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1.
Cell ; 174(3): 590-606.e21, 2018 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-29961574

RESUMO

Cerebral cortex size differs dramatically between reptiles, birds, and mammals, owing to developmental differences in neuron production. In mammals, signaling pathways regulating neurogenesis have been identified, but genetic differences behind their evolution across amniotes remain unknown. We show that direct neurogenesis from radial glia cells, with limited neuron production, dominates the avian, reptilian, and mammalian paleocortex, whereas in the evolutionarily recent mammalian neocortex, most neurogenesis is indirect via basal progenitors. Gain- and loss-of-function experiments in mouse, chick, and snake embryos and in human cerebral organoids demonstrate that high Slit/Robo and low Dll1 signaling, via Jag1 and Jag2, are necessary and sufficient to drive direct neurogenesis. Attenuating Robo signaling and enhancing Dll1 in snakes and birds recapitulates the formation of basal progenitors and promotes indirect neurogenesis. Our study identifies modulation in activity levels of conserved signaling pathways as a primary mechanism driving the expansion and increased complexity of the mammalian neocortex during amniote evolution.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/genética , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Córtex Cerebral/metabolismo , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1 , Proteína Jagged-2 , Mamíferos/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Neocórtex/fisiologia , Células-Tronco Neurais , Neurogênese/fisiologia , Neuroglia/fisiologia , Neurônios , Fator de Transcrição PAX6/metabolismo , Proteínas Repressoras , Transdução de Sinais , Serpentes/embriologia , Proteínas Roundabout
2.
Mol Cell ; 74(5): 951-965.e13, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31047794

RESUMO

RNA-binding proteins (RBPs) and long non-coding RNAs (lncRNAs) are key regulators of gene expression, but their joint functions in coordinating cell fate decisions are poorly understood. Here we show that the expression and activity of the RBP TDP-43 and the long isoform of the lncRNA Neat1, the scaffold of the nuclear compartment "paraspeckles," are reciprocal in pluripotent and differentiated cells because of their cross-regulation. In pluripotent cells, TDP-43 represses the formation of paraspeckles by enhancing the polyadenylated short isoform of Neat1. TDP-43 also promotes pluripotency by regulating alternative polyadenylation of transcripts encoding pluripotency factors, including Sox2, which partially protects its 3' UTR from miR-21-mediated degradation. Conversely, paraspeckles sequester TDP-43 and other RBPs from mRNAs and promote exit from pluripotency and embryonic patterning in the mouse. We demonstrate that cross-regulation between TDP-43 and Neat1 is essential for their efficient regulation of a broad network of genes and, therefore, of pluripotency and differentiation.


Assuntos
Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias Murinas/metabolismo , RNA Longo não Codificante/genética , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , MicroRNAs/genética , Células-Tronco Pluripotentes/metabolismo , Poliadenilação/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
3.
Blood ; 141(6): 645-658, 2023 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-36223592

RESUMO

The mechanisms of coordinated changes in proteome composition and their relevance for the differentiation of neutrophil granulocytes are not well studied. Here, we discover 2 novel human genetic defects in signal recognition particle receptor alpha (SRPRA) and SRP19, constituents of the mammalian cotranslational targeting machinery, and characterize their roles in neutrophil granulocyte differentiation. We systematically study the proteome of neutrophil granulocytes from patients with variants in the SRP genes, HAX1, and ELANE, and identify global as well as specific proteome aberrations. Using in vitro differentiation of human induced pluripotent stem cells and in vivo zebrafish models, we study the effects of SRP deficiency on neutrophil granulocyte development. In a heterologous cell-based inducible protein expression system, we validate the effects conferred by SRP dysfunction for selected proteins that we identified in our proteome screen. Thus, SRP-dependent protein processing, intracellular trafficking, and homeostasis are critically important for the differentiation of neutrophil granulocytes.


Assuntos
Células-Tronco Pluripotentes Induzidas , Proteoma , Animais , Humanos , Peixe-Zebra , Genética Humana , Mamíferos , Proteínas Adaptadoras de Transdução de Sinal
4.
Nucleic Acids Res ; 51(6): 2671-2690, 2023 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-36806742

RESUMO

The establishment of cellular identity is driven by transcriptional and epigenetic regulators of the chromatin proteome - the chromatome. Comprehensive analyses of the chromatome composition and dynamics can therefore greatly improve our understanding of gene regulatory mechanisms. Here, we developed an accurate mass spectrometry (MS)-based proteomic method called Chromatin Aggregation Capture (ChAC) followed by Data-Independent Acquisition (DIA) and analyzed chromatome reorganizations during major phases of pluripotency. This enabled us to generate a comprehensive atlas of proteomes, chromatomes, and chromatin affinities for the ground, formative and primed pluripotency states, and to pinpoint the specific binding and rearrangement of regulatory components. These comprehensive datasets combined with extensive analyses identified phase-specific factors like QSER1 and JADE1/2/3 and provide a detailed foundation for an in-depth understanding of mechanisms that govern the phased progression of pluripotency. The technical advances reported here can be readily applied to other models in development and disease.


Assuntos
Cromatina , Células-Tronco Embrionárias , Células-Tronco Pluripotentes , Proteômica , Cromatina/genética , Espectrometria de Massas/métodos , Proteoma/genética , Proteoma/metabolismo , Proteômica/métodos , Humanos , Animais , Camundongos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo
5.
Nucleic Acids Res ; 51(3): 1297-1316, 2023 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-36651277

RESUMO

The RNA-binding protein PURA has been implicated in the rare, monogenetic, neurodevelopmental disorder PURA Syndrome. PURA binds both DNA and RNA and has been associated with various cellular functions. Only little is known about its main cellular roles and the molecular pathways affected upon PURA depletion. Here, we show that PURA is predominantly located in the cytoplasm, where it binds to thousands of mRNAs. Many of these transcripts change abundance in response to PURA depletion. The encoded proteins suggest a role for PURA in immune responses, mitochondrial function, autophagy and processing (P)-body activity. Intriguingly, reduced PURA levels decrease the expression of the integral P-body components LSM14A and DDX6 and strongly affect P-body formation in human cells. Furthermore, PURA knockdown results in stabilization of P-body-enriched transcripts, whereas other mRNAs are not affected. Hence, reduced PURA levels, as reported in patients with PURA Syndrome, influence the formation and composition of this phase-separated RNA processing machinery. Our study proposes PURA Syndrome as a new model to study the tight connection between P-body-associated RNA regulation and neurodevelopmental disorders.


Assuntos
Proteínas de Ligação a RNA , Fatores de Transcrição , Humanos , Proteínas de Ligação a DNA/genética , Epilepsia , Corpos de Processamento , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo
6.
Int J Mol Sci ; 25(3)2024 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-38338800

RESUMO

Degenerative retinal diseases associated with photoreceptor loss are a leading cause of visual impairment worldwide, with limited treatment options. Phenotypic profiling coupled with medicinal chemistry were used to develop a small molecule with proliferative effects on retinal stem/progenitor cells, as assessed in vitro in a neurosphere assay and in vivo by measuring Msx1-positive ciliary body cell proliferation. The compound was identified as having kinase inhibitory activity and was subjected to cellular pathway analysis in non-retinal human primary cell systems. When tested in a disease-relevant murine model of adult retinal degeneration (MNU-induced retinal degeneration), we observed that four repeat intravitreal injections of the compound improved the thickness of the outer nuclear layer along with the regeneration of the visual function, as measured with ERG, visual acuity, and contrast sensitivity tests. This serves as a proof of concept for the use of a small molecule to promote endogenous regeneration in the eye.


Assuntos
Degeneração Retiniana , Humanos , Camundongos , Animais , Degeneração Retiniana/metabolismo , Metilnitrosoureia , Retina/metabolismo , Células Fotorreceptoras , Regeneração , Modelos Animais de Doenças , Mamíferos
7.
Angew Chem Int Ed Engl ; : e202416082, 2024 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-39315681

RESUMO

The master regulator transcription factor MYC is implicated in numerous human cancers, and its targeting is a long-standing challenge in drug development. MYC is a typical 'undruggable' target, with no binding pockets on its DNA binding domain and extensive intrinsically disordered regions. Rather than trying to target MYC directly with classical modalities, here we engineer synthetic miniproteins that can bind to MYC's target DNA, the enhancer box (E-Box), and potently inhibit MYC-driven transcription. We crafted the miniproteins via structure-based design and a combination of solid phase peptide synthesis and site-specific crosslinking. Our lead variant, DuoMYC, binds to E-Box DNA with high affinity (KD ~ 0.1 µM) and is able to enter cells and inhibit MYC-driven transcription with submicromolar potency (IC50 = 464 nM) as shown by reporter gene assay and confirmed by RNA sequencing. Notably, DuoMYC surpasses the efficacy of several other recently developed MYC inhibitors. Our results highlight the potential of engineered synthetic protein therapeutics for addressing challenging intracellular targets.

8.
J Cell Sci ; 132(5)2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30745340

RESUMO

The GGGGCC (G4C2) repeat expansion mutation in the C9ORF72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Transcription of the repeat and formation of nuclear RNA foci, which sequester specific RNA-binding proteins, is one of the possible pathological mechanisms. Here, we show that (G4C2) n repeat RNA predominantly associates with essential paraspeckle proteins SFPQ, NONO, RBM14, FUS and hnRNPH and colocalizes with known paraspeckle-associated RNA hLinc-p21. As formation of paraspeckles in motor neurons has been associated with early phases of ALS, we investigated the extent of similarity between paraspeckles and (G4C2) n RNA foci. Overexpression of (G4C2)72 RNA results in their increased number and colocalization with SFPQ-stained nuclear bodies. These paraspeckle-like (G4C2)72 RNA foci form independently of the known paraspeckle scaffold, the long non-coding RNA NEAT1 Moreover, the knockdown of SFPQ protein in C9ORF72 expansion mutation-positive fibroblasts significantly reduces the number of (G4C2) n RNA foci. In conclusion, (G4C2) n RNA foci have characteristics of paraspeckles, which suggests that both RNA foci and paraspeckles play roles in FTD and ALS, and implies approaches for regulation of their formation.


Assuntos
Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , Demência Frontotemporal/genética , Neurônios Motores/fisiologia , Complexos Multiproteicos/metabolismo , Mutação/genética , RNA Nuclear/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteína C9orf72/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Demência Frontotemporal/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Espaço Intranuclear , Camundongos , Fator de Processamento Associado a PTB/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Nuclear/genética , Proteína FUS de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ratos
9.
BMC Biol ; 18(1): 42, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321486

RESUMO

BACKGROUND: Many long noncoding RNAs (lncRNAs) have been implicated in general and cell type-specific molecular regulation. Here, we asked what underlies the fundamental basis for the seemingly random appearance of nuclear lncRNA condensates in cells, and we sought compounds that can promote the disintegration of lncRNA condensates in vivo. RESULTS: As a basis for comparing lncRNAs and cellular properties among different cell types, we screened lncRNAs in human pluripotent stem cells (hPSCs) that were differentiated to an atlas of cell lineages. We found that paraspeckles, which form by aggregation of the lncRNA NEAT1, are scaled by the size of the nucleus, and that small DNA-binding molecules promote the disintegration of paraspeckles and other lncRNA condensates. Furthermore, we found that paraspeckles regulate the differentiation of hPSCs. CONCLUSIONS: Positive correlation between the size of the nucleus and the number of paraspeckles exist in numerous types of human cells. The tethering and structure of paraspeckles, as well as other lncRNAs, to the genome can be disrupted by small molecules that intercalate in DNA. The structure-function relationship of lncRNAs that regulates stem cell differentiation is likely to be determined by the dynamics of nucleus size and binding site accessibility.


Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes/fisiologia , RNA Longo não Codificante/metabolismo , Núcleo Celular/genética , Núcleo Celular/fisiologia , DNA/genética , DNA/fisiologia , Humanos
10.
Proc Natl Acad Sci U S A ; 114(45): E9579-E9588, 2017 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-29078328

RESUMO

To elucidate the molecular basis of BMP4-induced differentiation of human pluripotent stem cells (PSCs) toward progeny with trophectoderm characteristics, we produced transcriptome, epigenome H3K4me3, H3K27me3, and CpG methylation maps of trophoblast progenitors, purified using the surface marker APA. We combined them with the temporally resolved transcriptome of the preprogenitor phase and of single APA+ cells. This revealed a circuit of bivalent TFAP2A, TFAP2C, GATA2, and GATA3 transcription factors, coined collectively the "trophectoderm four" (TEtra), which are also present in human trophectoderm in vivo. At the onset of differentiation, the TEtra factors occupy multiple sites in epigenetically inactive placental genes and in OCT4 Functional manipulation of GATA3 and TFAP2A indicated that they directly couple trophoblast-specific gene induction with suppression of pluripotency. In accordance, knocking down GATA3 in primate embryos resulted in a failure to form trophectoderm. The discovery of the TEtra circuit indicates how trophectoderm commitment is regulated in human embryogenesis.


Assuntos
Diferenciação Celular/fisiologia , Fator de Transcrição GATA2/metabolismo , Fator de Transcrição GATA3/metabolismo , Placenta/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fator de Transcrição AP-2/metabolismo , Animais , Proteína Morfogenética Óssea 4/metabolismo , Linhagem Celular , Desenvolvimento Embrionário/fisiologia , Células-Tronco Embrionárias/metabolismo , Feminino , Humanos , Macaca mulatta , Gravidez , Transcriptoma/fisiologia , Trofoblastos/metabolismo
11.
Glia ; 66(2): 413-427, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29119608

RESUMO

Astrocytes, the most abundant cells in the mammalian brain, perform key functions and are involved in several neurodegenerative diseases. The human immunodeficiency virus (HIV) can persist in astrocytes, contributing to the HIV burden and neurological dysfunctions in infected individuals. While a comprehensive approach to HIV cure must include the targeting of HIV-1 in astrocytes, dedicated tools for this purpose are still lacking. Here we report a novel Adeno-associated virus-based vector (AAV9P1) with a synthetic surface peptide for transduction of astrocytes. Analysis of AAV9P1 transduction efficiencies with single brain cell populations, including primary human brain cells, as well as human brain organoids demonstrated that AAV9P1 targeted terminally differentiated human astrocytes much more efficiently than neurons. We then investigated whether AAV9P1 can be used to deliver HIV-inhibitory genes to astrocytes. To this end we generated AAV9P1 vectors containing genes for HIV-1 proviral editing by CRISPR/Cas9. Latently HIV-1 infected astrocytes transduced with these vectors showed significantly diminished reactivation of proviruses, compared with untransduced cultures. Sequence analysis identified mutations/deletions in key HIV-1 transcriptional control regions. We conclude that AAV9P1 is a promising tool for gene delivery to astrocytes and may facilitate inactivation/destruction of persisting HIV-1 proviruses in astrocyte reservoirs.


Assuntos
Astrócitos/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Dependovirus/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Vetores Genéticos/administração & dosagem , HIV-1/fisiologia , Astrócitos/efeitos dos fármacos , Astrócitos/virologia , Linhagem Celular Transformada , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/efeitos dos fármacos , Prepúcio do Pênis/citologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Células HEK293 , HIV-1/efeitos dos fármacos , Humanos , Masculino
12.
Proc Natl Acad Sci U S A ; 110(9): 3405-10, 2013 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-23391730

RESUMO

A goal of regenerative medicine is to identify cardiovascular progenitors from human ES cells (hESCs) that can functionally integrate into the human heart. Previous studies to evaluate the developmental potential of candidate hESC-derived progenitors have delivered these cells into murine and porcine cardiac tissue, with inconclusive evidence regarding the capacity of these human cells to physiologically engraft in xenotransplantation assays. Further, the potential of hESC-derived cardiovascular lineage cells to functionally couple to human myocardium remains untested and unknown. Here, we have prospectively identified a population of hESC-derived ROR2(+)/CD13(+)/KDR(+)/PDGFRα(+) cells that give rise to cardiomyocytes, endothelial cells, and vascular smooth muscle cells in vitro at a clonal level. We observed rare clusters of ROR2(+) cells and diffuse expression of KDR and PDGFRα in first-trimester human fetal hearts. We then developed an in vivo transplantation model by transplanting second-trimester human fetal heart tissues s.c. into the ear pinna of a SCID mouse. ROR2(+)/CD13(+)/KDR(+)/PDGFRα(+) cells were delivered into these functioning fetal heart tissues: in contrast to traditional murine heart models for cell transplantation, we show structural and functional integration of hESC-derived cardiovascular progenitors into human heart.


Assuntos
Células-Tronco Embrionárias/citologia , Feto/citologia , Coração/embriologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/transplante , Transplante de Células-Tronco , Animais , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem da Célula , Separação Celular , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Endotélio Vascular/citologia , Feto/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mesoderma/citologia , Camundongos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Miocárdio/citologia , Miócitos Cardíacos/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Linha Primitiva/citologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Sobrevivência de Tecidos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
13.
Zoo Biol ; 35(4): 280-92, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27142508

RESUMO

With only three living individuals left on this planet, the northern white rhinoceros (Ceratotherium simum cottoni) could be considered doomed for extinction. It might still be possible, however, to rescue the (sub)species by combining novel stem cell and assisted reproductive technologies. To discuss the various practical options available to us, we convened a multidisciplinary meeting under the name "Conservation by Cellular Technologies." The outcome of this meeting and the proposed road map that, if successfully implemented, would ultimately lead to a self-sustaining population of an extremely endangered species are outlined here. The ideas discussed here, while centered on the northern white rhinoceros, are equally applicable, after proper adjustments, to other mammals on the brink of extinction. Through implementation of these ideas we hope to establish the foundation for reversal of some of the effects of what has been termed the sixth mass extinction event in the history of Earth, and the first anthropogenic one. Zoo Biol. 35:280-292, 2016. © 2016 The Authors. Zoo Biology published by Wiley Periodicals, Inc.


Assuntos
Conservação dos Recursos Naturais , Espécies em Perigo de Extinção , Perissodáctilos/fisiologia , Animais , Conservação dos Recursos Naturais/tendências , Extinção Biológica , Mamíferos , Especificidade da Espécie
14.
Proc Natl Acad Sci U S A ; 108(8): 3282-7, 2011 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-21300885

RESUMO

The promise of pluripotent stem cells as a research and therapeutic tool is partly undermined by the technical challenges of generating and maintaining these cells in culture. Human embryonic stem cells (hESCs) are exquisitely sensitive to culture conditions, and require constant signaling by growth factors and cell-cell and cell-matrix interactions to prevent apoptosis, senescence, and differentiation. Previous work from our laboratory demonstrated that overexpression of the prosurvival gene BCL2 in mouse embryonic stem cells overrode the requirement of serum factors and feeder cells to maintain mESCs in culture. To determine whether this prosurvival gene could similarly protect hESCs, we generated hESC lines that constitutively or inducibly express BCL2. We find that BCL2 overexpression significantly decreases dissociation-induced apoptosis, resulting in enhanced colony formation from sorted single cells, and enhanced embryoid body formation. In addition, BCL2-hESCs exhibit normal growth in the absence of serum, but require basic fibroblast growth factor to remain undifferentiated. Furthermore, they maintain their pluripotency markers, form teratomas in vivo, and differentiate into all three germ layers. Our data suggest that the BCL2 signaling pathway plays an important role in inhibiting hESC apoptosis, such that its overexpression in hESCs offers both a survival benefit in conditions of stress by resisting apoptosis and obviates the requirement for serum or a feeder layer for maintenance.


Assuntos
Células-Tronco Embrionárias/citologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular/genética , Células-Tronco Embrionárias/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Genes bcl-2/fisiologia , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Soro , Estresse Fisiológico
15.
Neuron ; 112(7): 1117-1132.e9, 2024 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-38266647

RESUMO

Mitochondria account for essential cellular pathways, from ATP production to nucleotide metabolism, and their deficits lead to neurological disorders and contribute to the onset of age-related diseases. Direct neuronal reprogramming aims at replacing neurons lost in such conditions, but very little is known about the impact of mitochondrial dysfunction on the direct reprogramming of human cells. Here, we explore the effects of mitochondrial dysfunction on the neuronal reprogramming of induced pluripotent stem cell (iPSC)-derived astrocytes carrying mutations in the NDUFS4 gene, important for Complex I and associated with Leigh syndrome. This led to the identification of the unfolded protein response as a major hurdle in the direct neuronal conversion of not only astrocytes and fibroblasts from patients but also control human astrocytes and fibroblasts. Its transient inhibition potently improves reprogramming by influencing the mitochondria-endoplasmic-reticulum-stress-mediated pathways. Taken together, disease modeling using patient cells unraveled novel general hurdles and ways to overcome these in human astrocyte-to-neuron reprogramming.


Assuntos
Células-Tronco Pluripotentes Induzidas , Doenças Mitocondriais , Humanos , Neurônios/fisiologia , Mitocôndrias/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Resposta a Proteínas não Dobradas , Astrócitos/metabolismo , Doenças Mitocondriais/metabolismo , Reprogramação Celular , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo
16.
iScience ; 26(11): 108205, 2023 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-38026193

RESUMO

In this study, we interrogate molecular mechanisms underlying the specification of lung progenitors from human pluripotent stem cells (hPSCs). We employ single-cell RNA-sequencing with high temporal precision, alongside an optimized differentiation protocol, to elucidate the transcriptional hierarchy of lung specification to chart the associated single-cell trajectories. Our findings indicate that Sonic hedgehog, TGF-ß, and Notch activation are essential within an ISL1/NKX2-1 trajectory, leading to the emergence of lung progenitors during the foregut endoderm phase. Additionally, the induction of HHEX delineates an alternate trajectory at the early definitive endoderm stage, preceding the lung pathway and giving rise to a significant hepatoblast population. Intriguingly, neither KDR+ nor mesendoderm progenitors manifest as intermediate stages in the lung and hepatic lineage development. Our multistep model offers insights into lung organogenesis and provides a foundation for in-depth study of early human lung development and modeling using hPSCs.

17.
Stem Cell Reports ; 17(4): 711-714, 2022 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-35334219

RESUMO

The manipulation of human leukocyte antigens (HLAs) and immune modulatory factors in "universal" human pluripotent stem cells (PSCs) holds promise for immunological tolerance without HLA matching. This paradigm raises concerns should "universal" grafts become virally infected. Furthermore, immunological manipulation might functionally impair certain progeny, such as hematopoietic stem cells. We discuss the risks and benefits of hypoimmunogenic PSCs, and the need to further advance HLA matching and autologous strategies.


Assuntos
Pandemias , Células-Tronco Pluripotentes , Antígenos HLA , Humanos , Transplante de Células-Tronco/efeitos adversos
18.
iScience ; 25(11): 105414, 2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36388963

RESUMO

Less than 80 Sumatran rhinos (SR, Dicerorhinus sumatrensis) are left on earth. Habitat loss and limited breeding possibilities are the greatest threats to the species and lead to a continuous population decline. To stop the erosion of genetic diversity, reintroduction of genetic material is indispensable. However, as the propagation rate of captive breeding is far too low, innovative technologies have to be developed. Induced pluripotent stem cells (iPSCs) are a powerful tool to fight extinction. They give rise to each cell within the body including gametes and provide a unique modality to preserve genetic material across time. Additionally, they enable studying species-specific developmental processes. Here, we generate iPSCs from the last male Malaysian SR Kertam, who died in 2019, and characterize them comprehensively. Differentiation in cells of the three germ layers and cerebral organoids demonstrate their high quality and great potential for supporting the rescue of this critically endangered species.

19.
Nat Cell Biol ; 24(11): 1666-1676, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36344775

RESUMO

Despite their fundamental role in assessing (patho)physiological cell states, conventional gene reporters can follow gene expression but leave scars on the proteins or substantially alter the mature messenger RNA. Multi-time-point measurements of non-coding RNAs are currently impossible without modifying their nucleotide sequence, which can alter their native function, half-life and localization. Thus, we developed the intron-encoded scarless programmable extranuclear cistronic transcript (INSPECT) as a minimally invasive transcriptional reporter embedded within an intron of a gene of interest. Post-transcriptional excision of INSPECT results in the mature endogenous RNA without sequence alterations and an additional engineered transcript that leaves the nucleus by hijacking the nuclear export machinery for subsequent translation into a reporter or effector protein. We showcase its use in monitoring interleukin-2 (IL2) after T cell activation and tracking the transcriptional dynamics of the long non-coding RNA (lncRNA) NEAT1 during CRISPR interference-mediated perturbation. INSPECT is a method for monitoring gene transcription without altering the mature lncRNA or messenger RNA of the target of interest.


Assuntos
RNA Longo não Codificante , Íntrons/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequência de Bases
20.
Sci Rep ; 12(1): 3100, 2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35260583

RESUMO

The northern white rhinoceros (NWR) is probably the earth's most endangered mammal. To rescue the functionally extinct species, we aim to employ induced pluripotent stem cells (iPSCs) to generate gametes and subsequently embryos in vitro. To elucidate the regulation of pluripotency and differentiation of NWR PSCs, we generated iPSCs from a deceased NWR female using episomal reprogramming, and observed surprising similarities to human PSCs. NWR iPSCs exhibit a broad differentiation potency into the three germ layers and trophoblast, and acquire a naïve-like state of pluripotency, which is pivotal to differentiate PSCs into primordial germ cells (PGCs). Naïve culturing conditions induced a similar expression profile of pluripotency related genes in NWR iPSCs and human ESCs. Furthermore, naïve-like NWR iPSCs displayed increased expression of naïve and PGC marker genes, and a higher integration propensity into developing mouse embryos. As the conversion process was aided by ectopic BCL2 expression, and we observed integration of reprogramming factors, the NWR iPSCs presented here are unsuitable for gamete production. However, the gained insights into the developmental potential of both primed and naïve-like NWR iPSCs are fundamental for in future PGC-specification in order to rescue the species from extinction using cryopreserved somatic cells.


Assuntos
Células-Tronco Pluripotentes Induzidas , Animais , Diferenciação Celular/genética , Feminino , Células Germinativas/metabolismo , Camadas Germinativas , Camundongos , Perissodáctilos/genética
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