RESUMEN
Natural killer T (NKT) cells are distinct glycolipid reactive innate lymphocytes that are implicated in the resistance to pathogens and tumors. Earlier attempts to mobilize NKT cells, specifically, in vivo in humans met with limited success. Here, we evaluated intravenous injection of monocyte-derived mature DCs that were loaded with a synthetic NKT cell ligand, alpha-galactosyl-ceramide (alpha-GalCer; KRN-7000) in five patients who had advanced cancer. Injection of alpha-GalCer-pulsed, but not unpulsed, dendritic cells (DCs) led to >100-fold expansion of several subsets of NKT cells in all patients; these could be detected for up to 6 mo after vaccination. NKT activation was associated with an increase in serum levels of interleukin-12 p40 and IFN-gamma inducible protein-10. In addition, there was an increase in memory CD8+ T cells specific for cytomegalovirus in vivo in response to alpha-GalCer-loaded DCs, but not unpulsed DCs. These data demonstrate the feasibility of sustained expansion of NKT cells in vivo in humans, including patients who have advanced cancer, and suggest that NKT activation might help to boost adaptive T cell immunity in vivo.
Asunto(s)
Proliferación Celular , Células Dendríticas/metabolismo , Galactosilceramidas/uso terapéutico , Inmunoterapia , Células Asesinas Naturales/inmunología , Neoplasias/terapia , Vacunación , Adulto , Análisis Químico de la Sangre , Linfocitos T CD8-positivos/inmunología , Quimiocina CXCL10 , Quimiocinas/metabolismo , Quimiocinas CXC/sangre , Citocinas/metabolismo , Citomegalovirus/inmunología , Células Dendríticas/inmunología , Citometría de Flujo , Galactosilceramidas/metabolismo , Humanos , Interleucina-12/sangre , Neoplasias/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Genome-wide association studies (GWAS) have highlighted a large number of genetic variants with potential disease association, but functional analysis remains a challenge. Here we describe an approach to functionally validate identified variants through differentiation of induced pluripotent stem cells (iPSCs) to study cellular pathophysiology. We collected peripheral blood cells from Framingham Heart Study participants and reprogrammed them to iPSCs. We then differentiated 68 iPSC lines into hepatocytes and adipocytes to investigate the effect of the 1p13 rs12740374 variant on cardiometabolic disease phenotypes via transcriptomics and metabolomic signatures. We observed a clear association between rs12740374 and lipid accumulation and gene expression in differentiated hepatocytes, in particular, expression of SORT1, CELSR2, and PSRC1, consistent with previous analyses of this variant using other approaches. Initial investigation of additional SNPs also highlighted correlations with gene expression. These findings suggest that iPSC-based population studies hold promise as tools for the functional validation of GWAS variants.
Asunto(s)
Diferenciación Celular/genética , Estudio de Asociación del Genoma Completo , Células Madre Pluripotentes Inducidas/citología , Enfermedades Metabólicas/genética , Adipocitos Blancos/citología , Adipocitos Blancos/metabolismo , Reprogramación Celular/genética , Cromosomas Humanos Par 1/genética , Estudios de Cohortes , Regulación hacia Abajo/genética , Genotipo , Hepatocitos/citología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/metabolismo , Metabolismo de los Lípidos/genética , Metabolómica , Modelos Genéticos , Fenotipo , Sitios de Carácter Cuantitativo/genética , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN , Donantes de Tejidos , Transcriptoma/genéticaRESUMEN
The utility of human pluripotent stem cells is dependent on efficient differentiation protocols that convert these cells into relevant adult cell types. Here we report the robust and efficient differentiation of human pluripotent stem cells into white or brown adipocytes. We found that inducible expression of PPARG2 alone or combined with CEBPB and/or PRDM16 in mesenchymal progenitor cells derived from pluripotent stem cells programmed their development towards a white or brown adipocyte cell fate with efficiencies of 85%-90%. These adipocytes retained their identity independent of transgene expression, could be maintained in culture for several weeks, expressed mature markers and had mature functional properties such as lipid catabolism and insulin-responsiveness. When transplanted into mice, the programmed cells gave rise to ectopic fat pads with the morphological and functional characteristics of white or brown adipose tissue. These results indicate that the cells could be used to faithfully model human disease.
Asunto(s)
Adipocitos Marrones/metabolismo , Adipocitos Blancos/metabolismo , Diferenciación Celular/genética , Células Madre Pluripotentes/metabolismo , Transgenes/genética , Células 3T3 , Adipocitos Marrones/citología , Adipocitos Blancos/citología , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/citología , Tejido Adiposo Blanco/metabolismo , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Células Cultivadas , Análisis por Conglomerados , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Inmunohistoquímica , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , PPAR gamma/genética , PPAR gamma/metabolismo , Células Madre Pluripotentes/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Trasplante HeterólogoRESUMEN
Incomplete urethral tubularization (hypospadias) and anorectal abnormalities are two common and poorly understood birth defects that affect the extreme caudal midline of the human embryo. We now show that cell surface molecules essential for proper axon pathfinding in the developing nervous system, namely ephrin-B2 and the ephrin receptors EphB2 and EphB3, also play major roles in cell adhesion events that tubularize the urethra and partition the urinary and alimentary tracts. Mice carrying mutations which disrupt the bidirectional signals that these molecules transduce develop with variably penetrant severe hypospadias and incomplete midline fusion of the primitive cloaca. We further show that animals completely lacking ephrin-B2 reverse signaling present a fully penetrant failure in cloacal septation. This results in severe anorectal malformations characterized by an absence of the terminal-most hindgut (rectum) and formation of a fistula that aberrantly connects the intestines to the urethra at the base of the bladder. Consistent with an apparent requisite for both forward and reverse signaling in these caudal remodeling events, EphB2 and ephrin-B2 are coexpressed at the midline in the fusing urethral/cloacal endoderm and underlying lateral mesoderm of the urorectal septum that migrates toward the caudal midline as the cloaca septates. Our data thus indicate that B-subclass Eph and ephrin molecules play an important role in these clinically significant midline cell-cell adhesion and fusion events.