Hipertrofia del cuerpo carotídeo en hipoxia crónica: mecanismos de activación, proliferación y diferenciación de los progenitores neurales en el sistema nervioso periférico

Tesis descargada de TESEOThe goal of this doctoral thesis is to achieve a better understanding of the biology and behavior of the recently discovered adult carotid body neural stem cells. The work focuses on two main objectives. First, to study from a physiological and cellular standpoint, the mechanisms involved in the activation of carotid body neural stem cells; and secondly, to unravel the genes and molecular pathways involved in stemness, maintenance and differentiation of these cells. An O2-sensitive glomus cell-stem cell synapse induces carotid body growth in chronic hypoxia Adult stem cells reside within specific ¿niches¿ which provide the appropriate environment to maintain their ability for self-renewal and multipotency. These cells are normally in a dormant state that protects them from stressors; the manner by which they are selectively activated to progress from quiescence to differentiated mature cells is still to be resolved (Suda et al., 2011; Chell and Frisen, 2012). Neural stem cells (NSC), which resemble embryonic radial glia-like cells, and are able to generate new neurons and glial cells, persist in two niches in the adult mammalian central nervous system: the subventricular zone (SVZ) and the subgranular layer of the hippocampus (SGZ) (Alvarez-Buylla and Lim, 2004; Zhao et al., 2008). Central neurogenesis is crucial for numerous brain functions and its impairment could be involved in some neuropsychiatric disorders (Kriegstein and Alvarez-Buylla, 2009; Ming and Song). NSC can sense neuronal activity, as a result of which adult neurogenesis is modulated by experience and environmental stimuli. However, the coupling of lineage progression to physiological demand remains poorly understood (Hoglinger et al., 2004; Liu et al., 2005; Ge et al., 2006; Song et al., 2012). Multipotent NSC of glial lineage also exist in the adult carotid body (CB), a neural crest-derived paired organ located in the carotid bifurcation (Pardal et al., 2007). The CB is composed of clusters (glomeruli) of neuron-like glomus (type I) cells that are electrically excitable and have numerous secretory vesicles containing neurotransmitters and neuropeptides. Glomus cells are surrounded by processes of glia-like sustentacular (type II) cells. This organ is the main arterial chemoreceptor that mediates reflex hyperventilation during hypoxemia. Glomus cells, the primary O2-sensing elements in the CB, depolarize in response to hypoxia, thereby releasing neurotransmitters that activate sensory nerve fibers terminating in the brainstem respiratory center (Lopez-Barneo et al., 2001). In addition to this fundamental role in acute oxygen sensing, the CB exhibits a remarkable structural plasticity that is uncommon for a neural tissue, which is manifested upon chronic exposure to hypoxia. The CB grows to several times its normal size during acclimatization in high altitude dwellers (Arias-Stella and Valcarcel, 1976) or in hypoxemic patients suffering cardiopulmonary disorders (Heath et al., 1982). We have shown that the glia-like type II cells, selectively expressing glial fibrillary acidic protein (GFAP), are NSC and contribute to CB growth in hypoxia. These cells form clonal colonies in vitro that are enriched in proliferating Nestin-positive(+) progenitors that give rise to mature glomus cells and other neural crest cell lineages. Similarly, cell fate experiments in vivo have demonstrated that NSC contribute to the generation of new glomus cells in animals exposed to sustained hypoxia (Pardal et al., 2007). Stem cells in the CB neurogenic center are quiescent under normoxic conditions. Nonetheless, they become activated upon lowering blood O2 tension (hypoxia), a well-defined and controllable variable. Therefore, the CB niche provides an ideal model in which to study activity-dependent neurogenesis and to explore the mechanisms whereby stem cells switch from dormancy to cycling. Herein we show that, unexpectedly, CB NSC proliferation in vitro is insensitive to hypoxia over a broad range of O2 tensions. We provide compelling structural and functional evidence supporting the existence of abundant direct ¿synaptic¿ contacts between mature neuron-like, O2-sensitive, glomus cells and glia-like progenitors, thus optimizing the activity-dependent stimulation of stem cells. The release of stored neurotransmitters from glomus cells during hypoxia induces the proliferation of progenitor cells and growth of the CB. Among the substances released by glomus cells we have identified endothelin-1 (ET-1), an agent involved in neural crest progenitor specification and migration (Shin et al., 1999; Bonano et al., 2008), as a powerful activator of CB stem cell proliferation in vitro and in vivo. In this way, O2-sensing glomus cells mediate both the acute activation of the respiratory center and the chronic induction of CB growth upon exposure to hypoxia. Gene expression profile in adult carotid body stem cells The molecular mechanisms underlying CB stem cell proliferation and differentiation are poorly known. We have set up conditions in vitro to enrich neurosphere cultures in undifferentiated (NSundiff) or differentiated (NSdiff) cells. We performed gene expression studies by microarray techniques to identify molecules and pathways involved in the biology of CB progenitor cells. Microarray results were validated by PCR analyses of individual molecules, thus confirming that proliferation genes and markers of stemness and undifferentiated state are mainly expressed in the NSundiff sample, whereas neuronal genes are highly expressed in differentiated cultures. The analysis in silico performed by IPA software shows that CB stem cells conserve the ability to give rise to several neural crest derivatives. The results confirm that multipotent CB progenitors are neural crest-derived stem cells that persist in the adult organ. CB stem cells cannot be prospectively isolated due to the lack of selective membrane markers suitable for cell sorting. We have identified CD10, a membrane metalloendopeptidase, as the most highly expressed surface marker in NSundiff. CD10 has allowed the sorting of a purified CB progenitor population, which is being currently studied and characterized in our laboratory. In summary, this study increases our knowledge on the molecular and cellular properties of CB stem cells, the only neurogenic niche known so far in the adult peripheral nervous system. Our results could be relevant for the understanding of hypoxia-associated pathologies, and for the use of CB stem cells in cell therapy against neurodegenerative disorders

Hipertrofia del cuerpo carotídeo en hipoxia crónica: mecanismos deactivación, proliferación y diferenciación de los progenitores neuralesen el sistema nervioso periférico

The goal of this doctoral thesis is to achieve a better understanding of the biology and behavior of the recently discovered adult carotid body neural stem cells. The work focuses on two main objectives. First, to study from a physiological and cellular standpoint, the mechanisms involved in the activation of carotid body neural stem cells; and secondly, to unravel the genes and molecular pathways involved in stemness, maintenance and differentiation of these cells. An O2-sensitive glomus cell-stem cell synapse induces carotid body growth in chronic hypoxia Adult stem cells reside within specific ¿niches¿ which provide the appropriate environment to maintain their ability for self-renewal and multipotency. These cells are normally in a dormant state that protects them from stressors; the manner by which they are selectively activated to progress from quiescence to differentiated mature cells is still to be resolved (Suda et al., 2011; Chell and Frisen, 2012). Neural stem cells (NSC), which resemble embryonic radial glia-like cells, and are able to generate new neurons and glial cells, persist in two niches in the adult mammalian central nervous system: the subventricular zone (SVZ) and the subgranular layer of the hippocampus (SGZ) (Alvarez-Buylla and Lim, 2004; Zhao et al., 2008). Central neurogenesis is crucial for numerous brain functions and its impairment could be involved in some neuropsychiatric disorders (Kriegstein and Alvarez-Buylla, 2009; Ming and Song). NSC can sense neuronal activity, as a result of which adult neurogenesis is modulated by experience and environmental stimuli. However, the coupling of lineage progression to physiological demand remains poorly understood (Hoglinger et al., 2004; Liu et al., 2005; Ge et al., 2006; Song et al., 2012). Multipotent NSC of glial lineage also exist in the adult carotid body (CB), a neural crest-derived paired organ located in the carotid bifurcation (Pardal et al., 2007). The CB is composed of clusters (glomeruli) of neuron-like glomus (type I) cells that are electrically excitable and have numerous secretory vesicles containing neurotransmitters and neuropeptides. Glomus cells are surrounded by processes of glia-like sustentacular (type II) cells. This organ is the main arterial chemoreceptor that mediates reflex hyperventilation during hypoxemia. Glomus cells, the primary O2-sensing elements in the CB, depolarize in response to hypoxia, thereby releasing neurotransmitters that activate sensory nerve fibers terminating in the brainstem respiratory center (Lopez-Barneo et al., 2001). In addition to this fundamental role in acute oxygen sensing, the CB exhibits a remarkable structural plasticity that is uncommon for a neural tissue, which is manifested upon chronic exposure to hypoxia. The CB grows to several times its normal size during acclimatization in high altitude dwellers (Arias-Stella and Valcarcel, 1976) or in hypoxemic patients suffering cardiopulmonary disorders (Heath et al., 1982). We have shown that the glia-like type II cells, selectively expressing glial fibrillary acidic protein (GFAP), are NSC and contribute to CB growth in hypoxia. These cells form clonal colonies in vitro that are enriched in proliferating Nestin-positive(+) progenitors that give rise to mature glomus cells and other neural crest cell lineages. Similarly, cell fate experiments in vivo have demonstrated that NSC contribute to the generation of new glomus cells in animals exposed to sustained hypoxia (Pardal et al., 2007).Stem cells in the CB neurogenic center are quiescent under normoxic conditions. Nonetheless, they become activated upon lowering blood O2 tension (hypoxia), a well-defined and controllable variable. Therefore, the CB niche provides an ideal model in which to study activity-dependent neurogenesis and to explore the mechanisms whereby stem cells switch from dormancy to cycling. Herein we show that, unexpectedly, CB NSC proliferation in vitro is insensitive to hypoxia over a broad range of O2 tensions. We provide compelling structural and functional evidence supporting the existence of abundant direct ¿synaptic¿ contacts between mature neuron-like, O2-sensitive, glomus cells and glia-like progenitors, thus optimizing the activity-dependent stimulation of stem cells. The release of stored neurotransmitters from glomus cells during hypoxia induces the proliferation of progenitor cells and growth of the CB. Among the substances released by glomus cells we have identified endothelin-1 (ET-1), an agent involved in neural crest progenitor specification and migration (Shin et al., 1999; Bonano et al., 2008), as a powerful activator of CB stem cell proliferation in vitro and in vivo. In this way, O2-sensing glomus cells mediate both the acute activation of the respiratory center and the chronic induction of CB growth upon exposure to hypoxia.Gene expression profile in adult carotid body stem cells The molecular mechanisms underlying CB stem cell proliferation and differentiation are poorly known. We have set up conditions in vitro to enrich neurosphere cultures in undifferentiated (NSundiff) or differentiated (NSdiff) cells. We performed gene expression studies by microarray techniques to identify molecules and pathways involved in the biology of CB progenitor cells. Microarray results were validated by PCR analyses of individual molecules, thus confirming that proliferation genes and markers of stemness and undifferentiated state are mainly expressed in the NSundiff sample, whereas neuronal genes are highly expressed in differentiated cultures. The analysis in silico performed by IPA software shows that CB stem cells conserve the ability to give rise to several neural crest derivatives. The results confirm that multipotent CB progenitors are neural crest-derived stem cells that persist in the adult organ. CB stem cells cannot be prospectively isolated due to the lack of selective membrane markers suitable for cell sorting. We have identified CD10, a membrane metalloendopeptidase, as the most highly expressed surface marker in NSundiff. CD10 has allowed the sorting of a purified CB progenitor population, which is being currently studied and characterized in our laboratory. In summary, this study increases our knowledge on the molecular and cellular properties of CB stem cells, the only neurogenic niche known so far in the adult peripheral nervous system. Our results could be relevant for the understanding of hypoxia-associated pathologies, and for the use of CB stem cells in cell therapy against neurodegenerative disorders.Peer Reviewe

Elixir of life thwarting aging with regenerative reprogramming

All living beings undergo systemic physiological decline after ontogeny, characterized as aging. Modern medicine has increased the life expectancy, yet this has created an aged society that has more predisposition to degenerative disorders. Therefore, novel interventions that aim to extend the healthspan in parallel to the life span are needed. Regeneration ability of living beings maintains their biological integrity and thus is the major leverage against aging. However, mammalian regeneration capacity is low and further declines during aging. Therefore, modalities that reinforce regeneration can antagonize aging. Recent advances in the field of regenerative medicine have shown that aging is not an irreversible process. Conversion of somatic cells to embryonic-like pluripotent cells demonstrated that the differentiated state and age of a cell is not fixed. Identification of the pluripotency-inducing factors subsequently ignited the idea that cellular features can be reprogrammed by defined factors that specify the desired outcome. The last decade consequently has witnessed a plethora of studies that modify cellular features including the hallmarks of aging in addition to cellular function and identity in a variety of cell types in vitro. Recently, some of these reprogramming strategies have been directly used in animal models in pursuit of rejuvenation and cell replacement. Here, we review these in vivo reprogramming efforts and discuss their potential use to extend the longevity by complementing or augmenting the regenerative capacity

The carotid body: a physiologically relevant germinal niche in the adult peripheral nervous system

Oxygen constitutes a vital element for the survival of every single cell in multicellular aerobic organisms like mammals. A complex homeostatic oxygen-sensing system has evolved in these organisms, including detectors and effectors, to guarantee a proper supply of the element to every cell. The carotid body represents the most important peripheral arterial chemoreceptor organ in mammals and informs about hypoxemic situations to the effectors at the brainstem cardiorespiratory centers. To optimize organismal adaptation to maintained hypoxemic situations, the carotid body has evolved containing a niche of adult tissue-specific stem cells with the capacity to differentiate into both neuronal and vascular cell types in response to hypoxia. These neurogenic and angiogenic processes are finely regulated by the niche and by hypoxia itself. Our recent data on the cellular and molecular mechanisms underlying the functioning of this niche might help to comprehend a variety of different diseases coursing with carotid body failure, and might also improve our capacity to use these stem cells for the treatment of neurological disease. Herein, we review those data about the recent characterization of the carotid body niche, focusing on the study of the phenotype and behavior of multipotent stem cells within the organ, comparing them with other well-documented neural stem cells within the adult nervous system

Carotid body oxygen sensing and adaptation to hypoxia

The carotid body (CB) is the principal arterial chemoreceptor that mediates the hyperventilatory response to hypoxia. Our understanding of CB function and its role in disease mechanisms has progressed considerably in the last decades, particularly in recent years. The sensory elements of the CB are the neuron-like glomus cells, which contain numerous transmitters and form synapses with afferent sensory fibers. The activation of glomus cells under hypoxia mainly depends on the modulation of O-sensitive K channels which leads to cell depolarization and the opening of Ca channels. This model of sensory transduction operates in all mammalian species studied thus far, including man. However, the molecular mechanisms underlying the modulation of ion channel function by changes in the O level are as yet unknown. The CB plays a fundamental role in acclimatization to sustained hypoxia. Mice with CB atrophy or patients who have undergone CB resection due to surgical treatments show a marked intolerance to even mild hypoxia. CB growth under hypoxia is supported by the existence of a resident population of neural crest-derived stem cells of glia-like phenotype. These stem cells are not highly affected by exposure to low O tension; however, there are abundant synapse-like contacts between the glomus cells and stem cells (chemoproliferative synapses), which may be needed to trigger progenitor cell proliferation and differentiation under hypoxia. CB hypo- or hyper-activation may also contribute to the pathogenesis of several prevalent human diseases.This work has been supported by grants from the Botín Foundation, the Spanish Ministries of Health and Science, and the European Research Council (European Union).Peer Reviewe

Interspecies chimerism with mammalian pluripotent stem cells

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos

Carotid body oxygen sensing and adaptation to hipoxia

The carotid body (CB) is the principal arterial chemoreceptor that mediates the hyperventilatory response to hypoxia. Our understanding of CB function and its role in disease mechanisms has progressed considerably in the last decades, particularly in recent years. The sensory elements of the CB are the neuron-like glomus cells, which contain numerous transmitters and form synapses with afferent sensory fibers. The activation of glomus cells under hypoxia mainly depends on the modulation of O2-sensitive K(+) channels which leads to cell depolarization and the opening of Ca(2+) channels. This model of sensory transduction operates in all mammalian species studied thus far, including man. However, the molecular mechanisms underlying the modulation of ion channel function by changes in the O2 level are as yet unknown. The CB plays a fundamental role in acclimatization to sustained hypoxia. Mice with CB atrophy or patients who have undergone CB resection due to surgical treatments show a marked intolerance to even mild hypoxia. CB growth under hypoxia is supported by the existence of a resident population of neural crest-derived stem cells of glia-like phenotype. These stem cells are not highly affected by exposure to low O2 tension; however, there are abundant synapse-like contacts between the glomus cells and stem cells (chemoproliferative synapses), which may be needed to trigger progenitor cell proliferation and differentiation under hypoxia. CB hypo- or hyper-activation may also contribute to the pathogenesis of several prevalent human diseases

In vivo amelioration of age-associated hallmarks by partial reprogramming

Aging is the major risk factor for many human diseases. In vitro studies have demonstrated that cellular reprogramming to pluripotency reverses cellular age, but alteration of the aging process through reprogramming has not been directly demonstrated in vivo. Here, we report that partial reprogramming by short-term cyclic expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in a mouse model of premature aging. Similarly, expression of OSKM in vivo improves recovery from metabolic disease and muscle injury in older wild-type mice. The amelioration of age-associated phenotypes by epigenetic remodeling during cellular reprogramming highlights the role of epigenetic dysregulation as a driver of mammalian aging. Establishing in vivo platforms to modulate age-associated epigenetic marks may provide further insights into the biology of aging

Gene expression profiling supports the neural crest origin of adult rodent carotid body stem cells and identifies CD10 as a marker for mesectoderm-committed progenitors

Neural stem cells (NSCs) are promising tools for understanding nervous system plasticity and repair, but their use is hampered by the lack of markers suitable for their prospective isolation and characterization. The carotid body (CB) contains a population of peripheral NSCs, which support organ growth during acclimatization to hypoxia. We have set up CB neurosphere (NS) cultures enriched in differentiated neuronal (glomus) cells versus undifferentiated progenitors to investigate molecular hallmarks of cell classes within the CB stem cell (CBSC) niche. Microarray gene expression analysis in NS is compatible with CBSCs being neural crest derived-multipotent progenitor cells able to sustain CB growth upon exposure to hypoxia. Moreover, we have identified CD10 as a marker suitable for isolation of a population of CB mesectoderm-committed progenitor cells. CD10 + cells are resting in normoxia, and during hypoxia they are activated to proliferate and to eventually complete maturation into mesectodermal cells, thus participating in the angiogenesis necessary for CB growth. Our results shed light into the molecular and cellular mechanisms involved in CBSC fate choice, favoring a potential use of these cells for cell therapy

Mitochondrial replacement in human oocytes carrying pathogenic mitochondrial DNA mutations

Erratum in • Author Correction: Mitochondrial replacement in human oocytes carrying pathogenic mitochondrial DNA mutations. Kang E, Wu J, Gutierrez NM, Koski A, Tippner-Hedges R, Agaronyan K, Platero-Luengo A, Martinez-Redondo P, Ma H, Lee Y, Hayama T, Van Dyken C, Wang X, Luo S, Ahmed R, Li Y, Ji D, Kayali R, Cinnioglu C, Olson S, Jensen J, Battaglia D, Lee D, Wu D, Huang T, Wolf DP, Temiakov D, Belmonte JCI, Amato P, Mitalipov S.Nature. 2019 Mar;567(7747):E5-E9. doi: 10.1038/s41586-019-0876-1.PMID: 30814738Maternally inherited mitochondrial (mt)DNA mutations can cause fatal or severely debilitating syndromes in children1,2,3, with disease severity dependent on the specific gene mutation and the ratio of mutant to wild-type mtDNA (heteroplasmy) in each cell and tissue4. Pathogenic mtDNA mutations are relatively common, with an estimated 778 affected children born each year in the United States5. Mitochondrial replacement therapies or techniques (MRT) circumventing mother–to–child mtDNA disease transmission involve replacement of oocyte maternal mtDNA6,7,8. Here we report MRT outcomes in several families with common mtDNA syndromes. The mother’s oocytes were of normal quality and mutation levels correlated with those in existing children. Efficient replacement of oocyte mutant mtDNA was performed by spindle transfer8, resulting in embryos containing >99% donor mtDNA. Donor mtDNA was stably maintained in embryonic stem cells (ES cells) derived from most embryos. However, some ES cell lines demonstrated gradual loss of donor mtDNA and reversal to the maternal haplotype. In evaluating donor–to–maternal mtDNA interactions, it seems that compatibility relates to mtDNA replication efficiency rather than to mismatch or oxidative phosphorylation dysfunction. We identify a polymorphism within the conserved sequence box II region of the D-loop as a plausible cause of preferential replication of specific mtDNA haplotypes. In addition, some haplotypes confer proliferative and growth advantages to cells. Hence, we propose a matching paradigm for selecting compatible donor mtDNA for MRT