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1.
Stem Cell Rev Rep ; 2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34476741

RESUMO

Metabolic rewiring and mitochondrial dynamics remodelling are hallmarks of cell reprogramming, but the roles of the reprogramming factors in these changes are not fully understood. Here we show that c-MYC induces biosynthesis of fatty acids and increases the rate of pentose phosphate pathway. Time-course profiling of fatty acids and complex lipids during cell reprogramming using lipidomics revealed a profound remodelling of the lipid content, as well as the saturation and length of their acyl chains, in a c-MYC-dependent manner. Pluripotent cells displayed abundant cardiolipins and scarce phosphatidylcholines, with a prevalence of monounsaturated acyl chains. Cells undergoing cell reprogramming showed an increase in mitochondrial membrane potential that paralleled that of mitochondrial-specific cardiolipins. We conclude that c-MYC controls the rewiring of somatic cell metabolism early in cell reprogramming by orchestrating cell proliferation, synthesis of macromolecular components and lipid remodelling, all necessary processes for a successful phenotypic transition to pluripotency. c-MYC promotes anabolic metabolism, mitochondrial fitness and lipid remodelling early in cell reprogramming. A high rate of aerobic glycolysis is crucial to provide intermediaries for biosynthetic pathways. To ensure the availability of nucleotides, amino acids and lipids for cell proliferation, cells must provide with a constant flux of the elemental building blocks for macromolecule assembly and fulfil the anabolic demands to reach the critical cellular mass levels to satisfactorily undergo cell division. A high rate of aerobic glycolysis is induced by c-MYC, increasing the amounts of intracellular Glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and glyceraldehyde-3-phosphate (GA3P), which can all enter pentose phosphate pathway (PPP) to produce Ribose-5-Phosphate (R5P) and NADPH, which are necessary for the biosynthesis of biomolecules such as proteins, nucleic acids, or lipids. C-MYC-dependent activation of glucose-6-phosphate dehydrogenase (G6PD) may play a critical role in the shunting of G6P to PPP and generation of NADPH. High glycolytic flux increases the amounts of dihydroxyacetone phosphate (DHAP), which is crucial for biosynthesis of phospholipids and triacylglycerols, and pyruvate (Pyr), which can be converted to citrate (Cit) in the mitochondria and enter the biosynthesis of fatty acids (FA). During cell reprogramming, c-MYC-dependent lipid remodelling leads to Polyunsaturated Fatty Acid (PUFA) downregulation and Monounsaturated Fatty Acid (MUFA) upregulation, which may play critical roles in cytoarchitectural remodelling of cell membrane or non-canonical autophagy, respectively. Cardiolipin (pink dots) rise early in cell reprogramming correlates with an increase in mitochondrial fitness, suggesting that c-MYC may restore proper levels of cardiolipins and antioxidant proteins, such as UCP2, to guarantee an optimal mitochondrial function while upholding ROS levels, reinforcing the idea of cell rejuvenation early in cell reprogramming.

3.
Nat Commun ; 12(1): 3330, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099664

RESUMO

Human pluripotent stem cell (hPSC)-derived pancreatic ß cells are an attractive cell source for treating diabetes. However, current derivation methods remain inefficient, heterogeneous, and cell line dependent. To address these issues, we first devised a strategy to efficiently cluster hPSC-derived pancreatic progenitors into 3D structures. Through a systematic study, we discovered 10 chemicals that not only retain the pancreatic progenitors in 3D clusters but also enhance their potentiality towards NKX6.1+/INS+ ß cells. We further systematically screened signaling pathway modulators in the three steps from pancreatic progenitors toward ß cells. The implementation of all these strategies and chemical combinations resulted in generating ß cells from different sources of hPSCs with high efficiency. The derived ß cells are functional and can reverse hyperglycemia in mice within two weeks. Our protocol provides a robust platform for studying human ß cells and developing hPSC-derived ß cells for cell replacement therapy.


Assuntos
Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/metabolismo , Pâncreas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Terapia Baseada em Transplante de Células e Tecidos , Diabetes Mellitus/metabolismo , Diabetes Mellitus Experimental , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transdução de Sinais
5.
Nat Commun ; 12(1): 3094, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035273

RESUMO

Short-term, systemic expression of the Yamanaka reprogramming factors (Oct-3/4, Sox2, Klf4 and c-Myc [OSKM]) has been shown to rejuvenate aging cells and promote tissue regeneration in vivo. However, the mechanisms by which OSKM promotes tissue regeneration are unknown. In this work, we focus on a specific tissue and demonstrate that local expression of OSKM, specifically in myofibers, induces the activation of muscle stem cells or satellite cells (SCs), which accelerates muscle regeneration in young mice. In contrast, expressing OSKM directly in SCs does not improve muscle regeneration. Mechanistically, expressing OSKM in myofibers regulates the expression of genes important for the SC microenvironment, including upregulation of p21, which in turn downregulates Wnt4. This is critical because Wnt4 is secreted by myofibers to maintain SC quiescence. Thus, short-term induction of the Yamanaka factors in myofibers may promote tissue regeneration by modifying the stem cell niche.


Assuntos
Diferenciação Celular/genética , Reprogramação Celular/genética , Miofibrilas/metabolismo , Regeneração/genética , Células Satélites de Músculo Esquelético/metabolismo , Nicho de Células-Tronco , Animais , Células Cultivadas , Feminino , Expressão Gênica , Fatores de Transcrição Kruppel-Like/genética , Camundongos Transgênicos , Miofibrilas/fisiologia , Fator 3 de Transcrição de Octâmero/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/genética , Células Satélites de Músculo Esquelético/citologia , Proteína Wnt4/genética
6.
Cell ; 184(8): 2020-2032.e14, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33861963

RESUMO

Interspecies chimera formation with human pluripotent stem cells (hPSCs) represents a necessary alternative to evaluate hPSC pluripotency in vivo and might constitute a promising strategy for various regenerative medicine applications, including the generation of organs and tissues for transplantation. Studies using mouse and pig embryos suggest that hPSCs do not robustly contribute to chimera formation in species evolutionarily distant to humans. We studied the chimeric competency of human extended pluripotent stem cells (hEPSCs) in cynomolgus monkey (Macaca fascicularis) embryos cultured ex vivo. We demonstrate that hEPSCs survived, proliferated, and generated several peri- and early post-implantation cell lineages inside monkey embryos. We also uncovered signaling events underlying interspecific crosstalk that may help shape the unique developmental trajectories of human and monkey cells within chimeric embryos. These results may help to better understand early human development and primate evolution and develop strategies to improve human chimerism in evolutionarily distant species.

7.
Med (N Y) ; 2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33821249

RESUMO

Background: Strategies for monitoring the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are crucial for combating the pandemic. Detection and mutation surveillance of SARS-CoV-2 and other respiratory viruses require separate and complex workflows that rely on highly-specialized facilities, personnel, and reagents. To date, no method can rapidly diagnose multiple viral infections and determine variants in a high-throughput manner. Methods: We describe a method for multiplex isothermal amplification-based sequencing and real-time analysis of multiple viral genomes, termed NIRVANA. It can simultaneously detect SARS-CoV-2, influenza A, human adenovirus, and human coronavirus, and monitor mutations for up to 96 samples in real-time. Findings: NIRVANA showed high sensitivity and specificity for SARS-CoV-2 in 70 clinical samples with a detection limit of 20 viral RNA copies per µl of extracted nucleic acid. It also detected the influenza A co-infection in two samples. The variant analysis results of SARS-CoV-2 positive samples mirror the epidemiology of COVID-19. Additionally, NIRVANA could simultaneously detect SARS-CoV-2 and PMMoV (an omnipresent virus and water quality indicator) in municipal wastewater samples. Conclusions: NIRVANA provides high-confidence detection of both SARS-CoV-2 and other respiratory viruses and mutation surveillance of SARS-CoV-2 on the fly. We expect it to offer a promising solution for rapid field-deployable detection and mutational surveillance of pandemic viruses. Funding: M.L. is supported by KAUST Office of Sponsored Research (BAS/1/1080-01). This work is supported by KAUST Competitive Research Grant (URF/1/3412-01-01, M.L. and J.C.I.B.) and Universidad Catolica San Antonio de Murcia (J.C.I.B.). A.M.H. is supported by Saudi Ministry of Education (project 436).

8.
Stem Cell Res ; 53: 102319, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33894548

RESUMO

The advent of cellular reprogramming technology converting somatic cells into induced pluripotent stem cells (iPSCs) has revolutionized our understandings of neurodegenerative diseases that are otherwise hard to access and model. Multiple Sclerosis (MS) is a chronic demyelinating, inflammatory disease of central nervous system eventually causing neuronal death and accompanied disabilities. Here, we report the generation of several relapsing-remitting MS (RRMS) and primary progressive MS (PPMS) iPSC lines from MS patients along with their age matched healthy controls from peripheral blood mononuclear cells (PBMC). These patient specific iPSC lines displayed characteristic embryonic stem cell (ESC) morphology and exhibited pluripotency marker expression. Moreover, these MS iPSC lines were successfully differentiated into neural progenitor cells (NPC) after subjecting to neural induction. Furthermore, we identified the elevated expression of cellular senescence hallmarks in RRMS and PPMS neural progenitors unveiling a novel drug target avenue of MS pathophysiology. Thus, our study altogether offers both RRMS and PPMS iPSC cellular models as a good tool for better understanding of MS pathologies and drug testing.


Assuntos
Células-Tronco Pluripotentes Induzidas , Esclerose Múltipla Crônica Progressiva , Esclerose Múltipla Recidivante-Remitente , Esclerose Múltipla , Humanos , Leucócitos Mononucleares
9.
Cell Stem Cell ; 28(1): 5-7, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33417872

RESUMO

The regeneration potential of axons projecting from retinal ganglion cells (RGCs) is lost shortly after birth. In Nature, Lu et al. (2020) demonstrate that epigenetic reprogramming of RGCs by overexpression of Oct4, Sox2, and Klf4 leads to axon regeneration and restoration of vision in a glaucoma model and aged mice.


Assuntos
Axônios , Regeneração Nervosa , Animais , Epigênese Genética , Camundongos , Células Ganglionares da Retina , Visão Ocular
10.
Trends Mol Med ; 27(3): 203-206, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33487569

RESUMO

Genome editing holds great promise for treating a range of human genetic diseases. While emerging clustered regularly interspaced short-palindromic repeats (CRISPR) technologies allow editing of the nuclear genome, it is still not possible to precisely manipulate mitochondrial DNA (mtDNA). Here, we summarize past developments and recent advances in nuclear and mitochondrial genome editing.

11.
Dev Cell ; 56(3): 383-397.e8, 2021 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-33238152

RESUMO

Skin undergoes constant self-renewal, and its functional decline is a visible consequence of aging. Understanding human skin aging requires in-depth knowledge of the molecular and functional properties of various skin cell types. We performed single-cell RNA sequencing of human eyelid skin from healthy individuals across different ages and identified eleven canonical cell types, as well as six subpopulations of basal cells. Further analysis revealed progressive accumulation of photoaging-related changes and increased chronic inflammation with age. Transcriptional factors involved in the developmental process underwent early-onset decline during aging. Furthermore, inhibition of key transcription factors HES1 in fibroblasts and KLF6 in keratinocytes not only compromised cell proliferation, but also increased inflammation and cellular senescence during aging. Lastly, we found that genetic activation of HES1 or pharmacological treatment with quercetin alleviated cellular senescence of dermal fibroblasts. These findings provide a single-cell molecular framework of human skin aging, providing a rich resource for developing therapeutic strategies against aging-related skin disorders.

12.
Stem Cell Reports ; 16(1): 212-223, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33338433

RESUMO

Most of our current knowledge regarding early lineage specification and embryo-derived stem cells comes from studies in rodent models. However, key gaps remain in our understanding of these developmental processes from nonrodent species. Here, we report the detailed characterization of pig extraembryonic endoderm (pXEN) cells, which can be reliably and reproducibly generated from primitive endoderm (PrE) of blastocyst. Highly expandable pXEN cells express canonical PrE markers and transcriptionally resemble rodent XENs. The pXEN cells contribute both to extraembryonic tissues including visceral yolk sac as well as embryonic gut when injected into host blastocysts, and generate live offspring when used as a nuclear donor in somatic cell nuclear transfer (SCNT). The pXEN cell lines provide a novel model for studying lineage segregation, as well as a source for genome editing in livestock.

13.
Genes (Basel) ; 11(9)2020 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-32842577

RESUMO

The development of novel genome editing tools has unlocked new opportunities that were not previously possible in basic and biomedical research. During the last two decades, several new genome editing methods have been developed that can be customized to modify specific regions of the genome. However, in the past couple of years, many newer and more exciting genome editing techniques have been developed that are more efficient, precise, and easier to use. These genome editing tools have helped to improve our understanding of genetic disorders by modeling them in cells and animal models, in addition to correcting the disease-causing mutations. Among the genome editing tools, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has proven to be the most popular one due to its versatility and has been successfully used in a wide variety of laboratory animal models and plants. In this review, we summarize the customizable nucleases currently used for genome editing and their uses beyond the modification of genome. We also discuss the potential future applications of gene editing tools for both basic research and clinical purposes.


Assuntos
Sistemas CRISPR-Cas , Endonucleases/metabolismo , Edição de Genes/métodos , Engenharia Genética , Genoma , Animais , Endonucleases/genética , Humanos
15.
EMBO J ; 39(16): e104324, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32614092

RESUMO

Full differentiation potential along with self-renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already-established murine and human PSCs. Short exposure to miR-203 in PSCs (miPSCs) induces a transient expression of 2C markers that later results in expanded differentiation potency to multiple lineages, as well as improved efficiency in tetraploid complementation and human-mouse interspecies chimerism assays. Mechanistically, these effects are at least partially mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasure of DNA methylation. These data support the use of transient exposure to miR-203 as a versatile method to reset the epigenetic memory in PSCs, and improve their effectiveness in regenerative medicine.


Assuntos
Diferenciação Celular , Metilação de DNA , Epigênese Genética , Células-Tronco Pluripotentes Induzidas/metabolismo , MicroRNAs/metabolismo , Animais , Linhagem Celular , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , MicroRNAs/genética
16.
Exp Gerontol ; 133: 110870, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32045634

RESUMO

Somatic cells can be reprogrammed to pluripotency by either ectopic expression of defined factors or exposure to chemical cocktails. During reprogramming, somatic cells undergo dramatic changes in a wide range of cellular processes, such as metabolism, mitochondrial morphology and function, cell signaling pathways or immortalization. Regulation of these processes during cell reprograming lead to the acquisition of a pluripotent state, which enables indefinite propagation by symmetrical self-renewal without losing the ability of reprogrammed cells to differentiate into all cell types of the adult. In this review, recent data from different laboratories showing how these processes are controlled during the phenotypic transformation of a somatic cell into a pluripotent stem cell will be discussed.


Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas , Adulto , Diferenciação Celular , Humanos , Dinâmica Mitocondrial , Transdução de Sinais
17.
Cell ; 180(3): 585-600.e19, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32004457

RESUMO

Molecular mechanisms of ovarian aging and female age-related fertility decline remain unclear. We surveyed the single-cell transcriptomic landscape of ovaries from young and aged non-human primates (NHPs) and identified seven ovarian cell types with distinct gene-expression signatures, including oocyte and six types of ovarian somatic cells. In-depth dissection of gene-expression dynamics of oocytes revealed four subtypes at sequential and stepwise developmental stages. Further analysis of cell-type-specific aging-associated transcriptional changes uncovered the disturbance of antioxidant signaling specific to early-stage oocytes and granulosa cells, indicative of oxidative damage as a crucial factor in ovarian functional decline with age. Additionally, inactivated antioxidative pathways, increased reactive oxygen species, and apoptosis were observed in granulosa cells from aged women. This study provides a comprehensive understanding of the cell-type-specific mechanisms underlying primate ovarian aging at single-cell resolution, revealing new diagnostic biomarkers and potential therapeutic targets for age-related human ovarian disorders.


Assuntos
Envelhecimento/genética , Ovário/fisiologia , Análise de Célula Única/métodos , Transcriptoma , Idoso , Animais , Antioxidantes/metabolismo , Apoptose/fisiologia , Atlas como Assunto , Biomarcadores , Linhagem Celular Tumoral , Feminino , Células da Granulosa/metabolismo , Humanos , Macaca fascicularis , Oócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia
18.
Semin Cell Dev Biol ; 97: 3-15, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31028854

RESUMO

Epigenetic regulation of gene expression is fundamental in the maintenance of cellular identity and the regulation of cellular plasticity during tissue repair. In fact, epigenetic modulation is associated with the processes of cellular de-differentiation, proliferation, and re-differentiation that takes place during tissue regeneration. In here we explore the epigenetic events that coordinate tissue repair in lower vertebrates with high regenerative capacity, and in mammalian adult stem cells, which are responsible for the homeostasis maintenance of most of our tissues. Finally we summarize promising CRISPR-based editing technologies developed during the last years, which look as promising tools to not only study but also promote specific events during tissue regeneration.


Assuntos
Cromatina/metabolismo , Epigênese Genética/genética , Regeneração/efeitos dos fármacos , Humanos
19.
Science ; 366(6467)2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31672917

RESUMO

The transition from peri-implantation to gastrulation in mammals entails the specification and organization of the lineage progenitors into a body plan. Technical and ethical challenges have limited understanding of the cellular and molecular mechanisms that underlie this transition. We established a culture system that enabled the development of cynomolgus monkey embryos in vitro for up to 20 days. Cultured embryos underwent key primate developmental stages, including lineage segregation, bilaminar disc formation, amniotic and yolk sac cavitation, and primordial germ cell-like cell (PGCLC) differentiation. Single-cell RNA-sequencing analysis revealed development trajectories of primitive endoderm, trophectoderm, epiblast lineages, and PGCLCs. Analysis of single-cell chromatin accessibility identified transcription factors specifying each cell type. Our results reveal critical developmental events and complex molecular mechanisms underlying nonhuman primate embryogenesis in the early postimplantation period, with possible relevance to human development.


Assuntos
Blastocisto/fisiologia , Técnicas de Cultura Embrionária , Implantação do Embrião/fisiologia , Gastrulação/fisiologia , Animais , Blastocisto/metabolismo , Cromatina/metabolismo , Implantação do Embrião/genética , Células Germinativas Embrionárias , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Macaca fascicularis , Análise de Célula Única , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt
20.
Cell ; 179(3): 687-702.e18, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31626770

RESUMO

A single mouse blastomere from an embryo until the 8-cell stage can generate an entire blastocyst. Whether laboratory-cultured cells retain a similar generative capacity remains unknown. Starting from a single stem cell type, extended pluripotent stem (EPS) cells, we established a 3D differentiation system that enabled the generation of blastocyst-like structures (EPS-blastoids) through lineage segregation and self-organization. EPS-blastoids resembled blastocysts in morphology and cell-lineage allocation and recapitulated key morphogenetic events during preimplantation and early postimplantation development in vitro. Upon transfer, some EPS-blastoids underwent implantation, induced decidualization, and generated live, albeit disorganized, tissues in utero. Single-cell and bulk RNA-sequencing analysis revealed that EPS-blastoids contained all three blastocyst cell lineages and shared transcriptional similarity with natural blastocysts. We also provide proof of concept that EPS-blastoids can be generated from adult cells via cellular reprogramming. EPS-blastoids provide a unique platform for studying early embryogenesis and pave the way to creating viable synthetic embryos by using cultured cells.


Assuntos
Blastocisto/citologia , Linhagem da Célula , Implantação do Embrião , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Embrionárias Murinas/citologia , Criação de Embriões para Pesquisa/métodos , Animais , Blastocisto/metabolismo , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Técnicas de Reprogramação Celular/métodos , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Células-Tronco Embrionárias Murinas/metabolismo , Transcriptoma
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