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1.
Int J Mol Sci ; 23(21)2022 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-36361956

RESUMO

In vitro models of corticogenesis from pluripotent stem cells (PSCs) have greatly improved our understanding of human brain development and disease. Among these, 3D cortical organoid systems are able to recapitulate some aspects of in vivo cytoarchitecture of the developing cortex. Here, we tested three cortical organoid protocols for brain regional identity, cell type specificity and neuronal maturation. Overall, all protocols gave rise to organoids that displayed a time-dependent expression of neuronal maturation genes such as those involved in the establishment of synapses and neuronal function. Comparatively, guided differentiation methods without WNT activation generated the highest degree of cortical regional identity, whereas default conditions produced the broadest range of cell types such as neurons, astrocytes and hematopoietic-lineage-derived microglia cells. These results suggest that cortical organoid models produce diverse outcomes of brain regional identity and cell type specificity and emphasize the importance of selecting the correct model for the right application.


Assuntos
Organoides , Células-Tronco Pluripotentes , Humanos , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Neurônios/metabolismo , Encéfalo
2.
Neurobiol Dis ; 146: 105140, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33065279

RESUMO

RUES2 cell lines represent the first collection of isogenic human embryonic stem cells (hESCs) carrying different pathological CAG lengths in the HTT gene. However, their neuronal differentiation potential has yet to be thoroughly evaluated. Here, we report that RUES2 during ventral telencephalic differentiation is biased towards medial ganglionic eminence (MGE). We also show that HD-RUES2 cells exhibit an altered MGE transcriptional signature in addition to recapitulating known HD phenotypes, with reduced expression of the neurodevelopmental regulators NEUROD1 and BDNF and increased cleavage of synaptically enriched N-cadherin. Finally, we identified the transcription factor SP1 as a common potential detrimental co-partner of muHTT by de novo motif discovery analysis on the LGE, MGE, and cortical genes differentially expressed in HD human pluripotent stem cells in our and additional datasets. Taken together, these observations suggest a broad deleterious effect of muHTT in the early phases of neuronal development that may unfold through its altered interaction with SP1.


Assuntos
Biomarcadores Tumorais/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Pluripotentes/citologia , Receptores Imunológicos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/patologia , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo
3.
Proc Natl Acad Sci U S A ; 114(7): E1234-E1242, 2017 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-28137879

RESUMO

Medium spiny neurons (MSNs) are a key population in the basal ganglia network, and their degeneration causes a severe neurodegenerative disorder, Huntington's disease. Understanding how ventral neuroepithelial progenitors differentiate into MSNs is critical for regenerative medicine to develop specific differentiation protocols using human pluripotent stem cells. Studies performed in murine models have identified some transcriptional determinants, including GS Homeobox 2 (Gsx2) and Early B-cell factor 1 (Ebf1). Here, we have generated human embryonic stem (hES) cell lines inducible for these transcription factors, with the aims of (i) studying their biological role in human neural progenitors and (ii) incorporating TF conditional expression in a developmental-based protocol for generating MSNs from hES cells. Using this approach, we found that Gsx2 delays cell-cycle exit and reduces Pax6 expression, whereas Ebf1 promotes neuronal differentiation. Moreover, we found that Gsx2 and Ebf1 combined overexpression in hES cells achieves high yields of MSNs, expressing Darpp32 and Ctip2, in vitro as well in vivo after transplantation. We show that hES-derived striatal progenitors can be transplanted in animal models and can differentiate and integrate into the host, extending fibers over a long distance.


Assuntos
Diferenciação Celular/genética , Proteínas de Homeodomínio/genética , Células-Tronco Embrionárias Humanas/metabolismo , Neurônios/metabolismo , Transativadores/genética , Animais , Ciclo Celular/genética , Linhagem Celular , Fosfoproteína 32 Regulada por cAMP e Dopamina/genética , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Células-Tronco Embrionárias Humanas/transplante , Humanos , Camundongos Nus , Neurônios/citologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transplante de Células-Tronco/métodos , Telencéfalo/citologia , Transativadores/metabolismo , Transplante Heterólogo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
4.
Nat Commun ; 15(1): 6534, 2024 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-39095390

RESUMO

Huntington's disease (HD) causes selective degeneration of striatal and cortical neurons, resulting in cell mosaicism of coexisting still functional and dysfunctional cells. The impact of non-cell autonomous mechanisms between these cellular states is poorly understood. Here we generated telencephalic organoids with healthy or HD cells, grown separately or as mosaics of the two genotypes. Single-cell RNA sequencing revealed neurodevelopmental abnormalities in the ventral fate acquisition of HD organoids, confirmed by cytoarchitectural and transcriptional defects leading to fewer GABAergic neurons, while dorsal populations showed milder phenotypes mainly in maturation trajectory. Healthy cells in mosaic organoids restored HD cell identity, trajectories, synaptic density, and communication pathways upon cell-cell contact, while showing no significant alterations when grown with HD cells. These findings highlight cell-type-specific alterations in HD and beneficial non-cell autonomous effects of healthy cells, emphasizing the therapeutic potential of modulating cell-cell communication in disease progression and treatment.


Assuntos
Doença de Huntington , Organoides , Fenótipo , Telencéfalo , Doença de Huntington/patologia , Doença de Huntington/genética , Doença de Huntington/metabolismo , Organoides/patologia , Organoides/metabolismo , Animais , Telencéfalo/patologia , Telencéfalo/citologia , Telencéfalo/metabolismo , Humanos , Camundongos , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/patologia , Análise de Célula Única , Comunicação Celular , Mosaicismo , Neurônios/metabolismo , Neurônios/patologia
5.
Cell Rep ; 43(4): 114031, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38583153

RESUMO

Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF), which is not expressed in guided cortical organoids, we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP, LIFR, and HOPX, closely matching human fetal oRG. Finally, incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely.


Assuntos
Diferenciação Celular , Ventrículos Laterais , Fator Inibidor de Leucemia , Organoides , Células-Tronco Pluripotentes , Humanos , Organoides/metabolismo , Organoides/citologia , Fator Inibidor de Leucemia/metabolismo , Fator Inibidor de Leucemia/farmacologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Ventrículos Laterais/citologia , Ventrículos Laterais/metabolismo , Fator de Transcrição STAT3/metabolismo , Neuroglia/metabolismo , Neuroglia/citologia , Transdução de Sinais
6.
bioRxiv ; 2023 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-36824730

RESUMO

Mammalian outer radial glia (oRG) emerge as cortical progenitor cells that directly support the development of an enlarged outer subventricular zone (oSVZ) and, in turn, the expansion of the neocortex. The in vitro generation of oRG is essential to model and investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 pathway using LIF, which is not produced in guided cortical organoids, we developed a cerebral organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The structured oSVZ is composed of progenitor cells expressing specific oRG markers such as GFAP, LIFR, HOPX , which closely matches human oRG in vivo . In this microenvironment, cortical neurons showed faster maturation with enhanced metabolic and functional activity. Incorporation of hPSC-derived brain vascular LIF- producing pericytes in cerebral organoids mimicked the effects of LIF treatment. These data indicate that the cellular complexity of the cortical microenvironment, including cell-types of the brain vasculature, favors the appearance of oRG and provides a platform to routinely study oRG in hPSC-derived brain organoids.

7.
Elife ; 122023 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-37272619

RESUMO

WDR62 is a spindle pole-associated scaffold protein with pleiotropic functions. Recessive mutations in WDR62 cause structural brain abnormalities and account for the second most common cause of autosomal recessive primary microcephaly (MCPH), indicating WDR62 as a critical hub for human brain development. Here, we investigated WDR62 function in corticogenesis through the analysis of a C-terminal truncating mutation (D955AfsX112). Using induced Pluripotent Stem Cells (iPSCs) obtained from a patient and his unaffected parent, as well as isogenic corrected lines, we generated 2D and 3D models of human neurodevelopment, including neuroepithelial stem cells, cerebro-cortical progenitors, terminally differentiated neurons, and cerebral organoids. We report that WDR62 localizes to the Golgi apparatus during interphase in cultured cells and human fetal brain tissue, and translocates to the mitotic spindle poles in a microtubule-dependent manner. Moreover, we demonstrate that WDR62 dysfunction impairs mitotic progression and results in alterations of the neurogenic trajectories of iPSC neuroderivatives. In summary, impairment of WDR62 localization and function results in severe neurodevelopmental abnormalities, thus delineating new mechanisms in the etiology of MCPH.


Assuntos
Proteínas de Ciclo Celular , Complexo de Golgi , Microcefalia , Proteínas do Tecido Nervoso , Polos do Fuso , Humanos , Microcefalia/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ciclo Celular/metabolismo , Masculino , Células-Tronco Pluripotentes Induzidas , Mitose , Criança , Adolescente
8.
Cell Rep Methods ; 2(12): 100367, 2022 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-36590694

RESUMO

Stem cell engineering of striatal medium spiny neurons (MSNs) is a promising strategy to understand diseases affecting the striatum and for cell-replacement therapies in different neurological diseases. Protocols to generate cells from human pluripotent stem cells (PSCs) are scarce and how well they recapitulate the endogenous fetal cells remains poorly understood. We have developed a protocol that modulates cell seeding density and exposure to specific morphogens that generates authentic and functional D1- and D2-MSNs with a high degree of reproducibility in 25 days of differentiation. Single-cell RNA sequencing (scRNA-seq) shows that our cells can mimic the cell-fate acquisition steps observed in vivo in terms of cell type composition, gene expression, and signaling pathways. Finally, by modulating the midkine pathway we show that we can increase the yield of MSNs. We expect that this protocol will help decode pathogenesis factors in striatal diseases and eventually facilitate cell-replacement therapies for Huntington's disease (HD).


Assuntos
Neurônios Espinhosos Médios , Células-Tronco Pluripotentes , Humanos , Reprodutibilidade dos Testes , Neurogênese , Corpo Estriado , Células-Tronco Pluripotentes/metabolismo
9.
Science ; 372(6542)2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33958447

RESUMO

Deciphering how the human striatum develops is necessary for understanding the diseases that affect this region. To decode the transcriptional modules that regulate this structure during development, we compiled a catalog of 1116 long intergenic noncoding RNAs (lincRNAs) identified de novo and then profiled 96,789 single cells from the early human fetal striatum. We found that D1 and D2 medium spiny neurons (D1- and D2-MSNs) arise from a common progenitor and that lineage commitment is established during the postmitotic transition, across a pre-MSN phase that exhibits a continuous spectrum of fate determinants. We then uncovered cell type-specific gene regulatory networks that we validated through in silico perturbation. Finally, we identified human-specific lincRNAs that contribute to the phylogenetic divergence of this structure in humans. This work delineates the cellular hierarchies governing MSN lineage commitment.


Assuntos
Atlas como Assunto , Corpo Estriado/citologia , Corpo Estriado/embriologia , Neurogênese/genética , RNA Longo não Codificante/genética , Análise de Célula Única , Fatores de Transcrição/genética , Feto , Neurônios GABAérgicos/metabolismo , Humanos , RNA-Seq , Transcrição Gênica
10.
Stem Cell Res ; 49: 102016, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33039807

RESUMO

GSX2 is a homeobox transcription factor (TF) controlling the specification of the ventral lateral ganglionic eminence and its major derivative, the corpus striatum. Medium spiny neurons (MSNs) represent the largest cell component of the striatum and they are primarily affected in Huntington disease (HD). Here, we used CRISPR technology to generate a pluripotent GSX2-reporter human embryonic stem cell (hESC) line that can be leveraged to monitor striatal differentiation in real-time and to enrich for MSN-committed progenitors.


Assuntos
Células-Tronco Embrionárias Humanas , Diferenciação Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Corpo Estriado , Células-Tronco Embrionárias , Humanos , Neurônios
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