

Section: Development Of Striatal and Midbrain Organoids Compatible Wi...mentioning

confidence: 99%

“…The following are a few example challenges related to mesolimbic cell types: Multiple distinct models have been developed without clear comparisons of how they differ in cellular composition and responsiveness [20][21][22][26][27][28]31,32,38,[40][41][42][43][44][45][46][47]; most ventral forebrain (e.g., striatal) organoids have not been characterized for their ability to generate MSNs [32,38]; it is unknown what the differences are between 2D [20][21][22]41,47] and 3D [38] models; it is a widely held but not validated assumption that 2D models are almost homogeneous systems, especially compared to 3D organoids [48]; it is unclear which cell types are driving the responses measured in bulk to perturbations such as substances of abuse or microenvironmental factors; and finally, though not exclusively, as more complex systems are built from hPSC-derived cells, such as assembloids to model connections between brain regions [32,38,[49][50][51], cell culture and media conditions will need to be engineered to be simultaneously compatible with the multiple neuronal or organoid systems being combined together. In all efforts to address these challenges, it will be important to rigorously characterize, compare, and establish baseline datasets for these systems; these datasets will support the efficient and rigorous investment, design, and interpretation of expensive functional genomics experiments as well as of association studies.…”

Section: Introductionmentioning

confidence: 99%

Single-Cell Assessment of Human Stem Cell-Derived Mesolimbic Models and Their Responses to Substances of Abuse

Rudibaugh,

Tam,

Estridge

et al. 2024

Organoids

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The mesolimbic pathway connects ventral tegmental area dopaminergic neurons and striatal medium spiny neurons, playing a critical role in reward and stress behaviors. Exposure to substances of abuse during development and adulthood has been linked to adverse outcomes and molecular changes. The rise of human cell repositories and whole-genome sequences enables human functional genomics ‘in a dish’, offering insights into human-specific responses to substances of abuse. Continued development of new models is needed, and the characterization of in vitro models is also necessary to ensure appropriate experimental designs and the accurate interpretation of results. This study introduces new culture conditions for generating medium spiny neurons and dopaminergic neurons with an early common media, allowing for coculture and assembloid generation. It then provides a comprehensive characterization of these and prior models and their responses to substances of abuse. Single-cell analysis reveals cell-type-specific transcriptomic responses to dopamine, cocaine, and morphine, including compound and cell-type-specific transcriptomic signatures related to neuroinflammation and alterations in signaling pathways. These findings offer a resource for future genomics studies leveraging human stem cell-derived models.

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“…Consequently, the findings obtained from these cell types may not fully represent the events occurring in striatal medium spiny neurons (MSNs), which are predominantly affected by the disease [46]. Recent studies have reported the successful generation of MSNs from iPSCs [60][61][62]. The employment of iPSC-derived MSNs would provide more comprehensive insights into how ZNF91 deletion impacts the molecular phenotype in the specific neuronal cell types affected by XDP.…”

Section: Figure 5 Proposed Model: Age-related Reduction Of Znf91 Unle...mentioning

confidence: 99%

ZNF91 is an endogenous repressor of the molecular phenotype associated with X-linked dystonia-parkinsonism (XDP)

Rosenkrantz,

Raghib,

Brandorff

et al. 2023

Preprint

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Background: X-linked dystonia-parkinsonism (XDP) is a severe neurodegenerative disorder resulting from the insertion of an intronic SINE-Alu-VNTR (SVA) retrotransposon in the TAF1 gene. Recent research has revealed that the pathogenic XDP-SVA insertion leads to dysregulation of TAF1 transcription, including increased intron retention and decreased expression of exons surrounding the insertion. The Kruppel-associated box (KRAB) zinc finger protein, ZNF91, is a critical repressor of SVA retrotransposons. However, it remains unclear whether ZNF91 is able to repress the XDP-SVA insertion and how this influences the XDP-associated molecular phenotype. In this study, we investigate the role of ZNF91 in repressing the XDP-SVA insertion and its impact on the molecular phenotype associated with XDP. Methods: Here, we used CRISPR/Cas9 to genetically delete ZNF91 in induced pluripotent stem cell (iPSC) lines derived from XDP patients, as well as isogenic control iPSC lines that lack the XDP-SVA insertion. Total RNA sequencing and capture RNA-sequencing were used to confirm ZNF91 deletion and to assess TAF1 transcriptional changes between conditions. Furthermore, publicly available transcriptomic data from whole blood and different brain regions were used to assess ZNF91 expression levels across ages. Results: We found that genetic deletion of ZNF91 exacerbates the molecular phenotype associated with the XDP-SVA insertion in patient cells, while no difference was observed when ZNF91 was deleted from isogenic control cells. Additionally, we observed a significant age-related reduction in ZNF91 expression in whole blood and brain, indicating a potential role of ZNF91 in the age-dependent onset of XDP. Conclusions: These findings indicate that ZNF91 plays a crucial role in controlling the molecular phenotype associated with XDP. Since ZNF91 is a critical epigenetic repressor of SVAs, this suggests that epigenetic silencing of the XDP-SVA minimizes the severity of the molecular phenotype. Our results showing that ZNF91 expression levels significantly decrease with age provide a potential explanation for the age-related progressive neurodegenerative character of XDP. Collectively, our study provides important insights into the protective role of ZNF91 in XDP pathogenesis and suggests that modulating ZNF91 levels or targeted repression of the XDP-SVA could be novel therapeutic strategies worth exploring.

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In vitro-derived medium spiny neurons recapitulate human striatal development and complexity at single-cell resolution

Cited by 9 publications

References 32 publications

Transcriptomic Characterization Reveals Disrupted Medium Spiny Neuron Trajectories in Huntington’s Disease and Possible Therapeutic Avenues

Transcriptomic Characterization Reveals Disrupted Medium Spiny Neuron Trajectories in Huntington’s Disease and Possible Therapeutic Avenues

Single-Cell Assessment of Human Stem Cell-Derived Mesolimbic Models and Their Responses to Substances of Abuse

ZNF91 is an endogenous repressor of the molecular phenotype associated with X-linked dystonia-parkinsonism (XDP)

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