RESUMO
Human natural killer (NK) cell-based therapies are under assessment for treating various cancers, but cryopreservation reduces both the recovery and function of NK cells, thereby limiting their therapeutic feasibility. Using cryopreservation protocols optimized for T cells, here we find that ~75% of NK cells die within 24 h post-thaw, with the remaining cells displaying reduced cytotoxicity. Using CRISPR-Cas9 gene editing and confocal microscopy, we find that cryopreserved NK cells largely die via apoptosis initiated by leakage of granzyme B from cytotoxic vesicles. Pretreatment of NK cells with a combination of Interleukins-15 (IL-15) and IL-18 prior to cryopreservation improves NK cell recovery to ~90-100% and enables equal tumour control in a xenograft model of disseminated Raji cell lymphoma compared to non-cryopreserved NK cells. The mechanism of IL-15 and IL-18-induced protection incorporates two mechanisms: a transient reduction in intracellular granzyme B levels via degranulation, and the induction of antiapoptotic genes.
Assuntos
Apoptose , Criopreservação , Granzimas , Interleucina-15 , Interleucina-18 , Células Matadoras Naturais , Granzimas/metabolismo , Interleucina-15/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Humanos , Interleucina-18/metabolismo , Animais , Criopreservação/métodos , Camundongos , Linhagem Celular Tumoral , Sistemas CRISPR-CasRESUMO
Splicing factor mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but how they alter cellular functions is unclear. We show that the pathogenic SRSF2P95H/+ mutation disrupts the splicing of mitochondrial mRNAs, impairs mitochondrial complex I function, and robustly increases mitophagy. We also identified a mitochondrial surveillance mechanism by which mitochondrial dysfunction modifies splicing of the mitophagy activator PINK1 to remove a poison intron, increasing the stability and abundance of PINK1 mRNA and protein. SRSF2P95H-induced mitochondrial dysfunction increased PINK1 expression through this mechanism, which is essential for survival of SRSF2P95H/+ cells. Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in SRSF2P95H/+ cells. These data reveal a homeostatic mechanism for sensing mitochondrial stress through PINK1 splicing and identify increased mitophagy as a disease marker and a therapeutic vulnerability in SRSF2P95H mutant MDS and AML.
RESUMO
Splicing factor mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but how they alter cellular functions is unclear. We show that the pathogenic SRSF2P95H/+ mutation disrupts the splicing of mitochondrial mRNAs, impairs mitochondrial complex I function, and robustly increases mitophagy. We also identified a mitochondrial surveillance mechanism by which mitochondrial dysfunction modifies splicing of the mitophagy activator PINK1 to remove a poison intron, increasing the stability and abundance of PINK1 mRNA and protein. SRSF2P95H-induced mitochondrial dysfunction increased PINK1 expression through this mechanism, which is essential for survival of SRSF2P95H/+ cells. Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in SRSF2P95H/+ cells. These data reveal a homeostatic mechanism for sensing mitochondrial stress through PINK1 splicing and identify increased mitophagy as a disease marker and a therapeutic vulnerability in SRSF2P95H mutant MDS and AML.
Assuntos
Leucemia Mieloide Aguda , Mitocôndrias , Mitofagia , Proteínas Quinases , Fatores de Processamento de Serina-Arginina , Animais , Humanos , Camundongos , Substituição de Aminoácidos , Linhagem Celular Tumoral , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitofagia/genética , Mutação de Sentido Incorreto , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Síndromes Mielodisplásicas/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Splicing de RNA , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismoRESUMO
Proper preimplantation development is essential to assemble a blastocyst capable of implantation. Live imaging has uncovered major events driving early development in mouse embryos; yet, studies in humans have been limited by restrictions on genetic manipulation and lack of imaging approaches. We have overcome this barrier by combining fluorescent dyes with live imaging to reveal the dynamics of chromosome segregation, compaction, polarization, blastocyst formation, and hatching in the human embryo. We also show that blastocyst expansion mechanically constrains trophectoderm cells, causing nuclear budding and DNA shedding into the cytoplasm. Furthermore, cells with lower perinuclear keratin levels are more prone to undergo DNA loss. Moreover, applying trophectoderm biopsy, a mechanical procedure performed clinically for genetic testing, increases DNA shedding. Thus, our work reveals distinct processes underlying human development compared with mouse and suggests that aneuploidies in human embryos may not only originate from chromosome segregation errors during mitosis but also from nuclear DNA shedding.
Assuntos
Diagnóstico Pré-Implantação , Gravidez , Feminino , Humanos , Animais , Camundongos , Diagnóstico Pré-Implantação/métodos , Blastocisto , Implantação do Embrião , Testes Genéticos/métodos , Aneuploidia , Biópsia/métodosRESUMO
During preimplantation development, contractile forces generated at the apical cortex segregate cells into inner and outer positions of the embryo, establishing the inner cell mass (ICM) and trophectoderm. To which extent these forces influence ICM-trophectoderm fate remains unresolved. Here, we found that the nuclear lamina is coupled to the cortex via an F-actin meshwork in mouse and human embryos. Actomyosin contractility increases during development, upregulating Lamin-A levels, but upon internalization cells lose their apical cortex and downregulate Lamin-A. Low Lamin-A shifts the localization of actin nucleators from nucleus to cytoplasm increasing cytoplasmic F-actin abundance. This results in stabilization of Amot, Yap phosphorylation and acquisition of ICM over trophectoderm fate. By contrast, in outer cells, Lamin-A levels increase with contractility. This prevents Yap phosphorylation enabling Cdx2 to specify the trophectoderm. Thus, forces transmitted to the nuclear lamina control actin organization to differentially regulate the factors specifying lineage identity.
Assuntos
Actinas , Proteínas Adaptadoras de Transdução de Sinal , Humanos , Animais , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Lâmina Nuclear/metabolismo , Proteínas de Ciclo Celular , Proteínas de Sinalização YAP , Blastocisto/metabolismo , LaminasRESUMO
During mammalian development, the first asymmetric cell divisions segregate cells into inner and outer positions of the embryo to establish the pluripotent and trophectoderm lineages. Typically, polarity components differentially regulate the mitotic spindle via astral microtubule arrays to trigger asymmetric division patterns. However, early mouse embryos lack centrosomes, the microtubule-organizing centres (MTOCs) that usually generate microtubule asters. Thus, it remains unknown whether spindle organization regulates lineage segregation. Here we find that heterogeneities in cell polarity in the early 8-cell-stage mouse embryo trigger the assembly of a highly asymmetric spindle organization. This spindle arises in an unusual modular manner, forming a single microtubule aster from an apically localized, non-centrosomal MTOC, before joining it to the rest of the spindle apparatus. When fully assembled, this 'monoastral' spindle triggers spatially asymmetric division patterns to segregate cells into inner and outer positions. Moreover, the asymmetric inheritance of spindle components causes differential cell polarization to determine pluripotent versus trophectoderm lineage fate.
Assuntos
Diferenciação Celular , Divisão Celular , Linhagem da Célula , Polaridade Celular , Embrião de Mamíferos/fisiologia , Fuso Acromático/fisiologia , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fuso Acromático/genética , Fuso Acromático/metabolismoRESUMO
Failure of neural tube closure during embryonic development can result in anencephaly, one of the most common birth defects in humans. A family with recurrent anencephalic fetuses was investigated to understand its etiology and pathogenesis. Exome sequencing revealed a recessive germline 21-bp in-frame deletion in NUAK2 segregating with the disease. In vitro kinase assays demonstrated that the 7-amino acid truncation in NUAK2, a serine/threonine kinase, completely abrogated its catalytic activity. Patient-derived disease models including neural progenitor cells and cerebral organoids showed that loss of NUAK2 activity led to decreased Hippo signaling via cytoplasmic YAP retention. In neural tube-like structures, endogenous NUAK2 colocalized apically with the actomyosin network, which was disrupted in patient cells, causing impaired nucleokinesis and apical constriction. Our results establish NUAK2 as an indispensable kinase for brain development in humans and suggest that a NUAK2-Hippo signaling axis regulates cytoskeletal processes that govern cell shape during neural tube closure.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Anencefalia/genética , Mutação com Perda de Função/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Agregação Celular , Consanguinidade , Regulação para Baixo/genética , Feminino , Feto/patologia , Genes Recessivos , Via de Sinalização Hippo , Humanos , Masculino , Células-Tronco Neurais/metabolismo , Tubo Neural/patologia , Organoides/patologia , Linhagem , Domínios Proteicos , Proteínas Serina-Treonina Quinases/química , Transdução de Sinais , Transcrição Gênica , Turquia , Proteínas de Sinalização YAPRESUMO
Patients lacking PYCR2, a mitochondrial enzyme that synthesizes proline, display postnatal degenerative microcephaly with hypomyelination. Here we report the crystal structure of the PYCR2 apo-enzyme and show that a novel germline p.Gly249Val mutation lies at the dimer interface and lowers its enzymatic activity. We find that knocking out Pycr2 in mice phenocopies the human disorder and depletes PYCR1 levels in neural lineages. In situ quantification of neurotransmitters in the brains of PYCR2 mutant mice and patients revealed a signature of encephalopathy driven by excessive cerebral glycine. Mechanistically, we demonstrate that loss of PYCR2 upregulates SHMT2, which is responsible for glycine synthesis. This hyperglycemia could be partially reversed by SHMT2 knockdown, which rescued the axonal beading and neurite lengths of cultured Pycr2 knockout neurons. Our findings identify the glycine metabolic pathway as a possible intervention point to alleviate the neurological symptoms of PYCR2-mutant patients.
Assuntos
Córtex Cerebral/metabolismo , Glicina Hidroximetiltransferase/metabolismo , Glicina/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Pirrolina Carboxilato Redutases/genética , Adolescente , Animais , Córtex Cerebral/patologia , Pré-Escolar , Feminino , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Humanos , Lactente , Masculino , Camundongos , Camundongos Knockout , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Linhagem , Pirrolina Carboxilato Redutases/deficiênciaRESUMO
Human mutations in KATNB1 (p80) cause severe congenital cortical malformations, which encompass the clinical features of both microcephaly and lissencephaly. Although p80 plays critical roles during brain development, the underlying mechanisms remain predominately unknown. Here, we demonstrate that p80 regulates microtubule (MT) remodeling in combination with NuMA (nuclear mitotic apparatus protein) and cytoplasmic dynein. We show that p80 shuttles between the nucleus and spindle pole in synchrony with the cell cycle. Interestingly, this striking feature is shared with NuMA. Importantly, p80 is essential for aster formation and maintenance in vitro. siRNA-mediated depletion of p80 and/or NuMA induced abnormal mitotic phenotypes in cultured mouse embryonic fibroblasts and aberrant neurogenesis and neuronal migration in the mouse embryonic brain. Importantly, these results were confirmed in p80-mutant harboring patient-derived induced pluripotent stem cells and brain organoids. Taken together, our findings provide valuable insights into the pathogenesis of severe microlissencephaly, in which p80 and NuMA delineate a common pathway for neurogenesis and neuronal migration via MT organization at the centrosome/spindle pole.
Assuntos
Adenosina Trifosfatases/metabolismo , Fibroblastos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Katanina/metabolismo , Microtúbulos/metabolismo , Malformações do Sistema Nervoso/metabolismo , Neurônios/fisiologia , Proteínas Nucleares/metabolismo , Adenosina Trifosfatases/genética , Animais , Proteínas de Ciclo Celular , Dineínas/metabolismo , Células HeLa , Humanos , Katanina/genética , Camundongos , Camundongos Endogâmicos , Mitose/genética , Mutação/genética , Malformações do Sistema Nervoso/genética , Neurogênese/genética , Proteínas Nucleares/genética , RNA Interferente Pequeno/genéticaRESUMO
Katanin is a microtubule-severing complex whose catalytic activities are well characterized, but whose in vivo functions are incompletely understood. Human mutations in KATNB1, which encodes the noncatalytic regulatory p80 subunit of katanin, cause severe microlissencephaly. Loss of Katnb1 in mice confirms essential roles in neurogenesis and cell survival, while loss of zebrafish katnb1 reveals specific roles for katnin p80 in early and late developmental stages. Surprisingly, Katnb1 null mutant mouse embryos display hallmarks of aberrant Sonic hedgehog signaling, including holoprosencephaly. KATNB1-deficient human cells show defective proliferation and spindle structure, while Katnb1 null fibroblasts also demonstrate a remarkable excess of centrioles, with supernumerary cilia but deficient Hedgehog signaling. Our results reveal unexpected functions for KATNB1 in regulating overall centriole, mother centriole, and cilia number, and as an essential gene for normal Hedgehog signaling during neocortical development.
Assuntos
Adenosina Trifosfatases/fisiologia , Centríolos/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Cílios/fisiologia , Adenosina Trifosfatases/genética , Animais , Estudos de Casos e Controles , Proliferação de Células/genética , Proliferação de Células/fisiologia , Centríolos/genética , Córtex Cerebral/anormalidades , Córtex Cerebral/metabolismo , Cílios/genética , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Fibroblastos/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Katanina , Camundongos , Microcefalia/genética , Mutação , Linhagem , Splicing de RNA/genética , População Branca/genética , Peixe-ZebraRESUMO
The ability to convert human somatic cells into induced pluripotent stem cells (iPSCs) is allowing the production of custom-tailored cells for drug discovery and for the study of disease phenotypes at the cellular and molecular level. IPSCs have been derived from patients suffering from a large variety of disorders with different severities. In many cases, disease related phenotypes have been observed in iPSCs or their lineage-specific progeny. Several proof of concept studies have demonstrated that these phenotypes can be reversed in vitro using approved drugs. However, several challenges must be overcome to take full advantage of this technology. Here, we highlight recent advances in the field and discuss the main challenges associated with this technology as it applies to disease modelling.
Assuntos
Doença , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Biológicos , Humanos , FenótipoRESUMO
Somatic tissues in female eutherian mammals are mosaic due to random X inactivation. In contrast to mice, X chromosome reactivation does not occur during the reprogramming of human female somatic cells to induced pluripotent stem cells (iPSCs), although this view is contested. Using balanced populations of female Rett patient and control fibroblasts, we confirm that all cells in iPSC colonies contain an inactive X, and additionally find that all colonies made from the same donor fibroblasts contain the same inactive X chromosome. Notably, this extreme "skewing" toward a particular dominant, active X is also a general feature of primary female fibroblasts during proliferation, and the skewing seen in reprogramming and fibroblast culture can be alleviated by overexpression of telomerase. These results have important implications for in vitro modeling of X-linked diseases and the interpretation of long-term culture studies in cancer and senescence using primary female fibroblast cell lines.
Assuntos
Reprogramação Celular/genética , Cromossomos Humanos X/metabolismo , Telomerase/metabolismo , Animais , Sequência de Bases , Proliferação de Células , Células Cultivadas , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Inativação do Cromossomo XRESUMO
Peripheral somatic sensory neurons (PSNs) are responsible for the critical function of transmitting multiple modalities of information from the outside world, including heat, touch, and pain, as well as the position of muscles required for coordinated voluntary movement to the central nervous system. Many peripheral neuropathies exist, including hereditary neurodegeneration in Familial Dysautonomia, infections of PSNs by viruses such as Varicella zoster and damage to PSNs and/or their process resulting from other disease conditions such as diabetes. Understanding of the etiology of these diseases and development of treatments is hampered by the lack of normal and healthy human PSNs for study, which are only available from abortuses or rare surgical procedures.Human embryonic stem cells (hESCs) are an ideal source of cells for generating normal PSNs for study of disease and drug development, since they can be grown virtually indefinitely in tissue culture and have the potential to form any cell type in the body. Several years ago, we generated human neurons with the molecular characteristics of PSNs from hESCs at low (less than 1%) yields (Pomp et al., Stem Cells 23:923-930, 2005). The present chapter details our most recently improved method that uses 2 rounds of PA6-induction to rapidly generate PSNs at more than 25% purity (Pomp et al., Br. Res. 1230: 50-60, 2008).The neural crest (NC) is a transient multipotent embryonic stem cell population that is the source of PSNs. NC cells give rise to diverse and important tissues in man, but human NC has not been studied because of the difficulty in obtaining 3-5 week human embryos. The methods described in this chapter can also be used to quickly generate large numbers of human NC for study.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Crista Neural/citologia , Células Receptoras Sensoriais/citologia , Animais , Sequência de Bases , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Separação Celular , Técnicas de Cocultura , Primers do DNA/genética , Células-Tronco Embrionárias/metabolismo , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Camundongos , Crista Neural/metabolismo , Inclusão em Parafina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Receptoras Sensoriais/metabolismoRESUMO
Adenovirus is an efficient vector for expression of transgenes in dividing and nondividing cells. However, very few studies of human embryonic stem cells (hESCs) have utilized adenoviral vectors. We examine here the ability of adenovirus to infect naive hESCs and the differentiated derivatives of multiple hESC lines. We found a striking variation in adenovirus infection rates between lines. The variability in infection rates was positively correlated with the expression of the coxsackievirus and adenovirus receptor, but not that of alpha(nu)-integrin. Adenoviral infection did not interfere with the expression of pluripotency markers, even after passaging. In addition, infection did not affect differentiation of hESC-derived neural precursors in vitro. We also found that green fluorescent protein expression mediated by adenovirus can be a useful marker for tracking hESC in xenografts. We conclude that adenovirus is a practical vector for genetic modification of naive hESC from most, but not all lines, but may be more generally useful for gene transfer into differentiated derivatives of hESC lines.
Assuntos
Adenoviridae , Células-Tronco Embrionárias , Vetores Genéticos , Receptores Virais/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Adenoviridae/patogenicidade , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Embrião de Galinha , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Células-Tronco Embrionárias/virologia , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Integrina alfaV/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Receptores Virais/genética , Transplante de Células-Tronco , Transplante HeterólogoRESUMO
Human embryonic stem cells (hESC) have been directed to differentiate into CNS cells with clinical importance. However, for study of development and regeneration of the human PNS, and peripheral neuropathies, it would be useful to have a source of human PNS derivatives. We have demonstrated that peripheral sensory neuron-like cells (PSN) can also be derived from hESC via neural crest-like (NC) intermediates, and from neural progenitors induced from hESC using noggin. Here we report the generation of higher purity PSN from passagable neurospheres (NSP) induced by murine PA6 stromal cells. hESC were cultured with PA6, and colonies that developed a specific morphology were cut from the plates. Culture of these colonies under non-adhesive conditions yielded NSPs. Several NC marker genes were expressed in the NSP, and these were also detected in 3-5week gestation human embryos containing migrating NC. These NSPs passaged for 2-8weeks and re-plated on PA6 gave rise to many Brn3a+/peripherin+ cells, characteristic of early sensory-like neurons. Re-culturing PA6-induced NSP cells with PA6 resulted in about 25% of the human cells in the co-cultures differentiating to PSN after 1week, compared to only about 10% PSN obtained after 3 weeks when noggin-induced NSP were used. Two month adherent cultures of PA6-induced NSP cells contained neurons expressing several PSN neuropeptides, and voltage-dependent currents and action potentials were obtained from a molecularly identified PSN. hESC-derived PA6-induced NSP cells are therefore an excellent potential source of human PSN for study of differentiation and modeling of PNS disease.
Assuntos
Células-Tronco Embrionárias/fisiologia , Crista Neural/fisiologia , Células Receptoras Sensoriais/fisiologia , Biomarcadores , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Adesão Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Análise Citogenética , Eletrofisiologia , Células-Tronco Embrionárias/metabolismo , Humanos , Imuno-Histoquímica , Crista Neural/citologia , Crista Neural/metabolismo , Neuropeptídeos/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Receptoras Sensoriais/metabolismoRESUMO
Neural precursors have been derived from human embryonic stem cells (hESC) using the bone morphogenetic protein antagonist noggin. These neural precursors can be further differentiated to produce neural cells that express central nervous system (CNS) markers. We have recently shown that naive hESC can be directed to differentiate into peripheral sensory (PS) neuron-like cells and putative neural crest precursors by co-culturing with PA6 stromal cells. In the present study, we examine whether hESC-derived neural precursors (NPC) can differentiate into the peripheral nervous system, as well as CNS cells. As little as 1 week after co-culture with PA6 cells, cells with the molecular characteristics of PS neurons and neural crest are observed in the cultures. With increased time in culture, more PS-like neurons appear, in parallel with a reduction in the neural crest-like cells. These results provide the first evidence that neural precursors derived from hESC have the potential to develop into PS neurons-like as well as CNS-like neuronal cells. About 10% of the cells in NPC-PA6 co-cultures express PS neuron markers after 3 weeks, compared with <1% of hESC cultured on PA6. This enrichment for peripheral neurons makes this an attractive system for generation of peripheral neurons for pathophysiology study and drug development for diseases of the peripheral nervous system such as Familial Dysautonomia and varicella virus infection.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Neurônios Aferentes/citologia , Animais , Proteínas de Transporte/metabolismo , Técnicas de Cocultura , Humanos , Camundongos , Nervos Periféricos/citologia , Células Estromais/metabolismoRESUMO
Human embryonic stem cells (hESCs) have been directed to differentiate into neuronal cells using many cell-culture techniques. Central nervous system cells with clinical importance have been produced from hESCs. To date, however, there have been no definitive reports of generation of peripheral neurons from hESCs. We used a modification of the method of Sasai and colleagues for mouse and primate embryonic stem cells to elicit neuronal differentiation from hESCs. When hESCs are cocultured with the mouse stromal line PA6 for 3 weeks, neurons are induced that coexpress (a) peripherin and Brn3a, and (b) peripherin and tyrosine hydroxylase, combinations characteristic of peripheral sensory and sympathetic neurons, respectively. In vivo, peripheral sensory and sympathetic neurons develop from the neural crest (NC). Analysis of expression of mRNAs identified in other species as NC markers reveals that the PA6 cells induce NC-like cells before neuronal differentiation takes place. Several NC markers, including SNAIL, dHAND, and Sox9, are increased at 1 week of coculture relative to naive cells. Furthermore, the expression of several NC marker genes known to be downregulated upon in vivo differentiation of NC derivatives, was observed to be present at lower levels at 3 weeks of PA6-hESC coculture than at 1 week. Our report is the first on the expression of molecular markers of NC-like cells in primates, in general, and in humans, specifically. Our results suggest that this system can be used for studying molecular and cellular events in the almost inaccessible human NC, as well as for producing normal human peripheral neurons for developing therapies for diseases such as familial dysautonomia.
Assuntos
Técnicas de Cultura , Embrião de Mamíferos/citologia , Crista Neural/citologia , Neurônios/metabolismo , Células-Tronco/citologia , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Regulação para Baixo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Dados de Sequência Molecular , Neurônios/citologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9 , Fatores de Transcrição da Família Snail , Sistema Nervoso Simpático/fisiologia , Fatores de Tempo , Fatores de Transcrição/metabolismo , Proteínas de Peixe-ZebraRESUMO
Fer is a nuclear and cytoplasmic tyrosine kinase that is ubiquitously expressed in mammalian cells. Herein we show that Fer sustains a key signaling step in hypoxic cells. Knock-down of the Fer protein using a specific siRNA decreased the production of VEGF by the hypoxic cells. Conversely, ectopic expression of this kinase led to an elevated production of VEGF under hypoxia. At the molecular level, Fer was found to associate with ERK1/2 and this interaction was intensified under hypoxia. Moreover, Fer increased the activation levels of ERK1/2, and reducing the level of Fer, impaired the activation of ERK1/2 in hypoxic cells. Blocking the MEK-ERK1/2 signaling pathway with the MEK inhibitors U0126, or PD98059 led to the abrogation of ERK1/2 activity in hypoxic cells, an effect that was counteracted by Fer. Hence, Fer sustains the activation of ERK1/2 and increases the production of VEGF in hypoxic cells, without affecting the MEK-ERK signaling pathway.