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1.
Development ; 138(5): 861-71, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21270052

RESUMO

The generation of insulin-producing ß-cells from human pluripotent stem cells is dependent on efficient endoderm induction and appropriate patterning and specification of this germ layer to a pancreatic fate. In this study, we elucidated the temporal requirements for TGFß family members and canonical WNT signaling at these developmental stages and show that the duration of nodal/activin A signaling plays a pivotal role in establishing an appropriate definitive endoderm population for specification to the pancreatic lineage. WNT signaling was found to induce a posterior endoderm fate and at optimal concentrations enhanced the development of pancreatic lineage cells. Inhibition of the BMP signaling pathway at specific stages was essential for the generation of insulin-expressing cells and the extent of BMP inhibition required varied widely among the cell lines tested. Optimal stage-specific manipulation of these pathways resulted in a striking 250-fold increase in the levels of insulin expression and yielded populations containing up to 25% C-peptide+ cells.


Assuntos
Células Secretoras de Insulina/citologia , Pâncreas/citologia , Células-Tronco Pluripotentes/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/metabolismo , Ativinas/metabolismo , Padronização Corporal , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Peptídeo C , Linhagem Celular , Linhagem da Célula , Endoderma , Humanos , Insulina/biossíntese , Transdução de Sinais/fisiologia
2.
Nat Methods ; 6(9): 659-62, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19701193

RESUMO

A human embryonic stem cell (hESC) line that enabled globin-expressing cells to be easily recognized would facilitate optimization of erythroid differentiation in vitro and aid in the identification of hESC-derived erythroid cells in transplanted animals. We describe a genetically modified hESC line, ErythRED, in which expression of RFP, controlled by regulatory sequences from the human beta-globin locus control region, is restricted to maturing erythroid cells.


Assuntos
Células-Tronco Embrionárias/citologia , Células Eritroides/citologia , Animais , Diferenciação Celular , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Células Eritroides/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Globinas beta/genética , Globinas beta/metabolismo
3.
Stem Cells ; 29(3): 462-73, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21425409

RESUMO

We have used homologous recombination in human embryonic stem cells (hESCs) to insert sequences encoding green fluorescent protein (GFP) into the NKX2.1 locus, a gene required for normal development of the basal forebrain. Generation of NKX2.1-GFP(+) cells was dependent on the concentration, timing, and duration of retinoic acid treatment during differentiation. NKX2.1-GFP(+) progenitors expressed genes characteristic of the basal forebrain, including SHH, DLX1, LHX6, and OLIG2. Time course analysis revealed that NKX2.1-GFP(+) cells could upregulate FOXG1 expression, implying the existence of a novel pathway for the generation of telencephalic neural derivatives. Further maturation of NKX2.1-GFP(+) cells gave rise to γ-aminobutyric acid-, tyrosine hydroxylase-, and somatostatin-expressing neurons as well as to platelet-derived growth factor receptor α-positive oligodendrocyte precursors. These studies highlight the diversity of cell types that can be generated from human NKX2.1(+) progenitors and demonstrate the utility of NKX2.1(GFP/w) hESCs for investigating human forebrain development and neuronal differentiation.


Assuntos
Linhagem da Célula/genética , Rastreamento de Células/métodos , Células-Tronco Embrionárias/metabolismo , Proteínas Nucleares/genética , Prosencéfalo/embriologia , Fatores de Transcrição/genética , Animais , Animais Recém-Nascidos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Células-Tronco Embrionárias/citologia , Citometria de Fluxo/métodos , Genes Reporter , Humanos , Camundongos , Camundongos Transgênicos , Terapia de Alvo Molecular/métodos , Neurogênese/genética , Neurogênese/fisiologia , Proteínas Nucleares/metabolismo , Prosencéfalo/citologia , Prosencéfalo/fisiologia , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/metabolismo
4.
Diabetes ; 54(2): 301-5, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15585742

RESUMO

We have generated an embryonic stem (ES) cell line in which sequences encoding green fluorescent protein (GFP) were targeted to the locus of the pancreatic-duodenal homeobox gene (Pdx1). Analysis of chimeric embryos derived from blastocyst injection of Pdx1(GFP/w) ES cells demonstrated that the pattern of GFP expression was consistent with that reported for the endogenous Pdx1 gene. By monitoring GFP expression during the course of ES cell differentiation, we have shown that retinoic acid (RA) can regulate the commitment of ES cells to form Pdx1(+) pancreatic endoderm. RA was most effective at inducing Pdx1 expression when added to cultures at day 4 of ES differentiation, a period corresponding to the end of gastrulation in the embryo. RT-PCR analysis showed that Pdx1-positive cells from day 8 cultures expressed the early endoderm markers Ptf1a, Foxa2, Hnf4alpha, Hnf1beta, and Hnf6, consistent with the notion that they corresponded to the early pancreatic endoderm present in the embryonic day 9.5 mouse embryo. These results demonstrate the utility of Pdx1(GFP/w) ES cells as a tool for monitoring the effects of factors that influence pancreatic differentiation from ES cells.


Assuntos
Endoderma/fisiologia , Proteínas de Homeodomínio/genética , Células-Tronco/citologia , Células-Tronco/fisiologia , Transativadores/genética , Tretinoína/farmacologia , Animais , Sequência de Bases , Blastocisto/citologia , Blastocisto/fisiologia , Diferenciação Celular , Quimera , Primers do DNA , Endoderma/efeitos dos fármacos , Genes Reporter , Camundongos , Camundongos Transgênicos , Pâncreas/citologia , Pâncreas/embriologia , Reação em Cadeia da Polimerase , Células-Tronco/efeitos dos fármacos
5.
Stem Cells Transl Med ; 4(6): 603-14, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25873746

RESUMO

UNLABELLED: Airway epithelial cells generated from pluripotent stem cells (PSCs) represent a resource for research into a variety of human respiratory conditions, including those resulting from infection with common human pathogens. Using an NKX2.1-GFP reporter human embryonic stem cell line, we developed a serum-free protocol for the generation of NKX2.1(+) endoderm that, when transplanted into immunodeficient mice, matured into respiratory cell types identified by expression of CC10, MUC5AC, and surfactant proteins. Gene profiling experiments indicated that day 10 NKX2.1(+) endoderm expressed markers indicative of early foregut but lacked genes associated with later stages of respiratory epithelial cell differentiation. Nevertheless, NKX2.1(+) endoderm supported the infection and replication of the common respiratory pathogen human rhinovirus HRV1b. Moreover, NKX2.1(+) endoderm upregulated expression of IL-6, IL-8, and IL-1B in response to infection, a characteristic of human airway epithelial cells. Our experiments provide proof of principle for the use of PSC-derived respiratory epithelial cells in the study of cell-virus interactions. SIGNIFICANCE: This report provides proof-of-principle experiments demonstrating, for the first time, that human respiratory progenitor cells derived from stem cells in the laboratory can be productively infected with human rhinovirus, the predominant cause of the common cold.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/virologia , Interações Hospedeiro-Patógeno , Proteínas Nucleares , Infecções por Picornaviridae/mortalidade , Mucosa Respiratória/virologia , Rhinovirus/fisiologia , Fatores de Transcrição , Animais , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Humanos , Camundongos , Camundongos Nus , Infecções por Picornaviridae/patologia , Mucosa Respiratória/metabolismo , Fator Nuclear 1 de Tireoide
6.
J Interferon Cytokine Res ; 24(7): 416-27, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15296653

RESUMO

The intestinal epithelium is a continuously renewing tissue. In the colon, stem cells are maintained at the base of highly organized crypts, where they undergo asymmetric division and give rise to daughter cells that proliferate and migrate up the crypt as they differentiate, then become senescent and are finally shed into the intestinal lumen. The growth factor requirements of fetal and prenatal colon cells for colony formation and that influence the establishment of cell lines from Immorto-mouse (Charles River, Wilmington, MA) transgenic embryos were explored. Single cell suspensions were isolated and cultured in a large range of growth factor combinations and conditions to determine their growth properties in soft agar. We report an important advance in the culture of mouse colonocytes by using macrophage colony-stimulating factor (CSF-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF). A substantial proportion of colonies grown under low oxygen tension in the presence of CSF-1 and GM-CSF express intestinal epithelial A33 antigen, have the expected gene expression profile, including c-fms and transcription factor c-myb, and show an appropriate epithelial cell morphology and undetectable CD45. Confocal microscopy on isolated crypts displays basolateral expression of c-Fms and E-cadherin on most epithelial cells. Fetal colon cultures from the Immorto-mouse with CSF-1 produced rapid outgrowth and readily established cell lines, in contrast to cultures without CSF-1. These observations have implications for the understanding of colon epithelial development and recovery following cytotoxic damage as well as providing a basis for the observation that some colon (and other epithelial) tumor cells respond to CSF-1 and GM-CSF.


Assuntos
Proliferação de Células/efeitos dos fármacos , Colo/fisiologia , Células Epiteliais/fisiologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Animais , Técnicas de Cultura de Células , Células Cultivadas , Colo/citologia , Ensaio de Unidades Formadoras de Colônias , Células Epiteliais/ultraestrutura , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Antígenos Comuns de Leucócito/biossíntese , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-myb/biossíntese , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese
7.
Rev Diabet Stud ; 11(1): 6-18, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25148364

RESUMO

Human embryonic stem cells have been advanced as a source of insulin-producing cells that could potentially replace cadaveric-derived islets in the treatment of type 1 diabetes. To this end, protocols have been developed that promote the formation of pancreatic progenitors and endocrine cells from human pluripotent stem cells, encompassing both embryonic stem cells and induced pluripotent stem cells. In this review, we examine these methods and place them in the context of the developmental and embryological studies upon which they are based. In particular, we outline the stepwise differentiation of cells towards definitive endoderm, pancreatic endoderm, endocrine lineages and the emergence of functional beta-cells. In doing so, we identify key factors common to many such protocols and discuss the proposed action of these factors in the context of cellular differentiation and ongoing development. We also compare strategies that entail transplantation of progenitor populations with those that seek to develop fully functional hormone expressing cells in vitro. Overall, our survey of the literature highlights the significant progress already made in the field and identifies remaining deficiencies in developing a pluripotent stem cell based treatment for type 1 diabetes.


Assuntos
Diferenciação Celular , Células Secretoras de Insulina/citologia , Modelos Biológicos , Células-Tronco Pluripotentes/citologia , Animais , Células Cultivadas , Células-Tronco Embrionárias/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células Secretoras de Insulina/metabolismo , Cultura Primária de Células/tendências
8.
Stem Cell Reports ; 2(6): 925-37, 2014 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-24936476

RESUMO

Thymic epithelial cells (TECs) play a critical role in T cell maturation and tolerance induction. The generation of TECs from in vitro differentiation of human pluripotent stem cells (PSCs) provides a platform on which to study the mechanisms of this interaction and has implications for immune reconstitution. To facilitate analysis of PSC-derived TECs, we generated hESC reporter lines in which sequences encoding GFP were targeted to FOXN1, a gene required for TEC development. Using this FOXN1 (GFP/w) line as a readout, we developed a reproducible protocol for generating FOXN1-GFP(+) thymic endoderm cells. Transcriptional profiling and flow cytometry identified integrin-ß4 (ITGB4, CD104) and HLA-DR as markers that could be used in combination with EpCAM to selectively purify FOXN1(+) TEC progenitors from differentiating cultures of unmanipulated PSCs. Human FOXN1(+) TEC progenitors generated from PSCs facilitate the study of thymus biology and are a valuable resource for future applications in regenerative medicine.


Assuntos
Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Epiteliais/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Antígenos HLA-DR/metabolismo , Integrina beta4/metabolismo , Células-Tronco Pluripotentes/citologia , Timo/citologia , Diferenciação Celular , Células Cultivadas , Molécula de Adesão da Célula Epitelial , Células Epiteliais/citologia , Humanos , Células-Tronco Pluripotentes/metabolismo
9.
Stem Cell Res ; 10(1): 103-17, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23164599

RESUMO

The limited availability of human vascular endothelial cells (ECs) hampers research into EC function whilst the lack of precisely defined culture conditions for this cell type presents problems for addressing basic questions surrounding EC physiology. We aimed to generate endothelial progenitors from human pluripotent stem cells to facilitate the study of human EC physiology, using a defined serum-free protocol. Human embryonic stem cells (hESC-ECs) differentiated under serum-free conditions generated CD34(+)KDR(+) endothelial progenitor cells after 6days that could be further expanded in the presence of vascular endothelial growth factor (VEGF). The resultant EC population expressed CD31 and TIE2/TEK, took up acetylated low-density lipoprotein (LDL) and up-regulated expression of ICAM-1, PAI-1 and ET-1 following treatment with TNFα. Immunofluorescence studies indicated that a key mediator of vascular tone, endothelial nitric oxide synthase (eNOS), was localised to a perinuclear compartment of hESC-ECs, in contrast with the pan-cellular distribution of this enzyme within human umbilical vein ECs (HUVECs). Further investigation revealed that that the serum-associated lipids, lysophosphatidic acid (LPA) and platelet activating factor (PAF), were the key molecules that affected eNOS localisation in hESC-ECs cultures. These studies illustrate the feasibility of EC generation from hESCs and the utility of these cells for investigating environmental cues that impact on EC phenotype. We have demonstrated a hitherto unrecognized role for LPA and PAF in the regulation of eNOS subcellular localization.


Assuntos
Meios de Cultura/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células Endoteliais/citologia , Lisofosfolipídeos/farmacologia , Óxido Nítrico Sintase Tipo III/análise , Fator de Ativação de Plaquetas/farmacologia , Antígenos CD34/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Colágeno/química , Combinação de Medicamentos , Células-Tronco Embrionárias/citologia , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Laminina/química , Óxido Nítrico Sintase Tipo III/metabolismo , Proteoglicanas/química , Fator de Necrose Tumoral alfa/farmacologia
10.
Stem Cells Dev ; 21(10): 1662-74, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22034992

RESUMO

To develop methods for the generation of insulin-producing ß-cells for the treatment of diabetes, we have used GFP-tagged embryonic stem cells (ESCs) to elucidate the process of pancreas development. Using the reporter Pdx1(GFP/w) ESC line, we have previously described a serum-free differentiation protocol in which Pdx1-GFP(+) cells formed GFP bright (GFP(br)) epithelial buds that resembled those present in the developing mouse pancreas. In this study we extend these findings to demonstrate that these cells can undergo a process of branching morphogenesis, similar to that seen during pancreatic development of the mid-gestation embryo. These partially disaggregated embryoid bodies containing GFP(br) buds initially form epithelial ring-like structures when cultured in Matrigel. After several days in culture, these rings undergo a process of proliferation and form a ramified network of epithelial branches. Comparative analysis of explanted dissociated pancreatic buds from E13.5 Pdx1(GFP/w) embryos and ESC-derived GFP(br) buds reveal a similar process of proliferation and branching, with both embryonic Pdx1(GFP/w) branching pancreatic epithelium and ESC-derived GFP(br) branching organoids expressing markers representing epithelial (EpCAM and E-Cadherin), ductal (Mucin1), exocrine (Amylase and Carboxypeptidase 1A), and endocrine cell types (Glucagon and Somatostatin). ESC-derived branching structures also expressed a suite of genes indicative of ongoing pancreatic differentiation, paralleling gene expression within similar structures derived from the E13.5 fetal pancreas. In summary, differentiating mouse ESCs can generate pancreatic material that has significant similarity to the fetal pancreatic anlagen, providing an in vitro platform for investigating the cellular and molecular mechanisms underpinning pancreatic development.


Assuntos
Corpos Embrioides/fisiologia , Desenvolvimento Embrionário , Organogênese , Pâncreas/embriologia , Animais , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Endoderma/citologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Pâncreas/citologia , Pâncreas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Técnicas de Cultura de Tecidos , Transativadores/genética , Transativadores/metabolismo
11.
Nat Protoc ; 3(10): 1550-8, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18802436

RESUMO

The first step in the generation of genetically tagged human embryonic stem cell (HESC) reporter lines is the isolation of cells that contain a stably integrated copy of the reporter vector. These cells are identified by their continued growth in the presence of a specific selective agent, usually conferred by a cassette encoding antibiotic resistance. In order to mitigate potential interference between the regulatory elements driving expression of the antibiotic resistance gene and those controlling the reporter gene, it is advisable to remove the positive selection cassette once the desired clones have been identified. This report describes a protocol for the removal of loxP-flanked selection cassettes from genetically modified HESCs by transient transfection with a vector expressing Cre recombinase. An integrated procedure for the clonal isolation of these genetically modified lines using single-cell deposition flow cytometry is also detailed. When performed sequentially, these protocols take approximately 1 month.


Assuntos
Resistência Microbiana a Medicamentos/genética , Células-Tronco Embrionárias/metabolismo , Citometria de Fluxo/métodos , Técnicas de Transferência de Genes , Mutagênese Insercional/métodos , Vetores Genéticos/genética , Humanos
12.
Curr Protoc Stem Cell Biol ; Chapter 1: Unit 1G.2, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18785172

RESUMO

This unit describes the derivation of pancreatic cells from mouse embryonic stem cells (ESCs). Mouse ESCs are pluripotent immortal cells derived from the inner cell mass of pre-implantation blastocyst-stage embryos that possess the ability to differentiate into any cell type within the adult animal. In vitro, ESCs can be differentiated into a variety of cell types representing derivatives of the three embryonic germ layers, mesoderm, endoderm, and ectoderm. Successfully differentiating ES cells to pancreatic cells has the potential to provide an alternative to cadaver-derived cells for treatment of type I diabetes. This unit outlines a method for the differentiation of ESCs toward pancreatic endoderm in serum-free medium from embryoid bodies (EBs) formed in suspension or spin EBs. In addition there is a protocol for maintaining ESC.


Assuntos
Células-Tronco Embrionárias/citologia , Pâncreas/citologia , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Meios de Cultura Livres de Soro , Camundongos , Células-Tronco Pluripotentes/citologia
13.
Stem Cell Res ; 1(1): 25-36, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19383384

RESUMO

We have examined factors affecting the in vitro differentiation of Pdx1(GFP/w) ESCs to pancreatic endocrine cells. Inclusion of Bone Morphogenetic Protein 4 (BMP4) during the first four days of differentiation followed by a 24-hour pulse of retinoic acid (RA) induced the formation of GFP(+) embryoid bodies (EBs). GFP expression was restricted to E-cadherin(+) tubes and GFP bright (GFP(br)) buds, reminiscent of GFP(+) early foregut endoderm and GFP(br) pancreatic buds observed in Pdx1(GFP/w) embryos. These organoid structures developed without further addition of exogenous factors between days 5 and 12, suggesting that day 5 EBs contained a template for the subsequent phase of development. EBs treated with nicotinamide after day 12 of differentiation expressed markers of endocrine and exocrine differentiation, but only in cells within the GFP(br) buds. Analysis of Pdx1(GFP/w) ESCs modified by targeting a dsRed1 gene to the Ins1 locus (Pdx1(GFP/w)Ins1(RFP/w) ESCs) provided corroborating evidence that insulin positive cells arose from GFP(br) buds, mirroring the temporal relationship between pancreatic bud development and the formation of endocrine cells in the developing embryo. The readily detectable co-expression of GFP and RFP in grafts derived from transplanted EBs demonstrated the utility of Pdx1(GFP/w)Ins1(RFP/w) ESCs for investigating pancreatic differentiation in vitro and in vivo.


Assuntos
Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células Endócrinas/citologia , Transplante de Células-Tronco , Tretinoína/farmacologia , Animais , Técnicas de Cultura de Células , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Camundongos , Pâncreas/citologia
14.
Genesis ; 44(6): 304-7, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16794995

RESUMO

The pancreatic and duodenal homeobox gene 1 (Pdx1) has multiple roles in the specification and development of foregut endoderm-derived tissues. We report the characterization of a mouse line in which the gene encoding green fluorescent protein (GFP) has been targeted to the Pdx1 locus, allowing the visualization of Pdx1 expressing cells. Analysis of GFP expression during development showed that the reporter faithfully reproduced the known expression pattern of Pdx1. We demonstrate the utility of this mouse line for the isolation of Pdx1(+) cells by fluorescence-activated cell sorting and for the real-time observation of Pdx1(+) cells in an ex vivo embryonic pancreas culture system. This mouse model should prove useful for the study of pancreas development and regeneration.


Assuntos
Proteínas de Fluorescência Verde/genética , Proteínas de Homeodomínio/genética , Camundongos Transgênicos/embriologia , Camundongos Transgênicos/metabolismo , Pâncreas/embriologia , Pâncreas/metabolismo , Transativadores/genética , Animais , Expressão Gênica , Marcação de Genes , Engenharia Genética , Controle Interno-Externo , Camundongos
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