RESUMEN
Fluid shear stress promotes the emergence of hematopoietic stem cells (HSCs) in the aorta-gonad-mesonephros (AGM) of the developing mouse embryo. We determined that the AGM is enriched for expression of targets of protein kinase A (PKA)-cAMP response element-binding protein (CREB), a pathway activated by fluid shear stress. By analyzing CREB genomic occupancy from chromatin-immunoprecipitation sequencing (ChIP-seq) data, we identified the bone morphogenetic protein (BMP) pathway as a potential regulator of CREB. By chemical modulation of the PKA-CREB and BMP pathways in isolated AGM VE-cadherin(+) cells from mid-gestation embryos, we demonstrate that PKA-CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor. Finally, we observed blunting of this signaling axis using Ncx1-null embryos, which lack a heartbeat and intravascular flow. Collectively, we have identified a novel PKA-CREB-BMP signaling pathway downstream of shear stress that regulates HSC emergence in the AGM via the endothelial-to-hematopoietic transition.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Embrión de Mamíferos/embriología , Células Madre Hematopoyéticas/metabolismo , Transducción de Señal/fisiología , Animales , Proteínas Morfogenéticas Óseas/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Embrión de Mamíferos/citología , Células Endoteliales/citología , Células Endoteliales/metabolismo , Células Madre Hematopoyéticas/citología , Mesonefro/citología , Mesonefro/embriología , Ratones , Ratones Mutantes , Intercambiador de Sodio-Calcio/genética , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
The first hematopoietic stem cells (HSCs) that engraft irradiated adult mice arise in the aorta-gonad-mesonephros (AGM) on embryonic day 11.5 (E11.5). However, at this stage, there is a discrepancy between the apparent frequency of HSCs depicted with imaging and their rarity when measured with limiting dilution transplant. We have attempted to reconcile this difference using neonatal recipients, which are more permissive for embryonic HSC engraftment. We found that embryonic HSCs from E9.5 and E10.5 preferentially engrafted neonates, whereas developmentally mature, definitive HSCs from E14.5 fetal liver or adult bone marrow (BM) more robustly engrafted adults. Neonatal engraftment was enhanced after treating adult BM-derived HSCs with interferon. Adult BM-derived HSCs preferentially homed to the liver in neonatal mice yet showed balanced homing to the liver and spleen in adults. These findings emphasize the functional differences between nascent and mature definitive HSCs.
Asunto(s)
Trasplante de Médula Ósea , Embrión de Mamíferos/citología , Células Madre Embrionarias/citología , Supervivencia de Injerto/fisiología , Células Madre Hematopoyéticas/citología , Hígado/fisiología , Bazo/fisiología , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Diferenciación Celular , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/metabolismo , Femenino , Perfilación de la Expresión Génica , Gónadas/citología , Gónadas/metabolismo , Células Madre Hematopoyéticas/metabolismo , Mesonefro/citología , Mesonefro/metabolismo , Ratones , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Human pluripotent stem cells (hPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens, and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor cells (HSPCs) has limited their characterization to in vitro assays. We report a strategy to respecify lineage-restricted CD34(+)CD45(+) myeloid precursors derived from hPSCs into multilineage progenitors that can be expanded in vitro and engrafted in vivo. HOXA9, ERG, and RORA conferred self-renewal and multilineage potential in vitro and maintained primitive CD34(+)CD38(-) cells. Screening cells via transplantation revealed that two additional factors, SOX4 and MYB, conferred engraftment. Progenitors specified with all five factors gave rise to reproducible short-term engraftment with myeloid and erythroid lineages. Erythroid precursors underwent hemoglobin switching in vivo, silencing embryonic and activating adult globin expression. Our combinatorial screening approach establishes a strategy for obtaining transcription-factor-mediated engraftment of blood progenitors from human pluripotent cells.
Asunto(s)
Linaje de la Célula , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Células Madre Multipotentes/citología , Células Madre Pluripotentes/citología , Antígenos CD34/metabolismo , Humanos , Antígenos Comunes de Leucocito/metabolismo , Células Madre Multipotentes/metabolismo , Células Madre Pluripotentes/metabolismoRESUMEN
Optimal surgical treatment of unstable sternal fractures is controversial. Wiring provides suboptimal fixation and adaptations of existing non-sternum specific plating systems may be dangerous when rapid sternal reentry is required. We present our experience with the sternal specific fixation system, SternaLock (Biomet Microfixation Inc, Jacksonville, FL), in the acute treatment of transverse sternal body fractures in 2 patients who sustained significant blunt anterior chest wall trauma. SternaLock provides the rigid sternal fixation necessary for reliable fracture healing while offering advantages over other systems with regards to ease of use and safety.
