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1.
Nat Cell Biol ; 21(12): 1518-1531, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31792383

RESUMO

The first lineage specification of pluripotent mouse epiblast segregates neuroectoderm (NE) from mesoderm and definitive endoderm (ME) by mechanisms that are not well understood. Here we demonstrate that the induction of ME gene programs critically relies on the T-box transcription factors Eomesodermin (also known as Eomes) and Brachyury, which concomitantly repress pluripotency and NE gene programs. Cells deficient in these T-box transcription factors retain pluripotency and differentiate to NE lineages despite the presence of ME-inducing signals transforming growth factor ß (TGF-ß)/Nodal and Wnt. Pluripotency and NE gene networks are additionally repressed by ME factors downstream of T-box factor induction, demonstrating a redundancy in program regulation to safeguard mutually exclusive lineage specification. Analyses of chromatin revealed that accessibility of ME enhancers depends on T-box factor binding, whereas NE enhancers are accessible and already activation primed at pluripotency. This asymmetry of the chromatin landscape thus explains the default differentiation of pluripotent cells to NE in the absence of ME induction that depends on activating and repressive functions of Eomes and Brachyury.


Assuntos
Cromatina/genética , Proteínas Fetais/genética , Camadas Germinativas/fisiologia , Células-Tronco Pluripotentes/fisiologia , Proteínas com Domínio T/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Separação Celular/métodos , Endoderma/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Placa Neural/fisiologia , Fator de Crescimento Transformador beta/genética
2.
Genes Dev ; 33(21-22): 1475-1490, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676735

RESUMO

A comprehensive understanding of mechanisms that underlie the development and function of human cells requires human cell models. For the pancreatic lineage, protocols have been developed to differentiate human pluripotent stem cells (hPSCs) into pancreatic endocrine and exocrine cells through intermediates resembling in vivo development. In recent years, this differentiation system has been employed to decipher mechanisms of pancreatic development, congenital defects of the pancreas, as well as genetic forms of diabetes and exocrine diseases. In this review, we summarize recent insights gained from studies of pancreatic hPSC models. We discuss how genome-scale analyses of the differentiation system have helped elucidate roles of chromatin state, transcription factors, and noncoding RNAs in pancreatic development and how the analysis of cells with disease-relevant mutations has provided insight into the molecular underpinnings of genetically determined diseases of the pancreas.


Assuntos
Modelos Biológicos , Pâncreas/citologia , Pâncreas/crescimento & desenvolvimento , Células-Tronco Pluripotentes/citologia , Diferenciação Celular , Estudo de Associação Genômica Ampla , Humanos , Pâncreas/patologia , Pancreatopatias/genética , Pancreatopatias/fisiopatologia , Células-Tronco Pluripotentes/fisiologia
3.
PLoS Comput Biol ; 15(11): e1007488, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31721764

RESUMO

Modeling cell differentiation from omics data is an essential problem in systems biology research. Although many algorithms have been established to analyze scRNA-seq data, approaches to infer the pseudo-time of cells or quantify their potency have not yet been satisfactorily solved. Here, we propose the Landscape of Differentiation Dynamics (LDD) method, which calculates cell potentials and constructs their differentiation landscape by a continuous birth-death process from scRNA-seq data. From the viewpoint of stochastic dynamics, we exploited the features of the differentiation process and quantified the differentiation landscape based on the source-sink diffusion process. In comparison with other scRNA-seq methods in seven benchmark datasets, we found that LDD could accurately and efficiently build the evolution tree of cells with pseudo-time, in particular quantifying their differentiation landscape in terms of potency. This study provides not only a computational tool to quantify cell potency or the Waddington potential landscape based on scRNA-seq data, but also novel insights to understand the cell differentiation process from a dynamic perspective.


Assuntos
Biologia Computacional/métodos , Análise de Sequência de RNA/métodos , Biologia de Sistemas/métodos , Algoritmos , Animais , Diferenciação Celular/fisiologia , Análise por Conglomerados , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Modelos Teóricos , Células-Tronco Pluripotentes/fisiologia , RNA/genética , Análise de Célula Única/métodos , Software
4.
Nat Protoc ; 14(12): 3303-3332, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31732721

RESUMO

Alveolar epithelial type II cells (AEC2s) are the facultative progenitors of lung alveoli and serve as the surfactant-producing cells of air-breathing organisms. Although primary human AEC2s are difficult to maintain stably in cell cultures, recent advances have facilitated the derivation of AEC2-like cells from human pluripotent stem cells (hPSCs) in vitro. Here, we provide a detailed protocol for the directed differentiation of hPSCs into self-renewing AEC2-like cells that can be maintained for up to 1 year in culture as epithelial-only spheres without the need for supporting mesenchymal feeder cells. The month-long protocol requires recapitulation of the sequence of milestones associated with in vivo development of the distal lung, beginning with differentiation of cells into anterior foregut endoderm, which is followed by their lineage specification into NKX2-1+ lung progenitors and then distal/alveolar differentiation to produce progeny that express transcripts and possess functional properties associated with AEC2s.


Assuntos
Células Epiteliais Alveolares/citologia , Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes/citologia , Células Epiteliais Alveolares/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Proliferação de Células , Autorrenovação Celular/genética , Autorrenovação Celular/fisiologia , Células Epiteliais/citologia , Células Alimentadoras , Humanos , Pulmão/citologia , Células-Tronco Pluripotentes/fisiologia
5.
Hum Genet ; 138(11-12): 1217-1225, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31606751

RESUMO

Pluripotent stem cell (PSC) cultures form an integral part of biomedical and medical research due to their capacity to rapidly proliferate and differentiate into hundreds of highly specialized cell types. This makes them a highly useful tool in exploring human physiology and disease. Genomic editing of PSC cultures is an essential method of attaining answers to basic physiological functions, developing in vitro models of human disease, and exploring potential therapeutic strategies and the identification of drug targets. Achieving reliable and efficient genomic editing is an important aspect of using large-scale PSC cultures. The CRISPR/Cas9 genomic editing tool has facilitated highly efficient gene knockout, gene correction, or gene modifications through the design and use of single-guide RNAs which are delivered to the target DNA via Cas9. CRISPR/Cas9 modification of PSCs has furthered the understanding of basic physiology and has been utilized to develop in vitro disease models, to test therapeutic strategies, and to facilitate regenerative or tissue repair approaches. In this review, we discuss the benefits of the CRISPR/Cas9 system in large-scale PSC cultures.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Técnicas de Inativação de Genes , Genômica/métodos , Células-Tronco Pluripotentes/fisiologia , Humanos , Células-Tronco Pluripotentes/citologia
6.
Elife ; 82019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31452509

RESUMO

Secreted Wnt proteins regulate development and adult tissue homeostasis by binding and activating cell-surface Frizzled receptors and co-receptors including LRP5/6. The hydrophobicity of Wnt proteins has complicated their purification and limited their use in basic research and as therapeutics. We describe modular tetravalent antibodies that can recruit Frizzled and LRP5/6 in a manner that phenocopies the activities of Wnts both in vitro and in vivo. The modular nature of these synthetic Frizzled and LRP5/6 Agonists, called FLAgs, enables tailored engineering of specificity for one, two or multiple members of the Frizzled family. We show that FLAgs underlie differentiation of pluripotent stem cells, sustain organoid growth, and activate stem cells in vivo. Activation of Wnt signaling circuits with tailored FLAgs will enable precise delineation of functional outcomes directed by distinct receptor combinations and could provide a new class of therapeutics to unlock the promise of regenerative medicine.


Assuntos
Anticorpos/metabolismo , Receptores Frizzled/agonistas , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Linhagem Celular , Humanos , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/agonistas , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/agonistas , Camundongos , Organoides/efeitos dos fármacos , Organoides/crescimento & desenvolvimento , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/fisiologia , Ligação Proteica
7.
Nat Genet ; 51(9): 1380-1388, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31427791

RESUMO

Chromatin architecture has been implicated in cell type-specific gene regulatory programs, yet how chromatin remodels during development remains to be fully elucidated. Here, by interrogating chromatin reorganization during human pluripotent stem cell (hPSC) differentiation, we discover a role for the primate-specific endogenous retrotransposon human endogenous retrovirus subfamily H (HERV-H) in creating topologically associating domains (TADs) in hPSCs. Deleting these HERV-H elements eliminates their corresponding TAD boundaries and reduces the transcription of upstream genes, while de novo insertion of HERV-H elements can introduce new TAD boundaries. The ability of HERV-H to create TAD boundaries depends on high transcription, as transcriptional repression of HERV-H elements prevents the formation of boundaries. This ability is not limited to hPSCs, as these actively transcribed HERV-H elements and their corresponding TAD boundaries also appear in pluripotent stem cells from other hominids but not in more distantly related species lacking HERV-H elements. Overall, our results provide direct evidence for retrotransposons in actively shaping cell type- and species-specific chromatin architecture.


Assuntos
Cromatina/genética , Retrovirus Endógenos/genética , Regulação da Expressão Gênica , Células-Tronco Pluripotentes/citologia , Elementos de Resposta , Retroelementos/genética , Transcrição Genética , Animais , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes/fisiologia , Primatas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
8.
Med Sci (Paris) ; 35(6-7): 549-555, 2019.
Artigo em Francês | MEDLINE | ID: mdl-31274085

RESUMO

The study of gut diseases is often limited by the access to human biological tissues and animal models that do not faithfully mimic the human pathologies. In this context, the development of intestinal organoids from human pluripotent stem cells is paving the way of gastrointestinal physiology and digestive disease study. In this review, we recall the embryonic development of the digestive tract and its translation to human pluripotent stem cell differentiation. We also present the different types of intestinal organoids that can be generated, as well as their applications in research.


Assuntos
Intestinos/citologia , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Gastroenteropatias/patologia , Gastroenteropatias/terapia , Trato Gastrointestinal/citologia , Trato Gastrointestinal/crescimento & desenvolvimento , Trato Gastrointestinal/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Intestinos/fisiologia , Organoides/fisiologia , Células-Tronco Pluripotentes/fisiologia , Regeneração/fisiologia , Técnicas de Cultura de Tecidos
9.
EMBO J ; 38(12)2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31088843

RESUMO

Human pluripotent stem cells (hPSCs) are being increasingly utilized worldwide in investigating human development, and modeling and discovering therapies for a wide range of diseases as well as a source for cellular therapy. Yet, since the first isolation of human embryonic stem cells (hESCs) 20 years ago, followed by the successful reprogramming of human-induced pluripotent stem cells (hiPSCs) 10 years later, various studies shed light on abnormalities that sometimes accumulate in these cells in vitro Whereas genetic aberrations are well documented, epigenetic alterations are not as thoroughly discussed. In this review, we highlight frequent epigenetic aberrations found in hPSCs, including alterations in DNA methylation patterns, parental imprinting, and X chromosome inactivation. We discuss the potential origins of these abnormalities in hESCs and hiPSCs, survey the different methods for detecting them, and elaborate on their potential consequences for the different utilities of hPSCs.


Assuntos
Epigênese Genética/fisiologia , Células-Tronco Pluripotentes/fisiologia , Diferenciação Celular/genética , Reprogramação Celular/genética , Metilação de DNA/fisiologia , Impressão Genômica/genética , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Inativação do Cromossomo X/fisiologia
10.
Ann Endocrinol (Paris) ; 80(2): 128-133, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30833018

RESUMO

In the modern world, type-2 diabetes mellitus has become a leading public healthcare problem, due to major risks of morbidity and mortality. Prevalence has increased significantly in recent decades. Treatment involves oral hypoglycemic agents or insulin replacement therapy. Development is ongoing for cell-based diabetes therapies using stem cells with the potential to differentiate into insulin-producing cells (IPCs): embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), and stem cells from adult pancreas, liver, central nervous system, bone marrow and adipose tissue. Successful induction of iPSCs, however, depends on the quantity and quality of available stem cells and the development of adapted protocols determining the environment of extrinsic factors and involvement of small molecules. Validating such new cell therapies must be founded on this experimental rationale.


Assuntos
Fatores Biológicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Animais , Fatores Biológicos/análise , Fatores Biológicos/isolamento & purificação , Técnicas de Cultura de Células , Reprogramação Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Pâncreas/citologia , Pâncreas/efeitos dos fármacos , Pâncreas/fisiologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Bibliotecas de Moléculas Pequenas/análise
11.
Nat Neurosci ; 22(4): 669-679, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30886407

RESUMO

Neural organoids have the potential to improve our understanding of human brain development and neurological disorders. However, it remains to be seen whether these tissues can model circuit formation with functional neuronal output. Here we have adapted air-liquid interface culture to cerebral organoids, leading to improved neuronal survival and axon outgrowth. The resulting thick axon tracts display various morphologies, including long-range projection within and away from the organoid, growth-cone turning, and decussation. Single-cell RNA sequencing reveals various cortical neuronal identities, and retrograde tracing demonstrates tract morphologies that match proper molecular identities. These cultures exhibit active neuronal networks, and subcortical projecting tracts can innervate mouse spinal cord explants and evoke contractions of adjacent muscle in a manner dependent on intact organoid-derived innervating tracts. Overall, these results reveal a remarkable self-organization of corticofugal and callosal tracts with a functional output, providing new opportunities to examine relevant aspects of human CNS development and disease.


Assuntos
Córtex Cerebral/crescimento & desenvolvimento , Neurônios/fisiologia , Organoides/crescimento & desenvolvimento , Técnicas de Cultura de Tecidos/métodos , Axônios/fisiologia , Sobrevivência Celular , Córtex Cerebral/citologia , Feminino , Humanos , Masculino , Vias Neurais/citologia , Vias Neurais/fisiologia , Neurônios/citologia , Organoides/citologia , Células-Tronco Pluripotentes/fisiologia
13.
J Orthop Res ; 37(6): 1246-1262, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30604468

RESUMO

Adult skeletal muscle can regenerate effectively after mild physical or chemical insult. Muscle trauma or disease can overwhelm this innate capacity for regeneration and result in heightened inflammation and fibrotic tissue deposition resulting in loss of structure and function. Recent studies have focused on biomaterial and stem cell-based therapies to promote skeletal muscle regeneration following injury and disease. Many stem cell populations besides satellite cells are implicated in muscle regeneration. These stem cells include but are not limited to mesenchymal stem cells, adipose-derived stem cells, hematopoietic stem cells, pericytes, fibroadipogenic progenitors, side population cells, and CD133+ stem cells. However, several challenges associated with their isolation, availability, delivery, survival, engraftment, and differentiation have been reported in recent studies. While acellular scaffolds offer a relatively safe and potentially off-the-shelf solution to cell-based therapies, they are often unable to stimulate host cell migration and activity to a level that would result in clinically meaningful regeneration of traumatized muscle. Combining stem cells and biomaterials may offer a viable therapeutic strategy that may overcome the limitations associated with these therapies when they are used in isolation. In this article, we review the stem cell populations that can stimulate muscle regeneration in vitro and in vivo. We also discuss the regenerative potential of combination therapies that utilize both stem cell and biomaterials for the treatment of skeletal muscle injury and disease. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1246-1262, 2019.


Assuntos
Materiais Biocompatíveis/uso terapêutico , Músculo Esquelético/fisiologia , Regeneração/fisiologia , Transplante de Células-Tronco , Animais , Células-Tronco Hematopoéticas/fisiologia , Humanos , Células-Tronco Mesenquimais/fisiologia , Pericitos/fisiologia , Células-Tronco Pluripotentes/fisiologia , Células Satélites de Músculo Esquelético/fisiologia
14.
SLAS Technol ; 24(1): 41-54, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29995450

RESUMO

Human pluripotent stem cells (hPSCs) are promising therapeutic tools for regenerative therapies and disease modeling. Differentiation of cultured hPSCs is influenced by both exogenous factors added to the cultures and endogenously secreted molecules. Optimization of protocols for the differentiation of hPSCs into different cell types is difficult because of the many variables that can influence cell fate. We present microfluidic devices designed to perform three- and four-factor, two-level full factorial experiments in parallel for investigating and directly optimizing hPSC differentiation. These devices feature diffusion-isolated, independent culture wells that allow for control of both exogenous and endogenous cellular signals and that allow for immunocytochemistry (ICC) and confocal microscopy in situ. These devices are fabricated by soft lithography in conjunction with 3D-printed molds and are operable with a single syringe pump, eliminating the need for specialized equipment or cleanroom facilities. Their utility was demonstrated by on-chip differentiation of hPSCs into the auditory neuron lineage. More broadly, these devices enable multiplexing for experimentation with any adherent cell type or even multiple cell types, allowing efficient investigation of the effects of medium conditions, pharmaceuticals, or other soluble reagents.


Assuntos
Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Microfluídica/instrumentação , Microfluídica/métodos , Células-Tronco Pluripotentes/fisiologia , Humanos , Imuno-Histoquímica , Microscopia Confocal , Células-Tronco Pluripotentes/citologia
15.
Exp Anim ; 68(1): 35-47, 2019 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-30089733

RESUMO

Stem cells are promising cell source for treatment of multiple diseases as well as myocardial infarction. Rabbit model has essentially used for cardiovascular diseases and regeneration but information on establishment of induced pluripotent stem cells (iPSCs) and differentiation potential is fairly limited. In addition, there is no report of cardiac differentiation from iPSCs in the rabbit model. In this study, we generated rabbit iPSCs by reprogramming rabbit fibroblasts using the 4 transcription factors (OCT3/4, SOX2, KLF4, and c-Myc). Three iPSC lines were established. The iPSCs from all cell lines expressed genes (OCT3/4, SOX2, KLF4 and NANOG) and proteins (alkaline phosphatase, OCT-3/4 and SSEA-4) essentially described for pluripotency (in vivo and in vitro differentiation). Furthermore, they also had ability to form embryoid body (EB) resulting in three-germ layer differentiation. However, ability of particular cell lines and cell numbers at seeding markedly influenced on EB formation and also their diameters. The cell density at 20,000 cells per EB was selected for cardiac differentiation. After plating, the EBs attached and cardiac-like beating areas were seen as soon as 11 days of culture. The differentiated cells expressed cardiac progenitor marker FLK1 (51 ± 1.48%) on day 5 and cardiac troponin-T protein (10.29 ± 1.37%) on day 14. Other cardiac marker genes (cardiac ryanodine receptors (RYR2), α-actinin and PECAM1) were also expressed. This study concluded that rabbit iPSCs remained their in vitro pluripotency with capability of differentiation into mature-phenotype cardiomyocytes. However, the efficiency of cardiac differentiation is still restricted.


Assuntos
Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Miócitos Cardíacos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Coelhos , Fosfatase Alcalina/fisiologia , Animais , Linhagem Celular , Fatores de Transcrição Kruppel-Like/fisiologia , Proteína Homeobox Nanog/fisiologia , Fator 3 de Transcrição de Octâmero/fisiologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Fatores de Transcrição SOXB1/fisiologia , Antígenos Embrionários Estágio-Específicos/fisiologia
16.
EMBO J ; 38(1)2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30257965

RESUMO

An intricate link is becoming apparent between metabolism and cellular identities. Here, we explore the basis for such a link in an in vitro model for early mouse embryonic development: from naïve pluripotency to the specification of primordial germ cells (PGCs). Using single-cell RNA-seq with statistical modelling and modulation of energy metabolism, we demonstrate a functional role for oxidative mitochondrial metabolism in naïve pluripotency. We link mitochondrial tricarboxylic acid cycle activity to IDH2-mediated production of alpha-ketoglutarate and through it, the activity of key epigenetic regulators. Accordingly, this metabolite has a role in the maintenance of naïve pluripotency as well as in PGC differentiation, likely through preserving a particular histone methylation status underlying the transient state of developmental competence for the PGC fate. We reveal a link between energy metabolism and epigenetic control of cell state transitions during a developmental trajectory towards germ cell specification, and establish a paradigm for stabilizing fleeting cellular states through metabolic modulation.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Células Germinativas/efeitos dos fármacos , Ácidos Cetoglutáricos/farmacologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Animais , Diferenciação Celular/genética , Células Cultivadas , Embrião de Mamíferos , Células-Tronco Embrionárias/fisiologia , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células Germinativas/fisiologia , Ácidos Cetoglutáricos/metabolismo , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Pluripotentes/fisiologia
17.
Curr Stem Cell Res Ther ; 14(1): 57-64, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30227822

RESUMO

Low back pain (LBP) is one of the world's most common musculoskeletal diseases and is frequently associated with intervertebral disc degeneration (IDD). While the main cause of IDD is commonly attributed to a reduced number of nucleus pulposus (NP) cells, current treatment strategies (both surgical and more conservative) fail to replenish NP cells or reverse the pathology. Cell replacement therapies are an attractive alternative for treating IDD. However, injecting intervertebral disc (IVD) cells, chondrocytes, or mesenchymal stem cells into various animal models of IDD indicate that transplanted cells generally fail to survive and engraft into the avascular IVD niche. Whereas pluripotent stem cells (PSCs), including induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), hold great potential for revolutionizing regenerative medicine, current protocols for differentiating these cells into NP-like cells are inadequate. Nucleus pulposus progenitor cells (NPPCs), which are derived from the embryonic notochord, can not only survive within the harsh hypoxic environment of the IVD, but they also efficiently differentiate into NP-like cells. Here we provide an overview of the latest progress in repairing degenerated IVDs using PSCs and NPPCs. We also discuss the molecular pathways by which PSCs differentiate into NPPCs in vitro and in vivo and propose a new, in vivo IDD therapy.


Assuntos
Diferenciação Celular , Disco Intervertebral/fisiologia , Núcleo Pulposo/fisiologia , Células-Tronco Pluripotentes/fisiologia , Regeneração , Medicina Regenerativa , Animais , Biomarcadores/metabolismo , Transdiferenciação Celular/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Degeneração do Disco Intervertebral/fisiopatologia , Degeneração do Disco Intervertebral/terapia , Dor Lombar/fisiopatologia , Dor Lombar/terapia
18.
EMBO J ; 38(1)2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30482756

RESUMO

During differentiation and reprogramming, new cell identities are generated by reconfiguration of gene regulatory networks. Here, we combined automated formal reasoning with experimentation to expose the logic of network activation during induction of naïve pluripotency. We find that a Boolean network architecture defined for maintenance of naïve state embryonic stem cells (ESC) also explains transcription factor behaviour and potency during resetting from primed pluripotency. Computationally identified gene activation trajectories were experimentally substantiated at single-cell resolution by RT-qPCR Contingency of factor availability explains the counterintuitive observation that Klf2, which is dispensable for ESC maintenance, is required during resetting. We tested 124 predictions formulated by the dynamic network, finding a predictive accuracy of 77.4%. Finally, we show that this network explains and predicts experimental observations of somatic cell reprogramming. We conclude that a common deterministic program of gene regulation is sufficient to govern maintenance and induction of naïve pluripotency. The tools exemplified here could be broadly applied to delineate dynamic networks underlying cell fate transitions.


Assuntos
Autorrenovação Celular/genética , Reprogramação Celular/genética , Células-Tronco Embrionárias/fisiologia , Epigênese Genética/fisiologia , Redes Reguladoras de Genes/fisiologia , Animais , Sistemas CRISPR-Cas , Diferenciação Celular/genética , Células Cultivadas , Biologia Computacional , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Células-Tronco Embrionárias Murinas/fisiologia , Células-Tronco Pluripotentes/fisiologia
19.
Curr Stem Cell Res Ther ; 14(3): 249-258, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30394215

RESUMO

Angiogenesis has always been a concern in the field of tissue engineering. Poor vascularization of engineered constructs is a problem for the clinical success of these structures. Among the various methods employed to induce angiogenesis, stem cells provide a promising tool for the future. The present review aims to present the application of stem cells in the induction of angiogenesis. Additionally, it summarizes recent advancements in stem cell-mediated angiogenesis of different tissue engineering constructs.


Assuntos
Células Endoteliais/citologia , Células-Tronco Mesenquimais/citologia , Neovascularização Patológica/genética , Neovascularização Fisiológica , Engenharia Tecidual/métodos , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Diferenciação Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Células Endoteliais/fisiologia , Regulação da Expressão Gênica , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Humanos , Células-Tronco Mesenquimais/fisiologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Transdução de Sinais , Transplante de Células-Tronco , Tecidos Suporte , Fator A de Crescimento do Endotélio Vascular/metabolismo
20.
Cell Mol Life Sci ; 76(5): 873-892, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30420999

RESUMO

Embryonic stem cells (ESCs) are immortal stem cells that own multi-lineage differentiation potential. ESCs are commonly derived from the inner cell mass (ICM) of pre-implantation embryos. Due to their tremendous developmental capacity and unlimited self-renewal, ESCs have diverse biomedical applications. Different culture media have been developed to procure and maintain ESCs in a state of naïve pluripotency, and to preserve a stable genome and epigenome during serial passaging. Chromatin modifications such as DNA methylation and histone modifications along with microRNA activity and different signaling pathways dynamically contribute to the regulation of the ESC gene regulatory network (GRN). Such modifications undergo remarkable changes in different ESC media and determine the quality and developmental potential of ESCs. In this review, we discuss the current approaches for derivation and maintenance of ESCs, and examine how differences in culture media impact on the characteristics of pluripotency via modulation of GRN during the course of ICM outgrowth into ESCs. We also summarize the current hypotheses concerning the origin of ESCs and provide a perspective about the relationship of these cells to their in vivo counterparts (early embryonic cells around the time of implantation). Finally, we discuss generation of ESCs from human embryos and domesticated animals, and offer suggestions to further advance this fascinating field.


Assuntos
Blastocisto/citologia , Células-Tronco Embrionárias/fisiologia , Animais , Reprogramação Celular , Metilação de DNA , Transição Epitelial-Mesenquimal , Feminino , Redes Reguladoras de Genes , Humanos , Masculino , Células-Tronco Pluripotentes/fisiologia , Caracteres Sexuais , Transdução de Sinais
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