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Proc Natl Acad Sci U S A ; 116(36): 17970-17979, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31420514


Single-cell sequencing technologies have revealed an unexpectedly broad repertoire of cells required to mediate complex functions in multicellular organisms. Despite the multiple roles of adipose tissue in maintaining systemic metabolic homeostasis, adipocytes are thought to be largely homogenous with only 2 major subtypes recognized in humans so far. Here we report the existence and characteristics of 4 distinct human adipocyte subtypes, and of their respective mesenchymal progenitors. The phenotypes of these distinct adipocyte subtypes are differentially associated with key adipose tissue functions, including thermogenesis, lipid storage, and adipokine secretion. The transcriptomic signature of "brite/beige" thermogenic adipocytes reveals mechanisms for iron accumulation and protection from oxidative stress, necessary for mitochondrial biogenesis and respiration upon activation. Importantly, this signature is enriched in human supraclavicular adipose tissue, confirming that these cells comprise thermogenic depots in vivo, and explain previous findings of a rate-limiting role of iron in adipose tissue browning. The mesenchymal progenitors that give rise to beige/brite adipocytes express a unique set of cytokines and transcriptional regulators involved in immune cell modulation of adipose tissue browning. Unexpectedly, we also find adipocyte subtypes specialized for high-level expression of the adipokines adiponectin or leptin, associated with distinct transcription factors previously implicated in adipocyte differentiation. The finding of a broad adipocyte repertoire derived from a distinct set of mesenchymal progenitors, and of the transcriptional regulators that can control their development, provides a framework for understanding human adipose tissue function and role in metabolic disease.

Nat Biotechnol ; 37(4): 461-468, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30936567


Recent single-cell RNA-sequencing studies have suggested that cells follow continuous transcriptomic trajectories in an asynchronous fashion during development. However, observations of cell flux along trajectories are confounded with population size effects in snapshot experiments and are therefore hard to interpret. In particular, changes in proliferation and death rates can be mistaken for cell flux. Here we present pseudodynamics, a mathematical framework that reconciles population dynamics with the concepts underlying developmental trajectories inferred from time-series single-cell data. Pseudodynamics models population distribution shifts across trajectories to quantify selection pressure, population expansion, and developmental potentials. Applying this model to time-resolved single-cell RNA-sequencing of T-cell and pancreatic beta cell maturation, we characterize proliferation and apoptosis rates and identify key developmental checkpoints, data inaccessible to existing approaches.

Diferenciação Celular/genética , Análise de Sequência de RNA/estatística & dados numéricos , Análise de Célula Única/estatística & dados numéricos , Animais , Apoptose/genética , Biotecnologia , Proliferação de Células/genética , Feminino , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Funções Verossimilhança , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Fatores de Tempo
Cell Rep ; 27(3): 708-718.e10, 2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-30995470


Studies in vertebrates have outlined conserved molecular control of definitive endoderm (END) development. However, recent work also shows that key molecular aspects of human END regulation differ even from rodents. Differentiation of human embryonic stem cells (ESCs) to END offers a tractable system to study the molecular basis of normal and defective human-specific END development. Here, we interrogated dynamics in chromatin accessibility during differentiation of ESCs to END, predicting DNA-binding proteins that may drive this cell fate transition. We then combined single-cell RNA-seq with parallel CRISPR perturbations to comprehensively define the loss-of-function phenotype of those factors in END development. Following a few candidates, we revealed distinct impairments in the differentiation trajectories for mediators of TGFß signaling and expose a role for the FOXA2 transcription factor in priming human END competence for human foregut and hepatic END specification. Together, this single-cell functional genomics study provides high-resolution insight on human END development.

Immunity ; 48(6): 1258-1270.e6, 2018 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-29884461


Thymus development is critical to the adaptive immune system, yet a comprehensive transcriptional framework capturing thymus organogenesis at single-cell resolution is still needed. We applied single-cell RNA sequencing (RNA-seq) to capture 8 days of thymus development, perturbations of T cell receptor rearrangement, and in vitro organ cultures, producing profiles of 24,279 cells. We resolved transcriptional heterogeneity of developing lymphocytes, and genetic perturbation confirmed T cell identity of conventional and non-conventional lymphocytes. We characterized maturation dynamics of thymic epithelial cells in vivo, classified cell maturation state in a thymic organ culture, and revealed the intrinsic capacity of thymic epithelium to preserve transcriptional regularity despite exposure to exogenous retinoic acid. Finally, by integrating the cell atlas with human genome-wide association study (GWAS) data and autoimmune-disease-related genes, we implicated embryonic thymus-resident cells as possible participants in autoimmune disease etiologies. This resource provides a single-cell transcriptional framework for biological discovery and molecular analysis of thymus organogenesis.

Development ; 141(1): 219-23, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24346702


The identification of the trans-acting factors and cis-regulatory modules that are involved in human pluripotent stem cell (hPSC) maintenance and differentiation is necessary to dissect the operating regulatory networks in these processes and thereby identify nodes where signal input will direct desired cell fate decisions in vitro or in vivo. To deconvolute these networks, we established a method to influence the differentiation state of hPSCs with a CRISPR-associated catalytically inactive dCas9 fused to an effector domain. In human embryonic stem cells, we find that the dCas9 effectors can exert positive or negative regulation on the expression of developmentally relevant genes, which can influence cell differentiation status when impinging on a key node in the regulatory network that governs the cell state. This system provides a platform for the interrogation of the underlying regulators governing specific differentiation decisions, which can then be employed to direct cellular differentiation down desired pathways.

Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Diferenciação Celular/genética , Endonucleases/genética , Redes Reguladoras de Genes/genética , Células-Tronco Pluripotentes/metabolismo , Ativação Transcricional/genética , Sequência de Aminoácidos , Proteína 9 Associada à CRISPR , Linhagem da Célula/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células HEK293 , Humanos , Dados de Sequência Molecular , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição SOXF/biossíntese , Transcrição Genética
Stem Cell Res ; 11(3): 1003-12, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23917481


Anterior foregut endoderm (AFE) gives rise to therapeutically relevant cell types in tissues such as the esophagus, salivary glands, lung, thymus, parathyroid and thyroid. Despite its importance, reports describing the generation of AFE from pluripotent stem cells (PSCs) by directed differentiation have mainly focused on the Nkx2.1(+) lung and thyroid lineages. Here, we describe a novel protocol to derive a subdomain of AFE, identified by expression of Pax9, from PSCs using small molecules and defined media conditions. We generated a reporter PSC line for isolation and characterization of Pax9(+) AFE cells, which when transplanted in vivo, can form several distinct complex AFE-derived epithelia, including mucosal glands and stratified squamous epithelium. Finally, we show that the directed differentiation protocol can be used to generate AFE from human PSCs. Thus, this work both broadens the range of PSC-derived AFE tissues and creates a platform enabling the study of AFE disorders.

Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem da Célula/efeitos dos fármacos , Meios de Cultura/farmacologia , Células-Tronco Embrionárias/citologia , Células Epiteliais/metabolismo , Células Epiteliais/transplante , Fator 3-beta Nuclear de Hepatócito/metabolismo , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Fator de Transcrição PAX9/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/metabolismo , Transcriptoma