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1.
Adv Sci (Weinh) ; : e2402287, 2024 May 06.
Artículo en Inglés | MEDLINE | ID: mdl-38711218

RESUMEN

Human stem cells and derivatives transplantation are widely used to treat nervous system diseases, while the fate determination of transplanted cells is not well elucidated. To explore cell fate changes of human brain organoids before and after transplantation, human brain organoids are transplanted into prefrontal cortex (PFC) and hippocampus (HIP), respectively. Single-cell sequencing is then performed. According to time-series sample comparison, transplanted cells mainly undergo neural development at 2 months post-transplantation (MPT) and then glial development at 4MPT, respectively. A different brain region sample comparison shows that organoids grafted to PFC have obtained cell fate close to those of host cells in PFC, other than HIP, which may be regulated by the abundant expression of dopamine (DA) and acetylcholine (Ach) in PFC. Meanwhile, morphological complexity of human astrocyte grafts is greater in PFC than in HIP. DA and Ach both activate the calcium activity and increase morphological complexity of astrocytes in vitro. This study demonstrates that human brain organoids receive host niche factor regulation after transplantation, resulting in the alignment of grafted cell fate with implanted brain regions, which may contribute to a better understanding of cell transplantation and regenerative medicine.

2.
Hum Cell ; 37(3): 607-624, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38498133

RESUMEN

Due to aging populations and changes in lifestyle, cardiovascular diseases including cardiomyopathy, hypertension, and atherosclerosis, are the leading causes of death worldwide. The heart is a complicated organ composed of multicellular types, including cardiomyocytes, fibroblasts, endothelial cells, vascular smooth muscle cells, and immune cells. Cellular specialization and complex interplay between different cell types are crucial for the cardiac tissue homeostasis and coordinated function of the heart. Mounting studies have demonstrated that dysfunctional cells and disordered cardiac microenvironment are closely associated with the pathogenesis of various cardiovascular diseases. In this paper, we discuss the composition and the homeostasis of cardiac tissues, and focus on the role of cardiac environment and underlying molecular mechanisms in various cardiovascular diseases. Besides, we elucidate the novel treatment for cardiovascular diseases, including stem cell therapy and targeted therapy. Clarification of these issues may provide novel insights into the prevention and potential targets for cardiovascular diseases.


Asunto(s)
Aterosclerosis , Enfermedades Cardiovasculares , Humanos , Enfermedades Cardiovasculares/terapia , Enfermedades Cardiovasculares/patología , Células Endoteliales/metabolismo , Miocitos Cardíacos/patología , Envejecimiento
3.
Elife ; 132024 Feb 23.
Artículo en Inglés | MEDLINE | ID: mdl-38390967

RESUMEN

Deciphering patterns of connectivity between neurons in the brain is a critical step toward understanding brain function. Imaging-based neuroanatomical tracing identifies area-to-area or sparse neuron-to-neuron connectivity patterns, but with limited throughput. Barcode-based connectomics maps large numbers of single-neuron projections, but remains a challenge for jointly analyzing single-cell transcriptomics. Here, we established a rAAV2-retro barcode-based multiplexed tracing method that simultaneously characterizes the projectome and transcriptome at the single neuron level. We uncovered dedicated and collateral projection patterns of ventromedial prefrontal cortex (vmPFC) neurons to five downstream targets and found that projection-defined vmPFC neurons are molecularly heterogeneous. We identified transcriptional signatures of projection-specific vmPFC neurons, and verified Pou3f1 as a marker gene enriched in neurons projecting to the lateral hypothalamus, denoting a distinct subset with collateral projections to both dorsomedial striatum and lateral hypothalamus. In summary, we have developed a new multiplexed technique whose paired connectome and gene expression data can help reveal organizational principles that form neural circuits and process information.


Asunto(s)
Neuritas , Neuronas , Neuronas/metabolismo , Encéfalo , Corteza Prefrontal , Vías Nerviosas/fisiología
4.
Cell Prolif ; 57(4): e13563, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-37881164

RESUMEN

Human midbrain dopaminergic progenitors (mDAPs) are one of the most representative cell types in both basic research and clinical applications. However, there are still many challenges for the preparation and quality control of mDAPs, such as the lack of standards. Therefore, the establishment of critical quality attributes and technical specifications for mDAPs is largely needed. "Human midbrain dopaminergic progenitor" jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research, is the first guideline for human mDAPs in China. This standard specifies the technical requirements, test methods, inspection rules, instructions for usage, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for human mDAPs, which is applicable to the quality control for human mDAPs. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that the publication of this guideline will facilitate the institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human mDAPs for clinical development and therapeutic applications.


Asunto(s)
Neuronas Dopaminérgicas , Mesencéfalo , Humanos , China , Neuronas Dopaminérgicas/metabolismo
5.
Cell Prolif ; 57(4): e13564, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-37853840

RESUMEN

'Human neural stem cells' jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research, is the first guideline for human neural stem cells (hNSCs) in China. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for hNSCs, which is applicable to the quality control for hNSCs. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that publication of the guideline will facilitate institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of hNSCs for clinical development and therapeutic applications.


Asunto(s)
Células-Madre Neurales , Trasplante de Células Madre , Humanos , Diferenciación Celular , China
6.
Front Chem ; 11: 1260099, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37927565

RESUMEN

Carbon nanotubes (CNTs) had potential applications in energy conversion and storage devices, and it could be prepared by expanded graphite loaded with catalyst at high temperature, however, the mechanism of carbon nanotube growth in expanded graphite need further confirmation. In this work, carbon nanotubes' in situ growth in expanded graphite (EG) were prepared via catalytic pyrolysis reaction using carbores P as a carbon source and Co(NO3)3•6H2O as a catalyst. The results of X-ray diffraction (XRD), scanning electron microscope (SEM) and energy dispersive X-ray spectroscope (EDS) indicated the carbon nanotubes could generate in, EG with the presence of carbores P as a carbon source and cobalt nitrate as a catalyst. More interestingly, the growth mechanism of carbon nanotubes could be concluded by the results of differential thermal analysis-thermogravimetry-mass spectrometry (DTA-TG-MS) and X-ray photoelectron spectroscopy (XPS) analysis. The pyrolysis products of carbores P were mainly hydrocarbon gas such as CH4 gas, which reacts with Co(NO3)3·6H2O catalyst to reduces CoOx to Co particles, then the carbon form pyrolysis was deposited the on the surface catalyst Co particles and, after continuous solid dissolution and precipitation, carbon nanotubes were at last generated in EG at last.

7.
Nat Methods ; 20(8): 1244-1255, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37460718

RESUMEN

A fundamental interest in developmental neuroscience lies in the ability to map the complete single-cell lineages within the brain. To this end, we developed a CRISPR editing-based lineage-specific tracing (CREST) method for clonal tracing in Cre mice. We then used two complementary strategies based on CREST to map single-cell lineages in developing mouse ventral midbrain (vMB). By applying snapshotting CREST (snapCREST), we constructed a spatiotemporal lineage landscape of developing vMB and identified six progenitor archetypes that could represent the principal clonal fates of individual vMB progenitors and three distinct clonal lineages in the floor plate that specified glutamatergic, dopaminergic or both neurons. We further created pandaCREST (progenitor and derivative associating CREST) to associate the transcriptomes of progenitor cells in vivo with their differentiation potentials. We identified multiple origins of dopaminergic neurons and demonstrated that a transcriptome-defined progenitor type comprises heterogeneous progenitors, each with distinct clonal fates and molecular signatures. Therefore, the CREST method and strategies allow comprehensive single-cell lineage analysis that could offer new insights into the molecular programs underlying neural specification.


Asunto(s)
Encéfalo , Células Madre , Ratones , Animales , Linaje de la Célula , Diferenciación Celular/fisiología , Neuronas Dopaminérgicas
8.
Cell Rep ; 42(6): 112652, 2023 06 27.
Artículo en Inglés | MEDLINE | ID: mdl-37335775

RESUMEN

Brain-derived transcriptomes are known to correlate with resting-state brain activity in humans. Whether this association holds in nonhuman primates remains uncertain. Here, we search for such molecular correlates by integrating 757 transcriptomes derived from 100 macaque cortical regions with resting-state activity in separate conspecifics. We observe that 150 noncoding genes explain variations in resting-state activity at a comparable level with protein-coding genes. In-depth analysis of these noncoding genes reveals that they are connected to the function of nonneuronal cells such as oligodendrocytes. Co-expression network analysis finds that the modules of noncoding genes are linked to both autism and schizophrenia risk genes. Moreover, genes associated with resting-state noncoding genes are highly enriched in human resting-state functional genes and memory-effect genes, and their links with resting-state functional magnetic resonance imaging (fMRI) signals are altered in the brains of patients with autism. Our results highlight the potential for noncoding RNAs to explain resting-state activity in the nonhuman primate brain.


Asunto(s)
Trastorno Autístico , Imagen por Resonancia Magnética , Animales , Humanos , Imagen por Resonancia Magnética/métodos , Encéfalo/fisiología , Primates/genética , Mapeo Encefálico/métodos , Red Nerviosa/fisiología
9.
Genes (Basel) ; 14(4)2023 04 14.
Artículo en Inglés | MEDLINE | ID: mdl-37107669

RESUMEN

Increasing evidence suggests that microRNAs' (miRNAs) abnormal expression is one of the main factors of chemotherapy resistance in various cancers. However, the role of miRNAs in lung adenocarcinoma (LUAD) resistance to cisplatin is still unclear. In this study, we analyzed a microarray dataset to investigate miRNAs related to cisplatin resistance in LUAD. The expression of miRNAs in LUAD tissues and cell lines was detected using real-time quantitative polymerase chain reaction (RT-qPCR). Special AT-Rich Sequence-Binding Protein 2 (SATB2) in LUAD cell lines was detected using RT-qPCR and Western blot. Cell proliferation was measured by CCK8 and colony formation assays, while cell cycle and apoptosis were measured by flow cytometry. A dual-luciferase reporter assay was performed to confirm that SATB2 is a target gene of microRNA-660 (miR-660). We showed that the expression of miR-660 was not only decreased in LUAD cells and tissues but also further decreased in the cisplatin-resistant A549 cell line. The overexpression of miR-660 increased cisplatin sensitivity in LUAD cells. In addition, we identified SATB2 as a direct target gene of miR-660. We also revealed that miR-660 increased cisplatin sensitivity in LUAD cells via targeting SATB2. In conclusion, miR-660/SATB2 axis is a key regulator of cisplatin resistance in LUAD.


Asunto(s)
Adenocarcinoma del Pulmón , Adenocarcinoma , Neoplasias Pulmonares , Proteínas de Unión a la Región de Fijación a la Matriz , MicroARNs , Humanos , Cisplatino/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/metabolismo , MicroARNs/metabolismo , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patología , Factores de Transcripción/genética , Proteínas de Unión a la Región de Fijación a la Matriz/genética
10.
Cell Stem Cell ; 30(4): 473-487.e9, 2023 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-36933556

RESUMEN

The cell lineages across developmental stages remain to be elucidated. Here, we developed single-cell split barcoding (SISBAR) that allows clonal tracking of single-cell transcriptomes across stages in an in vitro model of human ventral midbrain-hindbrain differentiation. We developed "potential-spective" and "origin-spective" analyses to investigate the cross-stage lineage relationships and mapped a multi-level clonal lineage landscape depicting the whole differentiation process. We uncovered many previously uncharacterized converging and diverging trajectories. Furthermore, we demonstrate that a transcriptome-defined cell type can arise from distinct lineages that leave molecular imprints on their progenies, and the multilineage fates of a progenitor cell-type represent the collective results of distinct rather than similar clonal fates of individual progenitors, each with distinct molecular signatures. Specifically, we uncovered a ventral midbrain progenitor cluster as the common clonal origin of midbrain dopaminergic (mDA) neurons, midbrain glutamatergic neurons, and vascular and leptomeningeal cells and identified a surface marker that can improve graft outcomes.


Asunto(s)
Mesencéfalo , Células Madre , Humanos , Diferenciación Celular/fisiología , Mesencéfalo/metabolismo , Neuronas/fisiología , Linaje de la Célula
11.
Brain ; 146(2): 612-628, 2023 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-36516880

RESUMEN

Perinatal hypoxic-ischaemic encephalopathy is the leading cause of neonatal death and permanent neurological deficits, while the basal ganglia is one of the major nuclei that is selectively and greatly affected in the brains of hypoxic-ischaemic encephalopathy patients, especially in severe cases. Human embryonic stem cell-derived neurons have shown great potential in different types of brain disorders in adults. However, it remains unknown whether and how grafted human embryonic stem cell-derived neurons can repair immature brains with hypoxic-ischaemic encephalopathy. Here, by administrating genetically labelled human embryonic stem cell-derived striatal neural progenitors into the ipsilateral striatum of hypoxic-ischaemic encephalopathy-injured mice, we found that the grafted cells gradually matured into GABA spiny projection neurons morphologically and electrophysiologically, and significantly rescued the area loss of hypoxic-ischaemic encephalopathy-injured brains. Intriguingly, using immunohistochemical staining combined with enhanced ascorbate peroxidase-based immunoelectron microscopy and rabies virus-mediated trans-synaptic tracing, we show that the grafts start to extend axonal projections to the endogenous target areas (globus pallidus externa, globus pallidus internus, substantia nigra), form synapses with host striatal, globus pallidus and nigra neurons, and receive extensive and stable synaptic inputs as early as 2 months post-transplantation. Importantly, we further demonstrated functional neural circuits re-established between the grafted neurons and host cortical, striatal and substantial nigra neurons at 3-6 months post-transplantation in the hypoxic-ischaemic encephalopathy-injured brain by optogenetics combined with electrophysiological recording. Finally, the transplanted striatal spiny projection neurons but not spinal GABA neurons restored the motor defects of hypoxic-ischaemic encephalopathy, which were reversed by clozapine-N-oxide-based inhibition of graft function. These findings demonstrate anatomical and functional reconstruction of the basal ganglia neural circuit including multiple loops by striatal spiny projection neurons in hypoxic-ischaemic encephalopathy-injured immature brains, which raises the possibility of such a cell replacement therapeutic strategy for hypoxic-ischaemic encephalopathy in neonates.


Asunto(s)
Hipoxia-Isquemia Encefálica , Femenino , Embarazo , Humanos , Ratones , Animales , Cuerpo Estriado/fisiología , Ganglios Basales , Neuronas/fisiología , Encéfalo
12.
iScience ; 25(10): 105172, 2022 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-36217550

RESUMEN

Oligodendrocyte spheroids (OL-spheroids) containing oligodendrocytes and neurons provide an accessible system to dissect demyelinating diseases and test therapeutic treatment. However, generation of human OL-spheroids is still technically challenging and time-consuming until now. Here, we presented evidence that overexpression of SOX10 and OLIG2 (SO) in human embryonic stem cells (hESCs)-derived ventral forebrain neural progenitors is sufficient to produce forebrain pre-oligodendrocytes (pre-OLs) and mature oligodendrocytes (OLs) within 20-40 days. More importantly, optimizing this procedure by overexpression of SO in ventral forebrain spheroids, we successfully generated OL-spheroids with pre-OLs, mature OLs, and neurons 40 days after OL-induction. We further demonstrated oligodendrocyte-neuron interactions and obvious axon myelination in OL-spheroids. Finally, over 30% cells developed into mature oligodendrocytes with forebrain identity and myelinate axons in mouse brain 3 months after transplantation. This study provides a strategy to generate forebrain OL-spheroids rapidly and efficiently which would facilitate development of new therapeutics for demyelinating disorders.

13.
PLoS One ; 17(9): e0273873, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36084059

RESUMEN

FastCloning, a reliable cloning technique for plasmid construction, is a widely used protocol in biomedical research laboratories. Only two-step molecular manipulations are required to add a gene (cDNA) of interest into the desired vector. However, parallel cloning of the gene into multiple vectors is still a labor-intensive operation, which requires a range of primers for different vectors in high-throughput cloning projects. The situation could even be worse if multiple fragments of DNA are required to be added into one plasmid. Here, we describe a high-throughput FastCloning (HTFC) method, a protocol for parallel cloning by adding an adaptor sequence into all vectors. The target gene and vectors were PCR amplified separately to obtain the insert product and linear vectors with 18-base overlapping at each end of the DNAs required for FastCloning. Furthermore, a method for generating polycistronic bacterial constructs based on the same strategy as that used for HTFC was developed. Thus, the HTFC technique is a simple, effective, reliable, and low-cost tool for parallel cloning.


Asunto(s)
Escherichia coli , Vectores Genéticos , Clonación Molecular , Escherichia coli/genética , Vectores Genéticos/genética , Plásmidos/genética , Reacción en Cadena de la Polimerasa/métodos
14.
J Clin Invest ; 132(14)2022 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-35700056

RESUMEN

Human pluripotent stem cell-based (hPSC-based) replacement therapy holds great promise for the treatment of Parkinson's disease (PD). However, the heterogeneity of hPSC-derived donor cells and the low yield of midbrain dopaminergic (mDA) neurons after transplantation hinder its broad clinical application. Here, we have characterized the single-cell molecular landscape during mDA neuron differentiation. We found that this process recapitulated the development of multiple but adjacent fetal brain regions including the ventral midbrain, the isthmus, and the ventral hindbrain, resulting in a heterogenous donor cell population. We reconstructed the differentiation trajectory of the mDA lineage and identified calsyntenin 2 (CLSTN2) and protein tyrosine phosphatase receptor type O (PTPRO) as specific surface markers of mDA progenitors, which were predictive of mDA neuron differentiation and could facilitate high enrichment of mDA neurons (up to 80%) following progenitor cell sorting and transplantation. Marker-sorted progenitors exhibited higher therapeutic potency in correcting motor deficits of PD mice. Different marker-sorted grafts had a strikingly consistent cellular composition, in which mDA neurons were enriched, while off-target neuron types were mostly depleted, suggesting stable graft outcomes. Our study provides a better understanding of cellular heterogeneity during mDA neuron differentiation and establishes a strategy to generate highly purified donor cells to achieve stable and predictable therapeutic outcomes, raising the prospect of hPSC-based PD cell replacement therapies.


Asunto(s)
Enfermedad de Parkinson , Animales , Antígenos de Diferenciación , Biomarcadores/metabolismo , Diferenciación Celular/fisiología , Tratamiento Basado en Trasplante de Células y Tejidos , Neuronas Dopaminérgicas/metabolismo , Humanos , Mesencéfalo/metabolismo , Ratones , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/terapia
15.
Elife ; 112022 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-35506651

RESUMEN

Brain organoids have been used to recapitulate the processes of brain development and related diseases. However, the lack of vasculatures, which regulate neurogenesis and brain disorders, limits the utility of brain organoids. In this study, we induced vessel and brain organoids, respectively, and then fused two types of organoids together to obtain vascularized brain organoids. The fused brain organoids were engrafted with robust vascular network-like structures and exhibited increased number of neural progenitors, in line with the possibility that vessels regulate neural development. Fusion organoids also contained functional blood-brain barrier-like structures, as well as microglial cells, a specific population of immune cells in the brain. The incorporated microglia responded actively to immune stimuli to the fused brain organoids and showed ability of engulfing synapses. Thus, the fusion organoids established in this study allow modeling interactions between the neuronal and non-neuronal components in vitro, particularly the vasculature and microglia niche.


Understanding how the organs form and how their cells behave is essential to finding the causes and treatment for developmental disorders, as well as understanding certain diseases. However, studying most organs in live animals or humans is technically difficult, expensive and invasive. To address this issue, scientists have developed models called 'organoids' that recapitulate the development of organs using stem cells in the lab. These models are easier to study and manipulate than the live organs. Brain organoids have been used to recapitulate brain formation as well as developmental, degenerative and psychiatric brain conditions such as microcephaly, autism and Alzheimer's disease. However, these brain organoids lack the vasculature (the network of blood vessels) that supplies a live brain with nutrients and regulates its development, and which has important roles in brain disorders. Partly due to this lack of blood vessels, brain organoids also do not develop a blood brain barrier, the structure that prevents certain contents of the blood, including pathogens, toxins and even certain drugs from entering the brain. These characteristics limit the utility of existing brain organoids. To overcome these limitations, Sun, Ju et al. developed brain organoids and blood vessel organoids independently, and then fused them together to obtain vascularized brain organoids. These fusion organoids developed a robust network of blood vessels that was well integrated with the brain cells, and produced more neural cell precursors than brain organoids that had not been fused. This result is consistent with the idea that blood vessels can regulate brain development. Analyzing the fusion organoids revealed that they contain structures similar to the blood-brain barrier, as well as microglial cells (immune cells specific to the brain). When exposed to lipopolysaccharide ­ a component of the cell wall of certain bacteria ­ these cells responded by initiating an immune response in the fusion organoids. Notably, the microglial cells were also able to engulf connections between brain cells, a process necessary for the brain to develop the correct structures and work normally. Sun, Ju et al. have developed a new organoid system that will be of broad interest to researchers studying interactions between the brain and the circulatory system. The development of brain-blood-barrier-like structures in the fusion organoids could also facilitate the development of drugs that can cross this barrier, making it easier to treat certain conditions that affect the brain. Refining this model to allow the fusion organoids to grow for longer times in the lab, and adding blood flow to the system will be the next steps to establish this system.


Asunto(s)
Encéfalo , Organoides , Barrera Hematoencefálica , Neurogénesis , Neuronas
16.
Front Cell Infect Microbiol ; 12: 1040248, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36683684

RESUMEN

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the seventh coronavirus (CoV) that has spread in humans and has become a global pandemic since late 2019. Efficient and accurate laboratory diagnostic methods are one of the crucial means to control the development of the current pandemic and to prevent potential future outbreaks. Although real-time reverse transcription-polymerase chain reaction (rRT-PCR) is the preferred laboratory method recommended by the World Health Organization (WHO) for diagnosing and screening SARS-CoV-2 infection, the versatile immunoassays still play an important role for pandemic control. They can be used not only as supplemental tools to identify cases missed by rRT-PCR, but also for first-line screening tests in areas with limited medical resources. Moreover, they are also indispensable tools for retrospective epidemiological surveys and the evaluation of the effectiveness of vaccination. In this review, we summarize the mainstream immunoassay methods for human coronaviruses (HCoVs) and address their benefits, limitations, and applications. Then, technical strategies based on bioinformatics and advanced biosensors were proposed to improve the performance of these methods. Finally, future suggestions and possibilities that can lead to higher sensitivity and specificity are provided for further research.


Asunto(s)
COVID-19 , Humanos , COVID-19/diagnóstico , SARS-CoV-2/genética , Estudios Retrospectivos , Inmunoensayo , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad
17.
Neurosci Bull ; 37(8): 1091-1106, 2021 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-33948885

RESUMEN

Genetic composition plays critical roles in the pathogenesis of autism spectrum disorder (ASD). Especially, inherited and de novo intronic variants are often seen in patients with ASD. However, the biological significance of intronic variants is difficult to address. Here, among a Chinese ASD cohort, we identified a recurrent inherited intronic variant in the CHD7 gene, which is specifically enriched in East Asian populations. CHD7 has been implicated in numerous developmental disorders including CHARGE syndrome and ASD. To investigate whether the ASD-associated CHD7 intronic variant affects neural development, we established human embryonic stem cells carrying this variant using CRISPR/Cas9 methods and found that the level of CHD7 mRNA significantly decreased compared to control. Upon differentiation towards the forebrain neuronal lineage, we found that neural cells carrying the CHD7 intronic variant exhibited developmental delay and maturity defects. Importantly, we found that TBR1, a gene also implicated in ASD, was significantly increased in neurons carrying the CHD7 intronic variant, suggesting the intrinsic relevance among ASD genes. Furthermore, the morphological defects found in neurons carrying CHD7 intronic mutations were rescued by knocking down TBR1, indicating that TBR1 may be responsible for the defects in CHD7-related disorders. Finally, the CHD7 intronic variant generated three abnormal forms of transcripts through alternative splicing, which all exhibited loss-of-function in functional assays. Our study provides crucial evidence supporting the notion that the intronic variant of CHD7 is potentially an autism susceptibility site, shedding new light on identifying the functions of intronic variants in genetic studies of autism.


Asunto(s)
Trastorno del Espectro Autista , Trastorno Autístico , Trastorno del Espectro Autista/genética , Trastorno Autístico/genética , Diferenciación Celular , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Humanos , Mutación/genética , Neuronas
18.
Environ Pollut ; 284: 117176, 2021 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-33901983

RESUMEN

Although crop residue return increases upland soil emissions of nitrous oxide (N2O), a potent greenhouse gas, the mechanisms responsible for the increase remain unclear. Here, we investigate N2O emission pathways, gross nitrogen (N)-cycling rates, and associated N-cycling gene abundances in an upland soil following the addition of various organic material under aerobic incubation using a combination of 15N tracing technique, acetylene (C2H2) inhibition, and real-time PCR (qPCR) methods. Increased total N2O emissions following organic material amendment was attributed to both increased nitrification-derived N2O emissions, following increased ammonia-oxidizing bacteria (AOB)-amoA abundance, and denitrification-derived N2O emissions, following increased nirS and decreased nosZ abundance. Increasing plant residue carbon (C)/N ratio decreased total N2O emissions by decreasing the contribution of denitrification to N2O emissions, potentially due to higher proportions of denitrified N emitted as N2O than nitrified N emitted as N2O. We further propose a novel conceptual framework for organic material input effects on denitrification-derived N2O emissions based on the decomposable characteristics of the added organic material. For slowly decomposing organic materials (e.g., plant residue) with insufficient available C, NO3--N immobilization surpassed denitrification, resulting in gradual decrease in denitrification-derived N2O emissions with an increase in mineralization of plant residue C losses. In contrast, available C provided by readily available C sources (e.g., glucose) seemed sufficient to support the co-occurrence of NO3--N immobilization and denitrification. Overall, for the first time, we offer a microbial process perspective of N2O emissions following organic material input. The findings could facilitate the improvement of process-orientated models of N2O emissions and the formulation of appropriate N2O mitigation strategies for crop residue-amended soils.


Asunto(s)
Desnitrificación , Suelo , Nitrificación , Óxido Nitroso/análisis , Microbiología del Suelo
19.
Nat Med ; 27(4): 632-639, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33649496

RESUMEN

Degeneration of dopamine (DA) neurons in the midbrain underlies the pathogenesis of Parkinson's disease (PD). Supplement of DA via L-DOPA alleviates motor symptoms but does not prevent the progressive loss of DA neurons. A large body of experimental studies, including those in nonhuman primates, demonstrates that transplantation of fetal mesencephalic tissues improves motor symptoms in animals, which culminated in open-label and double-blinded clinical trials of fetal tissue transplantation for PD1. Unfortunately, the outcomes are mixed, primarily due to the undefined and unstandardized donor tissues1,2. Generation of induced pluripotent stem cells enables standardized and autologous transplantation therapy for PD. However, its efficacy, especially in primates, remains unclear. Here we show that over a 2-year period without immunosuppression, PD monkeys receiving autologous, but not allogenic, transplantation exhibited recovery from motor and depressive signs. These behavioral improvements were accompanied by robust grafts with extensive DA neuron axon growth as well as strong DA activity in positron emission tomography (PET). Mathematical modeling reveals correlations between the number of surviving DA neurons with PET signal intensity and behavior recovery regardless autologous or allogeneic transplant, suggesting a predictive power of PET and motor behaviors for surviving DA neuron number.


Asunto(s)
Conducta Animal , Depresión/complicaciones , Trasplante de Tejido Fetal , Actividad Motora , Enfermedad de Parkinson/fisiopatología , Enfermedad de Parkinson/terapia , Animales , Dopamina/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Inflamación/patología , Modelos Lineales , Macaca mulatta , Masculino , Mesencéfalo/trasplante , Ratones , Enfermedad de Parkinson/complicaciones , Tomografía de Emisión de Positrones , Trasplante Autólogo , Trasplante Homólogo , Tirosina 3-Monooxigenasa/metabolismo
20.
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
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