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1.
Mol Psychiatry ; 26(7): 2964-2976, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33051604

RESUMEN

Numerous studies have used human pluripotent stem cell-derived cerebral organoids to elucidate the mystery of human brain development and model neurological diseases in vitro, but the potential for grafted organoid-based therapy in vivo remains unknown. Here, we optimized a culturing protocol capable of efficiently generating small human cerebral organoids. After transplantation into the mouse medial prefrontal cortex, the grafted human cerebral organoids survived and extended projections over 4.5 mm in length to basal brain regions within 1 month. The transplanted cerebral organoids generated human glutamatergic neurons that acquired electrophysiological maturity in the mouse brain. Importantly, the grafted human cerebral organoids functionally integrated into pre-existing neural circuits by forming bidirectional synaptic connections with the mouse host neurons. Furthermore, compared to control mice, the mice transplanted with cerebral organoids showed an increase in freezing time in response to auditory conditioned stimuli, suggesting the potentiation of the startle fear response. Our study showed that subcortical projections can be established by microtransplantation and may provide crucial insights into the therapeutic potential of human cerebral organoids for neurological diseases.


Asunto(s)
Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Animales , Encéfalo , Diferenciación Celular , Fenómenos Electrofisiológicos , Humanos , Ratones , Neuronas , Organoides
2.
J Biol Chem ; 293(23): 8722-8733, 2018 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-29628445

RESUMEN

Human pluripotent stem cell (hPSC)-based cell-replacement therapy has emerged as a promising approach for addressing numerous neurological diseases. However, hPSC transplantation has the potential to cause human cell overgrowth and cancer, which represents a major obstacle to implementing hPSC-based therapies. Inhibition of the overgrowth of transplanted cells could help reduce the risk for hPSC transplantation-induced tumorigenesis. In this study, we report that the telomerase inhibitor azidothymidine (3'-azido-3'-deoxythymidine; AZT) enhances the differentiation of cortical neurons and significantly suppresses the proliferation of hPSC-derived cortical progenitors. Using human embryonic stem cells and induced pluripotent stem cells in culture, we found that AZT effectively reduces the number of dividing progenitors without inducing cell death. Furthermore, AZT promoted differentiation of cortical progenitors and maturation of cortical neurons. Of note, AZT-pretreated, hPSC-derived neural progenitors exhibited decreased proliferation and increased differentiation into cortical neurons when transplanted into the mouse brain. In summary, our findings indicate that AZT prevents the overgrowth of hPSC-derived neural precursors and enhances the differentiation of cortical neurons in both cell cultures and hPSC-transplanted mouse brain. We propose that our work could inform clinical applications of hPSC-based cell therapy.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Inhibidores Enzimáticos/farmacología , Células-Madre Neurales/efectos de los fármacos , Células Madre Pluripotentes/efectos de los fármacos , Telomerasa/antagonistas & inhibidores , Zidovudina/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Humanos , Ratones SCID , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neurogénesis/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/trasplante , Telomerasa/metabolismo
3.
Biochim Biophys Acta Mol Basis Dis ; 1868(6): 166388, 2022 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-35301086

RESUMEN

Down syndrome (DS) is caused by trisomy 21, and it is characterized by developmental brain disorders and neurological dysfunction. Clinical studies and basic research have revealed that defects in mitochondrial function contribute to the pathogenesis of DS. However, the underlying mechanisms of mitochondrial dysfunction in DS remain unclear. In this study, we first generated GABAergic interneurons and medial ganglionic eminence (MGE) organoids from DS patients and control induced pluripotent stem cells. The mitochondria were abnormally clustered in the perinuclear region of GABA neurons and cell in MGE organoids from DS patients, which exhibited impaired mitochondrial function as assessed by seahorse oxidative phosphorylation assay. Inhibition of the DSCAM-PAK1 pathway by gene editing or treatment with a small molecule corrected mitochondrial perinuclear aggregation in cells from DS patients. Therefore, our study provides insight into the potential mechanism of mitochondrial dysfunction in DS.


Asunto(s)
Síndrome de Down , Células Madre Pluripotentes Inducidas , Síndrome de Down/genética , Síndrome de Down/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Interneuronas , Mitocondrias/metabolismo , Organoides/metabolismo
4.
J Clin Invest ; 131(12)2021 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-33945512

RESUMEN

Down syndrome (DS), caused by trisomy of chromosome 21, occurs in 1 of every 800 live births. Early defects in cortical development likely account for the cognitive impairments in DS, although the underlying molecular mechanism remains elusive. Here, we performed histological assays and unbiased single-cell RNA-Seq (scRNA-Seq) analysis on cerebral organoids derived from 4 euploid cell lines and from induced pluripotent stem cells (iPSCs) from 3 individuals with trisomy 21 to explore cell-type-specific abnormalities associated with DS during early brain development. We found that neurogenesis was significantly affected, given the diminished proliferation and decreased expression of layer II and IV markers in cortical neurons in the subcortical regions; this may have been responsible for the reduced size of the organoids. Furthermore, suppression of the DSCAM/PAK1 pathway, which showed enhanced activity in DS, using CRISPR/Cas9, CRISPR interference (CRISPRi), or small-molecule inhibitor treatment reversed abnormal neurogenesis, thereby increasing the size of organoids derived from DS iPSCs. Our study demonstrates that 3D cortical organoids developed in vitro are a valuable model of DS and provide a direct link between dysregulation of the DSCAM/PAK1 pathway and developmental brain defects in DS.


Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Corteza Cerebral/metabolismo , Síndrome de Down/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Neurogénesis , Organoides/metabolismo , Transducción de Señal , Quinasas p21 Activadas/metabolismo , Moléculas de Adhesión Celular/genética , Línea Celular , Síndrome de Down/genética , Humanos , Quinasas p21 Activadas/genética
5.
Acta Pharm Sin B ; 9(3): 557-564, 2019 May.
Artículo en Inglés | MEDLINE | ID: mdl-31193866

RESUMEN

Establishing an effective three-dimensional (3D) in vitro culture system to better model human neurological diseases is desirable, since the human brain is a 3D structure. Here, we demonstrated the development of a polydimethylsiloxane (PDMS) pillar-based 3D scaffold that mimicked the 3D microenvironment of the brain. We utilized this scaffold for the growth of human cortical glutamatergic neurons that were differentiated from human pluripotent stem cells. In comparison with the 2D culture, we demonstrated that the developed 3D culture promoted the maturation of human cortical glutamatergic neurons by showing significantly more MAP2 and less Ki67 expression. Based on this 3D culture system, we further developed an in vitro disease-like model of traumatic brain injury (TBI), which showed a robust increase of glutamate-release from the neurons, in response to mechanical impacts, recapitulating the critical pathology of TBI. The increased glutamate-release from our 3D culture model was attenuated by the treatment of neural protective drugs, memantine or nimodipine. The established 3D in vitro human neural culture system and TBI-like model may be used to facilitate mechanistic studies and drug screening for neurotrauma or other neurological diseases.

6.
Elife ; 72018 09 25.
Artículo en Inglés | MEDLINE | ID: mdl-30251953

RESUMEN

Human GABAergic interneurons (GIN) are implicated in normal brain function and in numerous mental disorders. However, the generation of functional human GIN subtypes from human pluripotent stem cells (hPSCs) has not been established. By expressing LHX6, a transcriptional factor that is critical for GIN development, we induced hPSCs to form GINs, including somatostatin (SST, 29%) and parvalbumin (PV, 21%) neurons. Our RNAseq results also confirmed the alteration of GIN identity with the overexpression of LHX6. Five months after transplantation into the mouse brain, the human GABA precursors generated increased population of SST and PV neurons by overexpressing LHX6. Importantly, the grafted human GINs exhibited functional electrophysiological properties and even fast-spiking-like action potentials. Thus, expression of the single transcription factor LHX6 under our GIN differentiation condition is sufficient to robustly induce human PV and SST subtypes.


Asunto(s)
Proteínas con Homeodominio LIM/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Parvalbúminas/metabolismo , Somatostatina/metabolismo , Factores de Transcripción/metabolismo , Potenciales de Acción , Animales , Animales Recién Nacidos , Tipificación del Cuerpo , Diferenciación Celular , Línea Celular , Perfilación de la Expresión Génica , Humanos , Interneuronas/citología , Interneuronas/metabolismo , Ratones SCID , Neuronas/citología , Neuronas/trasplante , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Prosencéfalo/citología , Ácido gamma-Aminobutírico/metabolismo
7.
Sci Rep ; 7(1): 3282, 2017 06 12.
Artículo en Inglés | MEDLINE | ID: mdl-28607372

RESUMEN

Human pluripotent stem cells (hPSCs) play important role in studying the function of human glutamatergic neurons and related disease pathogenesis. However, the current hPSC-derived cortical system produced a significant number of inhibitory GABAergic neurons that reduced the purity of excitatory neurons. In this study, we established a robust hPSC-derived cortical neurogenesis system by applying the SHH inhibitor cyclopamine. Cyclopamine specified the dorsal cortical fate in a dose-dependent manner and enhanced the generation of cortical glutamatergic neurons, expressing PAX6, TBR1, TBR2, CTIP2, SATB2, and vesicular glutamate transporters (vGLUT). In contrast, the ventral patterning was inhibited and the GABAergic neurons were significantly reduced to 12% with the treatment of cyclopamine. In addition, we applied our current method to generate trisomy 21 iPSC-derived glutamatergic neurons that showed a robust reduction of vesicular glutamate transporters in the glutamatergic neurons with trisomy 21, revealing the developmental deficits in cortical glutamatergic neurons. Our method enriched the generation of cortical glutamatergic neurons which may facilitate the study of human neurological diseases and cell therapy.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Corteza Cerebral/citología , Neuronas GABAérgicas/citología , Neuronas GABAérgicas/efectos de los fármacos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Células Cultivadas , Síndrome de Down , Descubrimiento de Drogas , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Neuronas GABAérgicas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Neurogénesis , Vesículas Transportadoras , Alcaloides de Veratrum/farmacología
9.
Sci Rep ; 5: 18550, 2015 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-26670131

RESUMEN

Human pluripotent stem cells (hPSCs) have potential to differentiate to unlimited number of neural cells, which provide powerful tools for neural regeneration. To date, most reported protocols were established with an animal feeder system. However, cells derived on this system are inappropriate for the translation to clinical applications because of the introduction of xenogenetic factors. In this study, we provided an optimized paradigm to generate region-specific forebrain neurons from hPSCs under a defined system. We assessed five conditions and found that a vitronectin-coated substrate was the most efficient method to differentiate hPSCs to neurons and astrocytes. More importantly, by applying different doses of purmorphamine, a small-molecule agonist of sonic hedgehog signaling, hPSCs were differentiated to different region-specific forebrain neuron subtypes, including glutamatergic neurons, striatal medium spiny neurons, and GABA interneurons. Our study offers a highly defined system without exogenetic factors to produce human neurons and astrocytes for translational medical studies, including cell therapy and stem cell-based drug discovery.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Neuronas/citología , Células Madre Pluripotentes/citología , Prosencéfalo/citología , Línea Celular , Humanos , Neuronas/metabolismo , Células Madre Pluripotentes/metabolismo , Prosencéfalo/metabolismo
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