RESUMO
Cardiogenesis relies on the precise spatiotemporal coordination of multiple progenitor populations. Understanding the specification and differentiation of these distinct progenitor pools during human embryonic development is crucial for advancing our knowledge of congenital cardiac malformations and designing new regenerative therapies. By combining genetic labelling, single-cell transcriptomics, and ex vivo human-mouse embryonic chimeras we uncovered that modulation of retinoic acid signaling instructs human pluripotent stem cells to form heart field-specific progenitors with distinct fate potentials. In addition to the classical first and second heart fields, we observed the appearance of juxta-cardiac field progenitors giving rise to both myocardial and epicardial cells. Applying these findings to stem-cell based disease modelling we identified specific transcriptional dysregulation in first and second heart field progenitors derived from stem cells of patients with hypoplastic left heart syndrome. This highlights the suitability of our in vitro differentiation platform for studying human cardiac development and disease.
Assuntos
Células-Tronco Pluripotentes , Tretinoína , Humanos , Animais , Camundongos , Tretinoína/farmacologia , Coração , Miocárdio , Diferenciação Celular , Miócitos CardíacosRESUMO
We identify the sodium leak channel non-selective protein (NALCN) as a key regulator of cancer metastasis and nonmalignant cell dissemination. Among 10,022 human cancers, NALCN loss-of-function mutations were enriched in gastric and colorectal cancers. Deletion of Nalcn from gastric, intestinal or pancreatic adenocarcinomas in mice did not alter tumor incidence, but markedly increased the number of circulating tumor cells (CTCs) and metastases. Treatment of these mice with gadolinium-a NALCN channel blocker-similarly increased CTCs and metastases. Deletion of Nalcn from mice that lacked oncogenic mutations and never developed cancer caused shedding of epithelial cells into the blood at levels equivalent to those seen in tumor-bearing animals. These cells trafficked to distant organs to form normal structures including lung epithelium, and kidney glomeruli and tubules. Thus, NALCN regulates cell shedding from solid tissues independent of cancer, divorcing this process from tumorigenesis and unmasking a potential new target for antimetastatic therapies.
Assuntos
Neoplasias , Humanos , Camundongos , Animais , Canais Iônicos/genética , Proteínas de Membrana/genéticaRESUMO
Large-scale cancer genome projects will soon be able to sequence many cancer genomes to comprehensively identify genetic changes in human cancer. Genome-wide association studies have also identified putative cancer associated loci. Functional validation of these genetic mutations in vivo is becoming a challenge. We describe here a DNA transposon-based platform that permits us to explore the oncogenic potential of genetic mutations in the mouse. Briefly, promoter-less human cancer gene cDNAs were first cloned into Sleeping Beauty (SB) transposons. DNA transposition in the mouse that carried both the transposons and the SB transposase made it possible for the cDNAs to be expressed from an appropriate endogenous genomic locus and in the relevant cell types for tumor development. Consequently, these mice developed a broad spectrum of tumors at very early postnatal stages. This technology thus complements the large-scale cancer genome projects.
Assuntos
Análise Mutacional de DNA/métodos , Elementos de DNA Transponíveis/genética , DNA de Neoplasias/genética , Neoplasias/genética , Oncogenes , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Teste de Complementação Genética , Genoma Humano , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Transposases/genéticaRESUMO
Mesoderm is induced at the primitive streak (PS) and patterns subsequently into mesodermal subtypes and organ precursors. It is unclear whether mesoderm induction generates a multipotent PS progenitor or several distinct ones with restricted subtype potentials. We induced mesoderm in human pluripotent stem cells with ACTIVIN and BMP or with GSK3-ß inhibition. Both approaches induced BRACHYURY(+) mesoderm of distinct PS-like identities, which had differing patterning potential. ACTIVIN and BMP-induced mesoderm patterned into cardiac but not somitic subtypes. Conversely, PS precursors induced by GSK3-ß inhibition did not generate lateral plate and cardiac mesoderm and favored instead somitic differentiation. The mechanism of these cell fate decisions involved mutual repression of NANOG and CDX2. Although NANOG was required for cardiac specification but blocked somitic subtypes, CDX2 was required for somitic mesoderm but blocked cardiac differentiation. In sum, rather than forming a common PS progenitor, separate induction mechanisms distinguish human mesoderm subtypes.
Assuntos
Proteínas de Homeodomínio/metabolismo , Mesoderma/embriologia , Mesoderma/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Ativinas/metabolismo , Padronização Corporal , Proteínas Morfogenéticas Ósseas/metabolismo , Fator de Transcrição CDX2 , Linhagem Celular , Linhagem da Célula , Proteínas Fetais/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Mesoderma/citologia , Miocárdio/citologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Proteína Homeobox Nanog , Linha Primitiva/citologia , Sequências Reguladoras de Ácido Nucleico/genética , Transdução de Sinais , Proteínas com Domínio T/metabolismoRESUMO
T cells develop in the thymus and are critical for adaptive immunity. Natural killer (NK) lymphocytes constitute an essential component of the innate immune system in tumor surveillance, reproduction, and defense against microbes and viruses. Here, we show that the transcription factor Bcl11b was expressed in all T cell compartments and was indispensable for T lineage development. When Bcl11b was deleted, T cells from all developmental stages acquired NK cell properties and concomitantly lost or decreased T cell-associated gene expression. These induced T-to-natural killer (ITNK) cells, which were morphologically and genetically similar to conventional NK cells, killed tumor cells in vitro, and effectively prevented tumor metastasis in vivo. Therefore, ITNKs may represent a new cell source for cell-based therapies.
Assuntos
Linhagem da Célula , Células Matadoras Naturais/fisiologia , Linfopoese , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Linfócitos T/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Citotoxicidade Imunológica , Deleção de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Linfopoese/genética , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Células Precursoras de Linfócitos T/citologia , Células Precursoras de Linfócitos T/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transdução de Sinais , Células Estromais/citologia , Células Estromais/fisiologia , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/transplante , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologiaRESUMO
Our laboratory recently identified a quiescent class of pluripotent hematopoietic stem cells (PHSCs) that are lineage negative (Linneg), lack c-Kit, and are able to give rise to c-Kit-positive (c-Kitpos) PHSCs in vivo. This population fails to proliferate in vitro but has delayed reconstituting activity in vivo. In this study, we purified these cells to enrich for the PHSCs and we identified in vitro conditions capable of supporting their maturation. The c-Kit-negative (c-Kitneg) cells exhibited differential expression of Sca-1, CD34, CD43, CD45, and Thy 1.2. We purified the cells based on Sca-1, as it is expressed on active PHSCs. We detected pre-colony-forming unit spleen (pre-CFU-s) activity in both the Sca-1neg and Sca-1pos populations, indicating the presence of primitive PHSCs in both populations. However, our in vitro studies suggest that the Sca-1pos population is enriched for PHSCs. The in vitro systems that support the growth of these dormant cells include a modified long-term marrow culture and various stromal cell lines. In modified long-term bone marrow cultures, c-Kitneg cells gave rise to c-Kitpos PHSCs, with long-term reconstitution activity in vivo. Thus we have established an in vitro system to examine PHSC maturation that will allow us to study the mediators of the c-Kitneg to c-Kitpos transition.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Pluripotentes/citologia , Proteínas Proto-Oncogênicas c-kit/genética , Animais , Antígenos Ly/análise , Células da Medula Óssea/citologia , Divisão Celular , Separação Celular , Técnicas de Cocultura , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Pluripotentes/metabolismo , Células Estromais/citologiaRESUMO
CCAAT enhancer binding protein-alpha (C/EBPalpha) inhibits proliferation in multiple cell types; therefore, we evaluated whether C/EBPalpha-deficient hematopoietic progenitor cells (HPCs) have an increased proliferative potential in vitro and in vivo. In this study we demonstrate that C/EBPalpha(-/-) fetal liver (FL) progenitors are hyperproliferative, show decreased differentiation potential, and show increased self-renewal capacity in response to hematopoietic growth factors (HGFs). There are fewer committed bipotential progenitors in C/EBPalpha(-/-) FL, whereas multipotential progenitors are unaffected. HGF-dependent progenitor cell lines can be derived by directly culturing C/EBPalpha(-/-) FL cells in vitro Hyperproliferative spleen colonies and myelodysplastic syndrome (MDS) are observed in mice reconstituted with C/EBPalpha(-/-) FL cells, indicating progenitor hyperproliferation in vitro and in vivo. C/EBPalpha(-/-) FL lacked macrophage progenitors in vitro and had impaired ability to generate macrophages in vivo. These findings show that C/EBPalpha deficiency results in hyperproliferation of HPCs and a block in the ability of multipotential progenitors to differentiate into bipotential granulocyte/macrophage progenitors and their progeny.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/deficiência , Diferenciação Celular , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Transplante de Células , Células Cultivadas , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Interleucina-3/metabolismo , Fígado/embriologia , Fígado/metabolismo , Fígado/patologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Taxa de SobrevidaRESUMO
Bcl11a (also called Evi9) functions as a myeloid or B cell proto-oncogene in mice and humans, respectively. Here we show that Bcl11a is essential for postnatal development and normal lymphopoiesis. Bcl11a mutant embryos lack B cells and have alterations in several types of T cells. Phenotypic and expression studies show that Bcl11a functions upstream of the transcription factors Ebf1 and Pax5 in the B cell pathway. Transplantation studies show that these defects in Bcl11a mutant mice are intrinsic to fetal liver precursor cells. Mice transplanted with Bcl11a-deficient cells died from T cell leukemia derived from the host. Thus, Bcl11a may also function as a non-autonomous T cell tumor suppressor gene.