Physiological Plasticity of Neural-Crest-Derived Stem Cells in the Adult Mammalian Carotid Body

Adult stem cell plasticity, or the ability of somatic stem cells to cross boundaries and differentiate into unrelated cell types, has been amatter of debate in the last decade. Neural-crest-derived stem cells (NCSCs) display a remarkable plasticity during development. Whether adult populations of NCSCs retain this plasticity is largely unknown. Herein, we describe that neural-crest-derived adult carotid body stem cells (CBSCs) are able to undergo endothelial differentiation in addition to their reported role in neurogenesis, contributing to both neurogenic and angiogenic processes taking place in the organ during acclimatization to hypoxia. Moreover, CBSC conversion into vascular cell types is hypoxia inducible factor (HIF) dependent and sensitive to hypoxiareleased vascular cytokines such as erythropoietin. Our data highlight a remarkable physiological plasticity in an adult population of tissue-specific stem cells and could have impact on the use of these cells for cell therapy.ERC Starting Grant: CBSCsMinisterio de Economía y Competitividad SAF2013-48535-P and SAF2016-80412-

Rotenone selectively occludes sensitivity to hypoxia in rat carotid body glomus cells

Carotid body glomus cells release transmitters in response to hypoxia due to the increase of excitability resulting from inhibition of O2 -regulated K+ channels. However, the mechanisms involved in the detection of changes of O2 tension are unknown. We have studied the interaction between glomus cell O2 sensitivity and inhibition of the mitochondrial electron transport chain (ETC) in a carotid body thin slice preparation in which catecholamine release from intact single glomus cells can be monitored by amperometry. Inhibition of the mitochondrial ETC at proximal and distal complexes induces external Ca2+-dependent catecholamine secretion. At saturating concentration of the ETC inhibitors, the cellular response to hypoxia is maintained. However, rotenone, a complex I blocker, selectively occludes the responsiveness to hypoxia of glomus cells in a dose-dependent manner. The effect of rotenone is mimicked by 1-methyl-4-phenylpyridinium ion(MPP+), an agent that binds to the same site as rotenone, but not by complex I inhibitors acting on different sites. In addition, the effect of rotenone is not prevented by incubation of the cells with succinate, a substrate of complex II. These data strongly suggest that sensitivity to hypoxia of carotid body glomus cells is not linked in a simple way to mitochondrial electron flow and that a rotenone (and MPP+)-sensitive molecule critically participates in acute oxygen sensing in the carotid body.Ministerio de Ciencia y Tecnología 1FD97–161

Hypoxia in the Initiation and Progression of Neuroblastoma Tumours

Neuroblastoma is the most frequent extracranial solid tumour in children, causing 10% of all paediatric oncology deaths. It arises in the embryonic neural crest due to an uncontrolled behaviour of sympathetic nervous system progenitors, giving rise to heterogeneous tumours. Low local or systemic tissue oxygen concentration has emerged as a cellular stimulus with important consequences for tumour initiation, evolution and progression. In neuroblastoma, several evidences point towards a role of hypoxia in tumour initiation during development, tumour cell differentiation, survival and metastatic spreading. However, the heterogeneous nature of the disease, its developmental origin and the lack of suitable experimental models have complicated a clear understanding of the effect of hypoxia in neuroblastoma tumour progression and the molecular mechanisms implicated. In this review, we have compiled available evidences to try to shed light onto this important field. In particular, we explore the effect of hypoxia in neuroblastoma cell transformation and differentiation. We also discuss the experimental models available and the emerging alternatives to study this problem, and we present hypoxia-related therapeutic avenues being explored in the field.Spanish Ministry of Science and Innovation SAF program (grant number SAF2016-80412-P

Trophic restoration of the nigrostriatal dopaminergic pathway in long-term carotid body-grafted parkinsonian rats

We studied the mechanisms underlying long-term functional recovery of hemiparkinsonian rats grafted intrastriatally with carotid body (CB) cell aggregates. Amelioration of their motor syndrome is a result of the trophic actions of these grafts on the remaining ipsilateral substantia nigra neurons rather than of the release of dopamine from the CB grafts. The grafts maintain a stable morphological appearance and differentiated cell phenotype for the duration of the life of the host. Adult CB expresses high levels of glial cell line-derived neurotrophic factor (GDNF) and the multicomponent GDNF receptor complex. These properties may contribute to the trophic actions of the CB transplants on nigrostriatal neurons and to their extraordinary longevity. We show that CB glomus cells, although highly dopaminergic, are protected from dopamine-mediated oxidative damage because of the absence of the high-affinity dopamine transporter. Thus, intrastriatal CB grafts are uniquely suited for long-term delivery of trophic factors capable of promoting restoration of the nigrostriatal pathway

A protocol to enrich in undifferentiated cells from neuroblastoma tumor tissue samples and cell lines

The existence of a subpopulation of undifferentiated cells with stem-like properties has been suggested in neuroblastoma tumors, but a definitive biomarker for their successful isolation is missing. Here we describe an in vitro culture system for the enrichment in undifferentiated stem-like tumor cells for subsequent functional assays. We make use of clonal non-adherent cell culture conditions together with cell sorting with specific expression markers. This protocol allows for the differential study of heterogeneous cell population in neuroblastoma tumors. For complete details on the use and execution of this protocol, please refer to Vega et al. (2019)

CD44-high neural crest stem-like cells are associated with tumour aggressiveness and poor survival in neuroblastoma tumours

BACKGROUND: Neuroblastoma is a paediatric tumour originated from sympathoadrenal precursors and characterized by its heterogeneity and poor outcome in advanced stages. Intra-tumoral cellular heterogeneity has emerged as an important feature in neuroblastoma, with a potential major impact on tumour aggressiveness and response to therapy. CD44 is an adhesion protein involved in tumour progression, metastasis and stemness in different cancers; however, there has been controversies about the significance of CD44 expression in neuroblastoma and its relationship with tumour progression. METHODS: We have performed transcriptomic analysis on patient tumour samples studying the outcome of patients with high CD44 expression. Adhesion, invasion and proliferation assays were performed in sorted CD44high neuroblastoma cells. Tumoursphere cultures have been used to enrich in undifferentiated stem-like cells and to asses self-renewal and differentiation potential. We have finally performed in vivo tumorigenic assays on cell line-derived or Patient-derived xenografts. FINDINGS: We show that high CD44 expression is associated with low survival in high-grade human neuroblastoma, independently of MYCN amplification. CD44 is expressed in a cell population with neural crest stem-like features, and with the capacity to generate multipotent, undifferentiated tumourspheres in culture. These cells are more invasive and proliferative in vitro. CD44 positive cells obtained from tumours are more tumorigenic and metastatic, giving rise to aggressive neuroblastic tumours at high frequency upon transplantation. INTERPRETATION: We describe an unexpected intra-tumoural heterogeneity within cellular entities expressing CD44 in neuroblastoma, and propose that CD44 has a role in neural crest stem-like undifferentiated cells, which can contribute to tumorigenesis and malignancy in this type of cancer. FUNDING: Research supported by grants from the "Asociación Española contra el Cáncer" (AECC), the Spanish Ministry of Science and Innovation SAF program (SAF2016-80412-P), and the European Research Council (ERC Starting Grant to RP).Spanish Ministry of Science and Innovation SAF program (SAF2016-80412-P

Physiology of the adult carotid body germinal niche

Trabajo presentado en las CABIMER Confenrece Series 2020-2021.Peer reviewe

Sensibilidad a hipoxia e hipoglucemia de células glómicas en rodajas de cuerpo carotídeo

Los objetivos generales del presente trabajo fueron los siguientes: 1. Puesta a punto de la preparación de rodajas finas de cuerpo carotídeo. Nos pareció importante el desarrollo de esta nueva técnica porque, al igual que ocurre con las rodajas finas de tejido cerebral, esta preparación permitiría el estudio de la actividad secretora y de las características electrofisiológicas en células individuales identificadas en las rodajas bajo control visual. Por otra parte, la preparación evitaría el trauma que supone para las células el tratamiento enzimático y la dispersión mecánica. 2. Caracterización electrofisiológica general de las células glómicas en las rodajas y comparación de sus propiedades (tipos de corrientes, actividad secretora, sensibilidad a hipoxia, etc) con las descritas en células dispersas enzimáticamente. 3. Estudio del efecto de bloqueantes de canales de potasio sobre la actividad secretora de las células glómicas en las rodajas. Con este análisis se pretendió demostrar si, como predice el modelo de la membrana descrito en “Introducción”, la inhibición de la actividad de los canales de potasio voltaje-dependientes participa de forma determinante en el mecanismo de quimiotransducción de la hipoxia en el cuerpo carotídeo de rata. 4. Análisis sistemático del efecto del bloqueo mitocondrial a diferentes niveles sobre la actividad secretora de células glómicas intactas y relaciones entre la sensibilidad a la hipoxia y la intoxicación mitocondrial. Se pretendió explorar si algún componente de la cadena de transporte de electrones podría actuar como sensor de O2 y si la hipoxia debida a la disminución de la pO2 es equivalente a la estimulación celular resultante de la intoxicación metabólica. 5. Estudio del posible papel glucosensor del cuerpo carotídeo, analizando la sensibilidad de las células glómicas a la hipoglucemia y el posible mecanismo de la detección de glucosa. Nos planteamos este objetivo dado el importante papel como quimiorreceptor arterial del cuerpo carotídeo y la extremada sensibilidad de las neuronas centrales a la isquemia (hipoxia más hipoglucemia). Se postuló, por tanto, que el cuerpo carotídeo podría participar en la regulación de la glucemia