RESUMO
We have generated an induced pluripotent stem cell (iPSC) line KCLi002-A (iOP107) from a female donor, heterozygous for the loss-of-function mutation p.R2447X in the filaggrin gene (FLG). Epidermal keratinocytes were reprogrammed using non-integrating Sendai virus vectors. The entire process of derivation and expansion of iPSCs were performed under xeno-free culture conditions. Characterization of KCLi002-A line included molecular karyotyping, mutation screening using restriction enzyme digestion Sanger sequencing and next generation sequencing (NGS), whereas pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and in vivo teratoma assay.
Assuntos
Heterozigoto , Células-Tronco Pluripotentes Induzidas , Mutação com Perda de Função , Mutação de Sentido Incorreto , Proteínas S100 , Substituição de Aminoácidos , Linhagem Celular , Feminino , Proteínas Filagrinas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas S100/genética , Proteínas S100/metabolismoRESUMO
Stem cells are unique cell populations able to copy themselves exactly as well as specialize into new cell types. Stem cells isolated from early stages of embryo development are pluripotent, i.e., can be differentiated into multiple different cell types. In addition, scientists have found a way of reverting specialized cells from an adult into an embryonic-like state. These cells, that are as effective as cells isolated from early embryos, are termed induced pluripotent stem cells (iPSCs). The potency of iPSC technology is recently being employed by researchers aimed at helping wildlife and environmental conservation efforts. Ambitious attempts using iPSCs are being made to preserve endangered animals as well as reanimate extinct species, merging science fiction with reality. Other research to sustain natural resources and promote animal welfare are exploring iPSCs for laboratory grown animal products without harm to animals offering unorthodox options for creating meat, leather, and fur. There is great potential in iPSC technology and what can be achieved in consumerism, animal welfare, and environmental protection and conservation. Here, we discuss current research in the field of iPSCs and how these research groups are attempting to achieve their goals. Stem Cells Translational Medicine 2019;8:7-13.
Assuntos
Bem-Estar do Animal , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Biotecnologia , Conservação dos Recursos Naturais , Células-Tronco Embrionárias/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismoRESUMO
We have generated an induced pluripotent stem cell (iPSC) line KCLi001-A (iOP118) from a female atopic dermatitis (AD) patient, heterozygous for the loss-of-function mutation c.2282del4 in the filaggrin gene (FLG). Epidermal keratinocytes were reprogrammed using non-integrating Sendai virus vectors. The entire process of derivation and expansion of AD-iPSCs were performed under xeno-free culture conditions. Characterization of KCLi001-A line included molecular karyotyping, mutation screening using restriction enzyme digestion and Sanger sequencing, while pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and by in vivo teratoma assay.
Assuntos
Dermatite Atópica/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Dermatite Atópica/patologia , Feminino , Proteínas Filagrinas , Heterozigoto , Humanos , MutaçãoRESUMO
Loss of the CDKN2A tumor suppressor is associated with melanoma metastasis, but the mechanisms connecting the phenomena are unknown. Using CRISPR-Cas9 to engineer a cellular model of melanoma initiation from primary human melanocytes, we discovered that a lineage-restricted transcription factor, BRN2, is downstream of CDKN2A and directly regulated by E2F1. In a cohort of melanocytic tumors that capture distinct progression stages, we observed that CDKN2A loss coincides with both the onset of invasive behavior and increased BRN2 expression. Loss of the CDKN2A protein product p16INK4A permitted metastatic dissemination of human melanoma lines in mice, a phenotype rescued by inhibition of BRN2. These results demonstrate a mechanism by which CDKN2A suppresses the initiation of melanoma invasion through inhibition of BRN2.
Assuntos
Movimento Celular , Inibidor p16 de Quinase Dependente de Ciclina/genética , Proteínas de Homeodomínio/genética , Perda de Heterozigosidade , Neoplasias Pulmonares/genética , Melanócitos/metabolismo , Melanoma/genética , Fatores do Domínio POU/genética , Neoplasias Cutâneas/genética , Ativação Transcricional , Animais , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Masculino , Melanócitos/patologia , Melanoma/metabolismo , Melanoma/secundário , Camundongos Endogâmicos NOD , Invasividade Neoplásica , Fatores do Domínio POU/metabolismo , Mutação Puntual , Proteínas Proto-Oncogênicas B-raf/genética , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologiaRESUMO
Telomeres are short in type II alveolar epithelial cells (AECs) of patients with idiopathic pulmonary fibrosis (IPF). Whether dysfunctional telomeres contribute directly to development of lung fibrosis remains unknown. The objective of this study was to investigate whether telomere dysfunction in type II AECs, mediated by deletion of the telomere shelterin protein TRF1, leads to pulmonary fibrosis in mice (SPC-Cre TRF1fl/fl mice). Deletion of TRF1 in type II AECs for 2 weeks increased γH2AX DNA damage foci, but not histopathologic changes in the lung. Deletion of TRF1 in type II AECs for up to 9 months resulted in short telomeres and lung remodeling characterized by increased numbers of type II AECs, α-smooth muscle actin+ mesenchymal cells, collagen deposition, and accumulation of senescence-associated ß-galactosidase+ lung epithelial cells. Deletion of TRF1 in collagen-expressing cells caused pulmonary edema, but not fibrosis. These results demonstrate that prolonged telomere dysfunction in type II AECs, but not collagen-expressing cells, leads to age-dependent lung remodeling and fibrosis. We conclude that telomere dysfunction in type II AECs is sufficient to cause lung fibrosis, and may be a dominant molecular defect causing IPF. SPC-Cre TRF1fl/fl mice will be useful for assessing cellular and molecular mechanisms of lung fibrosis mediated by telomere dysfunction.
Assuntos
Células Epiteliais Alveolares/citologia , Pulmão/patologia , Fibrose Pulmonar/patologia , Telômero/patologia , Animais , Células Cultivadas , Células Epiteliais , Fibrose Pulmonar Idiopática , Camundongos , Encurtamento do TelômeroAssuntos
Vírus da Influenza A Subtipo H1N1/metabolismo , Infecções por Orthomyxoviridae , Alvéolos Pulmonares , Mucosa Respiratória , Animais , Camundongos , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Alvéolos Pulmonares/virologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Mucosa Respiratória/virologiaRESUMO
Mechanisms of initial cell fate decisions differ among species. To gain insights into lineage allocation in humans, we derived ten human embryonic stem cell lines (designated UCSFB1-10) from single blastomeres of four 8-cell embryos and one 12-cell embryo from a single couple. Compared with numerous conventional lines from blastocysts, they had unique gene expression and DNA methylation patterns that were, in part, indicative of trophoblast competence. At a transcriptional level, UCSFB lines from different embryos were often more closely related than those from the same embryo. As predicted by the transcriptomic data, immunolocalization of EOMES, T brachyury, GDF15 and active ß-catenin revealed differential expression among blastomeres of 8- to 10-cell human embryos. The UCSFB lines formed derivatives of the three germ layers and CDX2-positive progeny, from which we derived the first human trophoblast stem cell line. Our data suggest heterogeneity among early-stage blastomeres and that the UCSFB lines have unique properties, indicative of a more immature state than conventional lines.
Assuntos
Blastômeros/citologia , Técnicas de Cultura Embrionária , Células-Tronco Embrionárias/citologia , Trofoblastos/citologia , Blastocisto/citologia , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Metilação de DNA , Endoderma/metabolismo , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Fator 15 de Diferenciação de Crescimento/metabolismo , Humanos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Células-Tronco Neurais/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Transcrição Gênica , Transcriptoma , beta Catenina/metabolismoRESUMO
A technique for tracing stem cells and their descendants reveals how the lining of the airways is maintained, and how this process is altered in smokers.
Assuntos
Células-Tronco/metabolismo , Processos Estocásticos , Traqueia/metabolismo , HumanosRESUMO
Cellular stress responses are frequently governed by the subcellular localization of critical effector proteins. Apoptosis-inducing Factor (AIF) or Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH), for example, can translocate from mitochondria to the nucleus, where they modulate apoptotic death pathways. Hypoxia-inducible gene domain 1A (HIGD1A) is a mitochondrial protein regulated by Hypoxia-inducible Factor-1α (HIF1α). Here we show that while HIGD1A resides in mitochondria during physiological hypoxia, severe metabolic stress, such as glucose starvation coupled with hypoxia, in addition to DNA damage induced by etoposide, triggers its nuclear accumulation. We show that nuclear localization of HIGD1A overlaps with that of AIF, and is dependent on the presence of BAX and BAK. Furthermore, we show that AIF and HIGD1A physically interact. Additionally, we demonstrate that nuclear HIGD1A is a potential marker of metabolic stress in vivo, frequently observed in diverse pathological states such as myocardial infarction, hypoxic-ischemic encephalopathy (HIE), and different types of cancer. In summary, we demonstrate a novel nuclear localization of HIGD1A that is commonly observed in human disease processes in vivo.
Assuntos
Núcleo Celular/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Neoplasias/metabolismo , Estresse Fisiológico , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Glioblastoma/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia-Isquemia Encefálica/metabolismo , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Infarto do Miocárdio/metabolismo , Ligação Proteica , Transporte Proteico , Transplante Heterólogo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Mucous cell hyperplasia and airway smooth muscle (ASM) hyperresponsiveness are hallmark features of inflammatory airway diseases, including asthma. Here, we show that the recently identified calcium-activated chloride channel (CaCC) TMEM16A is expressed in the adult airway surface epithelium and ASM. The epithelial expression is increased in asthmatics, particularly in secretory cells. Based on this and the proposed functions of CaCC, we hypothesized that TMEM16A inhibitors would negatively regulate both epithelial mucin secretion and ASM contraction. We used a high-throughput screen to identify small-molecule blockers of TMEM16A-CaCC channels. We show that inhibition of TMEM16A-CaCC significantly impairs mucus secretion in primary human airway surface epithelial cells. Furthermore, inhibition of TMEM16A-CaCC significantly reduces mouse and human ASM contraction in response to cholinergic agonists. TMEM16A-CaCC blockers, including those identified here, may positively impact multiple causes of asthma symptoms.
Assuntos
Canais de Cloreto/metabolismo , Mucinas/metabolismo , Contração Muscular/fisiologia , Músculo Liso/fisiologia , Sistema Respiratório/citologia , Sistema Respiratório/metabolismo , Animais , Anoctamina-1 , Células Cultivadas , Células Epiteliais/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Microscopia de FluorescênciaRESUMO
Placental trophoblasts are key determinants of in utero development. Mouse trophoblast (TB) stem cells, which were first derived over a decade ago, are a powerful cell culture model for studying their self-renewal or differentiation. Our attempts to isolate an equivalent population from the trophectoderm of human blastocysts generated colonies that quickly differentiated in vitro. This finding suggested that the human placenta has another progenitor niche. Here, we show that the chorion is one such site. Initially, we immunolocalized pluripotency factors and TB fate determinants in the early gestation placenta, amnion, and chorion. Immunoreactive cells were numerous in the chorion. We isolated these cells and plated them in medium containing fibroblast growth factor which is required for human embryonic stem cell self-renewal, and an inhibitor of activin/nodal signaling. Colonies of polarized cells with a limited lifespan emerged. Trypsin dissociation yielded continuously self-replicating monolayers. Colonies and monolayers formed the two major human TB lineages-multinucleate syncytiotrophoblasts and invasive cytotrophoblasts (CTBs). Transcriptional profiling experiments revealed the factors associated with the self-renewal or differentiation of human chorionic TB progenitor cells (TBPCs). They included imprinted genes, NR2F1/2, HMGA2, and adhesion molecules that were required for TBPC differentiation. Together, the results of these experiments suggested that the chorion is one source of epithelial CTB progenitors. These findings explain why CTBs of fully formed chorionic villi have a modest mitotic index and identify the chorionic mesoderm as a niche for TBPCs that support placental growth.