RESUMO
The dorsal and ventral human telencephalons contain different neuronal subtypes, including glutamatergic, GABAergic, and cholinergic neurons, and how these neurons are generated during early development is not well understood. Using scRNA-seq and stringent validations, we reveal here a developmental roadmap for human telencephalic neurons. Both dorsal and ventral telencephalic radial glial cells (RGs) differentiate into neurons via dividing intermediate progenitor cells (IPCs_div) and early postmitotic neuroblasts (eNBs). The transcription factor ASCL1 plays a key role in promoting fate transition from RGs to IPCs_div in both regions. RGs from the regionalized neuroectoderm show heterogeneity, with restricted glutamatergic, GABAergic, and cholinergic differentiation potencies. During neurogenesis, IPCs_div gradually exit the cell cycle and branch into sister eNBs to generate distinct neuronal subtypes. Our findings highlight a general RGs-IPCs_div-eNBs developmental scheme for human telencephalic progenitors and support that the major neuronal fates of human telencephalon are predetermined during dorsoventral regionalization with neuronal diversity being further shaped during neurogenesis and neural circuit integration.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/genética , Neurônios/metabolismo , Telencéfalo/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ciclo Celular/genética , Diferenciação Celular , Colina/metabolismo , Proteína Duplacortina/genética , Proteína Duplacortina/metabolismo , Feto , Ontologia Genética , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Anotação de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/classificação , Neurônios/citologia , Fatores de Transcrição SOXC/genética , Fatores de Transcrição SOXC/metabolismo , Transdução de Sinais , Estatmina/genética , Estatmina/metabolismo , Telencéfalo/citologia , Telencéfalo/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Allogeneic immune rejection is a major barrier for the application of human pluripotent stem cells (hPSCs) in regenerative medicine. A broad spectrum of immune cells, including T cells, natural killer (NK) cells, and antigen-presenting cells, which either cause direct cell killing or constitute an immunogenic environment, are involved in allograft immune rejection. A strategy to protect donor cells from cytotoxicity while decreasing the secretion of inflammatory cytokines of lymphocytes is still lacking. Here, we engineered hPSCs with no surface expression of classical human leukocyte antigen (HLA) class I proteins via beta-2 microglobulin (B2M) knockout or biallelic knockin of HLA-G1 within the frame of endogenous B2M loci. Elimination of the surface expression of HLA class I proteins protected the engineered hPSCs from cytotoxicity mediated by T cells. However, this lack of surface expression also resulted in missing-self response and NK cell activation, which were largely compromised by expression of ß2m-HLA-G1 fusion proteins. We also proved that the engineered ß2m-HLA-G5 fusion proteins were soluble, secretable, and capable of safeguarding low immunogenic environments by lowering inflammatory cytokines secretion in allografts. Our current study reveals a novel strategy that may offer unique advantages to construct hypoimmunogenic hPSCs via the expression of membrane-bound and secreted ß2m-HLA-G fusion proteins. These engineered hPSCs are expected to serve as an unlimited cell source for generating universally compatible "off-the-shelf" cell grafts in the future.
Assuntos
Proteínas de Fusão de Membrana/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Técnicas de Cultura de Células , Humanos , Masculino , CamundongosRESUMO
The anteroposterior patterning of the central nervous system follows an activation/transformation model, which proposes that a prospective telencephalic fate will be activated by default during the neural induction stage, while this anterior fate could be transformed posteriorly according to caudalization morphogens. Although both extrinsic signals and intrinsic transcription factors have been implicated in dorsoventral (DV) specification of vertebrate telencephalon, the DV patterning model remains elusive. This is especially true in human considering its evolutionary trait and uniqueness of gene regulatory networks during neural induction. Here, we point to a model that human forebrain DV patterning also follows an activation/transformation paradigm. Human neuroectoderm (NE) will activate a forebrain dorsal fate automatically and this default anterior dorsal fate does not depend on Wnts activation or Pax6 expression. Forced expression of Pax6 in human NE hinders its ventralization even under sonic hedgehog (Shh) treatment, suggesting that the ventral fate is repressed by dorsal genes. Genetic manipulation of Nkx2.1, a key gene for forebrain ventral progenitors, shows that Nkx2.1 is neither necessary nor sufficient for Shh-driven ventralization. We thus propose that Shh represses dorsal genes of human NE and subsequently transforms the primitively activated dorsal fate ventrally in a repression release manner.
Assuntos
Padronização Corporal/fisiologia , Diferenciação Celular/fisiologia , Modelos Biológicos , Prosencéfalo/fisiologia , Transdução de Sinais/fisiologia , Linhagem Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Prosencéfalo/citologia , Prosencéfalo/embriologia , RNA Mensageiro/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
BACKGROUND: Ubiquitin-conjugating enzyme E2S (UBE2S), an E2 enzyme, is associated with the development of various tumors and exerts oncogenic activities. UBE2S is overexpressed in tumors, including hepatocellular carcinoma (HCC). However, the key molecular mechanisms of UBE2S in HCC still need additional research. The aim of this study was to explore the role of UBE2S in HCC. METHODS: The expression levels of UBE2S in HCC tissues and cells were detected by western blot analysis, quantitative real-time polymerase chain reaction analysis (qRT-PCR), and immunohistochemistry (IHC). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, wound healing assay, colony formation assay transwell assay, and animal models were used to detect the proliferation and migration ability of HCC cells. Western blot analysis, qRT-PCR, immunofluorescence, small-interfering RNA (siRNA), and plasmid transfection and coimmunoprecipitation (Co-IP) assays were performed to detect the interaction among UBE2S, von Hippel-Lindau (VHL), hypoxia-inducible factor 1-alpha (HIF-1α), Janus kinase-2 (JAK2), and signal transducer and activator of transcription 3 (STAT3). RESULTS: In this study, we found that high UBE2S expression was associated with poor prognosis in HCC patients. In addition, UBE2S expression was upregulated in HCC tissues and cell lines. Knockdown of UBE2S inhibited the proliferation and migration of HCC cells in vitro and in vivo by directly interacting with VHL to downregulate the HIF-1α and JAK2/STAT3 signaling pathways. Accordingly, overexpression of UBE2S significantly enhanced the proliferation and migration of HCC cells in vitro via VHL to upregulate HIF-1α and JAK2/STAT3 signaling pathways. Furthermore, we found that downregulation of UBE2S expression enhanced the sensitivity of HCC cells to sorafenib in vivo and in vitro. CONCLUSION: UBE2S enhances malignant properties via the VHL/HIF-1α and VHL/JAK2/STAT3 signaling pathways and reduces sensitivity to sorafenib in HCC. The findings of this study may open a new approach for HCC diagnosis and provide a potential option for the treatment of HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Sorafenibe/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismoRESUMO
Congenital hydrocephalus is a major neurological disorder with high rates of morbidity and mortality; however, the underlying cellular and molecular mechanisms remain largely unknown. Reproducible animal models mirroring both embryonic and postnatal hydrocephalus are also limited. Here, we describe a new mouse model of congenital hydrocephalus through knockout of ß-catenin in Nkx2.1-expressing regional neural progenitors. Progressive ventriculomegaly and an enlarged brain were consistently observed in knockout mice from embryonic day 12.5 through to adulthood. Transcriptome profiling revealed severe dysfunctions in progenitor maintenance in the ventricular zone and therefore in cilium biogenesis after ß-catenin knockout. Histological analyses also revealed an aberrant neuronal layout in both the ventral and dorsal telencephalon in hydrocephalic mice at both embryonic and postnatal stages. Thus, knockout of ß-catenin in regional neural progenitors leads to congenital hydrocephalus and provides a reproducible animal model for studying pathological changes and developing therapeutic interventions for this devastating disease.
Assuntos
Hidrocefalia , beta Catenina , Animais , Modelos Animais de Doenças , Hidrocefalia/genética , Camundongos , Camundongos Knockout , Neurônios , beta Catenina/genéticaRESUMO
Understanding the biological processes that determine the entry of three germ layers of human pluripotent stem cells (hPSCs) is a central question in developmental and stem cell biology. Here, we genetically engineered hPSCs with the germ layer reporter and inducible CRISPR/Cas9 knockout system, and a genome-scale screening was performed to define pathways restricting germ layer specification. Genes clustered in the key biological processes, including embryonic development, mRNA processing, metabolism, and epigenetic regulation, were centered in the governance of pluripotency and lineage development. Other than typical pluripotent transcription factors and signaling molecules, loss of function of mesendodermal specifiers resulted in advanced neuroectodermal differentiation, given their inter-germ layer antagonizing effect. Regarding the epigenetic superfamily, microRNAs enriched in hPSCs showed clear germ layer-targeting specificity. The cholesterol synthesis pathway maintained hPSCs via retardation of neuroectoderm specification. Thus, in this study, we identified a full landscape of genetic wiring and biological processes that control hPSC self-renewal and trilineage specification.
RESUMO
Medial ganglionic eminence (MGE)-like cells yielded from human pluripotent stem cells (hPSCs) hold great potentials for cell therapies of related neurological disorders. However, cues that orchestrate the maintenance versus differentiation of human MGE progenitors, and ways for large-scale expansion of these cells have not been investigated. Here, we report that WNT/CTNNB1 signaling plays an essential role in maintaining MGE-like cells derived from hPSCs. Ablation of CTNNB1 in MGE cells led to precocious cell-cycle exit and advanced neuronal differentiation. Activation of WNT signaling through genetic or chemical approach was sufficient to maintain MGE cells in an expandable manner with authentic neuronal differentiation potencies through activation of endogenous NOTCH signaling. Our findings reveal that WNT/NOTCH signaling cascade is a key player in governing the maintenance versus terminal differentiation of MGE progenitors in humans. Large-scale expansion of functional MGE progenitors for cell therapies can therefore be achieved by modifying WNT/NOTCH pathway.
Assuntos
Proliferação de Células/fisiologia , Eminência Mediana/citologia , Células-Tronco Pluripotentes/citologia , Receptores Notch/metabolismo , Via de Sinalização Wnt/fisiologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células/genética , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Humanos , Interneurônios/citologia , Interneurônios/metabolismo , Células-Tronco Pluripotentes/metabolismo , Receptores Notch/genética , Via de Sinalização Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
It is highly desirable to specify human developmental principles in an appropriate human model with advanced genetic tools. However, genetically engineering human cells with lineage-tracing systems has not been achieved. Here we introduce strategies to construct lineage-tracing systems in human embryonic stem cells (hESCs). The AAVS1 locus was suitable for the integration of the conditional reporter. The Cre-LoxP and Flp-FRT systems were highly sensitive, which may cause inaccurate lineage labeling in human cells. The recombination sensitivity and tracing fidelity could be finely tuned by modification of the LoxP recombination site. Moreover, tamoxifen-controllable CreERT2-LoxP and FlpERT2-FRT systems showed compelling advantages in tightly tracing human lineages temporally. In proof-of-principle experiments, we traced human PAX6+ neuroectoderm cells and revealed their full neural lineage differentiation potency both in vitro and in vivo. Devising and optimizing of lineage-tracing systems in hESCs will thus set up a solid foundation for human developmental studies.
Assuntos
Linhagem da Célula , Engenharia Genética , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Ectoderma/metabolismo , Loci Gênicos , Humanos , Integrases/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação/genética , Células-Tronco Neurais/metabolismo , Fator de Transcrição PAX6/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fatores de TempoRESUMO
Both environmental cues and intracellular bioenergetic states profoundly affect intracellular pH (pHi). How a cell responds to pHi changes to maintain bioenergetic homeostasis remains elusive. Here we show that Smad5, a well-characterized downstream component of bone morphogenetic protein (BMP) signaling responds to pHi changes. Cold, basic or hypertonic conditions increase pHi, which in turn dissociates protons from the charged amino acid clusters within the MH1 domain of Smad5, prompting its relocation from the nucleus to the cytoplasm. On the other hand, heat, acidic or hypotonic conditions decrease pHi, blocking the nuclear export of Smad5, and thus causing its nuclear accumulation. Active nucleocytoplasmic shuttling of Smad5 induced by environmental changes and pHi fluctuation is independent of BMP signaling, carboxyl terminus phosphorylation and Smad4. In addition, ablation of Smad5 causes chronic and irreversible dysregulation of cellular bioenergetic homeostasis and disrupted normal neural developmental processes as identified in a differentiation model of human pluripotent stem cells. Importantly, these metabolic and developmental deficits in Smad5-deficient cells could be rescued only by cytoplasmic Smad5. Cytoplasmic Smad5 physically interacts with hexokinase 1 and accelerates glycolysis. Together, our findings indicate that Smad5 acts as a pHi messenger and maintains the bioenergetic homeostasis of cells by regulating cytoplasmic metabolic machinery.
Assuntos
Metabolismo Energético , Homeostase , Espaço Intracelular/metabolismo , Proteína Smad5/metabolismo , Transporte Ativo do Núcleo Celular , Aminoácidos/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Respiração Celular , Regulação para Baixo , Técnicas de Inativação de Genes , Glicólise , Células HEK293 , Hexoquinase/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/ultraestrutura , Humanos , Concentração de Íons de Hidrogênio , Carioferinas/metabolismo , Mitocôndrias/metabolismo , Concentração Osmolar , Ligação Proteica , Domínios Proteicos , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais , Proteína Smad5/química , Proteína Smad5/deficiência , Relação Estrutura-Atividade , Temperatura , Proteína Exportina 1RESUMO
The transforming growth factor beta (TGFß) related signaling is one of the most important signaling pathways regulating early developmental events. Smad2 and Smad3 are structurally similar and it is mostly considered that they are equally important in mediating TGFß signals. Here, we show that Smad3 is an insensitive TGFß transducer as compared with Smad2. Smad3 preferentially localizes within the nucleus and is thus sequestered from membrane signaling. The ability of Smad3 in oligomerization with Smad4 upon agonist stimulation is also impaired given its unique linker region. Smad2 mediated TGFß signaling plays a crucial role in epiblast development and patterning of three germ layers. However, signaling unrelated nuclear localized Smad3 is dispensable for TGFß signaling-mediated epiblast specification, but important for early neural development, an event blocked by TGFß/Smad2 signaling. Both Smad2 and Smad3 bind to the conserved Smads binding element (SBE), but they show nonoverlapped target gene binding specificity and differential transcriptional activity. We conclude that Smad2 and Smad3 possess differential sensitivities in relaying TGFß signaling and have distinct roles in regulating early developmental events.
Assuntos
Proteína Smad2/fisiologia , Proteína Smad3/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Sequência de Bases , Padronização Corporal , Carcinogênese/metabolismo , Linhagem da Célula , Núcleo Celular/metabolismo , Sequência Conservada , Células HEK293 , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Ligação Proteica , Multimerização Proteica , Elementos de Resposta , Transdução de Sinais , Teratoma/metabolismo , Teratoma/patologiaRESUMO
Loss-of-function studies in human pluripotent stem cells (hPSCs) require efficient methodologies for lesion of genes of interest. Here, we introduce a donor-free paired gRNA-guided CRISPR/Cas9 knockout strategy (paired-KO) for efficient and rapid gene ablation in hPSCs. Through paired-KO, we succeeded in targeting all genes of interest with high biallelic targeting efficiencies. More importantly, during paired-KO, the cleaved DNA was repaired mostly through direct end joining without insertions/deletions (precise ligation), and thus makes the lesion product predictable. The paired-KO remained highly efficient for one-step targeting of multiple genes and was also efficient for targeting of microRNA, while for long non-coding RNA over 8 kb, cleavage of a short fragment of the core promoter region was sufficient to eradicate downstream gene transcription. This work suggests that the paired-KO strategy is a simple and robust system for loss-of-function studies for both coding and non-coding genes in hPSCs.
Assuntos
Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Células-Tronco Pluripotentes/metabolismo , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias , Marcação de Genes , Humanos , Células-Tronco Pluripotentes/citologia , RNA Guia de Cinetoplastídeos , RNA Longo não Codificante/genética , Proteína Smad3/deficiência , Proteína Smad3/genética , beta Catenina/deficiência , beta Catenina/genéticaRESUMO
Embryoid body (EB) formation and adherent culture (AD) paradigms are equivalently thought to be applicable for neural specification of human pluripotent stem cells. Here, we report that sonic hedgehog-induced ventral neuroprogenitors under EB conditions are fated to medial ganglionic eminence (MGE), while the AD cells mostly adopt a floor-plate (FP) fate. The EB-MGE later on differentiates into GABA and cholinergic neurons, while the AD-FP favors dopaminergic neuron specification. Distinct developmental, metabolic, and adhesion traits in AD and EB cells may potentially account for their differential patterning potency. Gene targeting combined with small-molecule screening experiments identified that concomitant inhibition of Wnts, STAT3, and p38 pathways (3i) could largely convert FP to MGE under AD conditions. Thus, differentiation paradigms and signaling regulators can be integrated together to specify distinct neuronal subtypes for studying and treating related neurological diseases, such as epilepsy, Alzheimer's disease, and Parkinson's disease.