RESUMO
In order to support bone marrow regeneration after myeloablation, hematopoietic stem cells (HSCs) actively divide to provide both stem and progenitor cells. However, the mechanisms regulating HSC function and cell fate choice during hematopoietic recovery remain unclear. We herein provide novel insights into HSC regulation during regeneration by focusing on mitochondrial metabolism and ATP citrate lyase (ACLY). After 5-fluorouracil-induced myeloablation, HSCs highly expressing endothelial protein C receptor (EPCRhigh ) were enriched within the stem cell fraction at the expense of more proliferative EPCRLow HSCs. These EPCRHigh HSCs were initially more primitive than EPCRLow HSCs and enabled stem cell expansion by enhancing histone acetylation, due to increased activity of ACLY in the early phase of hematopoietic regeneration. In the late phase of recovery, HSCs enhanced differentiation potential by increasing the accessibility of cis-regulatory elements in progenitor cell-related genes, such as CD48. In conditions of reduced mitochondrial metabolism and ACLY activity, these HSCs maintained stem cell phenotypes, while ACLY-dependent histone acetylation promoted differentiation into CD48+ progenitor cells. Collectively, these results indicate that the dynamic control of ACLY-dependent metabolism and epigenetic alterations is essential for HSC regulation during hematopoietic regeneration.
Assuntos
ATP Citrato (pro-S)-Liase , Medula Óssea , ATP Citrato (pro-S)-Liase/genética , ATP Citrato (pro-S)-Liase/metabolismo , Receptor de Proteína C Endotelial/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Histonas/metabolismoRESUMO
Human endogenous retroviruses (HERVs) occupy approximately 8% of the human genome. HERVs, transcribed in early embryos, are epigenetically silenced in somatic cells, except under pathological conditions. HERV-K is thought to protect embryos from exogenous viral infection. However, uncontrolled HERV-K expression in somatic cells has been implicated in several diseases. Here, we show that SOX2, which plays a key role in maintaining the pluripotency of stem cells, is critical for HERV-K LTR5Hs. HERV-K undergoes retrotransposition within producer cells in the absence of Env expression. Furthermore, we identified new HERV-K integration sites in long-term culture of induced pluripotent stem cells that express SOX2. These results suggest that the strict dependence of HERV-K on SOX2 has allowed HERV-K to protect early embryos during evolution while limiting the potentially harmful effects of HERV-K retrotransposition on host genome integrity in these early embryos. IMPORTANCE Human endogenous retroviruses (HERVs) account for approximately 8% of the human genome; however, the physiological role of HERV-K remains unknown. This study found that HERV-K LTR5Hs and LTR5B were transactivated by SOX2, which is essential for maintaining and reestablishing pluripotency. HERV-K can undergo retrotransposition within producer cells without env expression, and new integration sites may affect cell proliferation. In induced pluripotent stem cells (iPSCs), genomic impairment due to HERV-K retrotransposition has been identified, but it is a rare event. Considering the retention of SOX2-responsive elements in the HERV-K long terminal repeat (LTR) for over 20 million years, we conclude that HERV-K may play important physiological roles in SOX2-expressing cells.
Assuntos
Retrovirus Endógenos , Células-Tronco Pluripotentes Induzidas , Fatores de Transcrição SOXB1 , Retrovirus Endógenos/genética , Humanos , Células-Tronco Pluripotentes Induzidas/virologia , Fatores de Transcrição SOXB1/genética , Sequências Repetidas Terminais/genética , Integração ViralRESUMO
The diagnosis of mosaicism is challenging in patients with neurofibromatosis type 2 (NF2) subset due to low variant allele frequency. In this study, we generated induced pluripotent stem cells (iPSCs) were generated from a patient clinically diagnosed with NF2 based on multiple schwannomas, including bilateral vestibular schwannomas and meningiomas. Genetic analysis of the patient's mononuclear cells (MNCs) from peripheral blood failed to detect NF2 alteration but successfully found p.Q65X (c.193C>T) mutation in all separate tumors with three intracranial meningiomas and one intraorbital schwannoma, and confirming mosaicism diagnosis in NF2 alteration using deep sequencing. Five different clones with patient-derived iPSCs were established from MNCs in peripheral blood, which showed sufficient expression of pluripotent markers. Genetic analysis showed that one of five generated iPSC lines from MNCs had the same p.Q65X mutation as that found in NF2. There was no significant difference in the expression of genes related to NF2 between iPSC clones with the wild-type and mutant NF2. In this case, clonal expansion of mononuclear bone marrow-derived stem cells recapitulated mosaicism's genetic alteration in NF2. Patient-derived iPSCs from mosaic NF2 would contribute to further functional research of NF2 alteration.
Assuntos
Células-Tronco Pluripotentes Induzidas , Neoplasias Meníngeas , Meningioma , Neurofibromatose 2 , Células Clonais/patologia , Genes da Neurofibromatose 2 , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Neoplasias Meníngeas/genética , Meningioma/genética , Mutação , Neurofibromatose 2/diagnóstico , Neurofibromatose 2/genéticaRESUMO
Sialidosis is a neuropathic lysosomal storage disease caused by a deficiency in the NEU1 gene-encoding lysosomal neuraminidase and characterized by abnormal accumulation of undigested sialyl-oligoconjugates in systemic organs including brain. Although patients exhibit neurological symptoms, the underlying neuropathological mechanism remains unclear. Here, we generated induced pluripotent stem cells (iPSCs) from skin fibroblasts with sialidosis and induced the differentiation into neural progenitor cells (NPCs) and neurons. Sialidosis NPCs and neurons mimicked the disease-like phenotypes including reduced neuraminidase activity, accumulation of sialyl-oligoconjugates and lysosomal expansions. Functional analysis also revealed that sialidosis neurons displayed two distinct abnormalities, defective exocytotic glutamate release and augmented α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR)-mediated Ca2+ influx. These abnormalities were restored by overexpression of the wild-type NEU1 gene, demonstrating causative role of neuraminidase deficiency in functional impairments of disease neurons. Comprehensive proteomics analysis revealed the significant reduction of SNARE proteins and glycolytic enzymes in synaptosomal fraction, with downregulation of ATP production. Bypassing the glycolysis by treatment of pyruvate, which is final metabolite of glycolysis pathway, improved both the synaptsomal ATP production and the exocytotic function. We also found that upregulation of AMPAR and L-type voltage dependent Ca2+ channel (VDCC) subunits in disease neurons, with the restoration of AMPAR-mediated Ca2+ over-load by treatment of antagonists for the AMPAR and L-type VDCC. Our present study provides new insights into both the neuronal pathophysiology and potential therapeutic strategy for sialidosis.
Assuntos
Sinalização do Cálcio/fisiologia , Mucolipidoses/fisiopatologia , Neurônios/patologia , Neurônios/fisiologia , Exocitose/fisiologia , Glicólise/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas , Sinapses/patologia , Sinapses/fisiologiaRESUMO
Mesenchymal stem cells (MSCs) are somatic stem cells that can be derived from adult bone marrow (BM) and white adipose tissue (WAT), and that display multipotency and self-renewal capacity. Although MSCs are essential for tissue formation and have already been used in clinical therapy, the origins and markers of these cells remain unknown. In this study, we first investigated the developmental process of MSCs in mouse embryos using the gene encoding platelet-derived growth factor receptor α (Pdgfra) as a marker. We then traced cells expressing Pdgfra and other genes (brachyury, Sox1 and Pmx1) in various mutant mouse embryos until the adult stage. This tracing of MSC origins and destinies indicates that embryonic MSCs emerge in waves and that almost all adult BM MSCs and WAT MSCs originate from mesoderm and embryonic Pdgfrα-positive cells. Furthermore, we demonstrate that adult Pdgfrα-positive cells are involved in some pathological conditions.
Assuntos
Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Tecido Adiposo Branco/crescimento & desenvolvimento , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula , Feminino , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Gravidez , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismoRESUMO
Lysosomal storage diseases (LSDs) are a group of metabolism inborn errors caused by defective enzymes in the lysosome, resulting in the accumulation of undegraded substrates. Many characteristic cell features have been revealed in LSDs, including abnormal autophagy and mitochondrial dysfunction. The development of induced pluripotent stem cells (iPSCs) dramatically boosted research on LSDs, particularly regarding novel opportunities to clarify the disease etiology based on the storage of macromolecules, such as sphingolipids in lysosomes. iPSCs made from LSD patients (LSD-iPSCs) have been differentiated into neurons, endothelial cells, cardiomyocytes, hepatocytes, and macrophages, with each cell type closely resembling the primary disease phenotypes, providing new tools to probe the disease pathogenesis and to test therapeutic strategies. Abnormally accumulated substrates impaired autophagy and mitochondrial and synapse functions in LSD-iPSC-derived neurons. Reducing the accumulation with the treatment of drug candidates improved LSD-iPSC-derived neuron functions. Additionally, iPSC technology can help probe the gene expressions, proteomics, and metabolomics of LSDs. Further, gene repair and the generation of new mutations in causative genes in LSD-iPSCs can be used to understand both the specific roles of causative genes and the contributions of other genetic factors to these phenotypes. Moreover, the development of iPSC-derived organoids as disease models has bridged the gap between studies using cell lines and in vivo animal models. There are some reproducibility issues in iPSC research, however, including genetic and epigenetic abnormalities, such as chromosomal abnormalities, DNA mutations, and gene modifications via methylation. In this review, we present the disease and treatment concepts gathered using selected LSD-iPSCs, discuss iPSC research limitations, and set our future research visions. Such studies are expected to further inform and generate insights into LSDs and are important in research and clinical practice.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Doenças por Armazenamento dos Lisossomos/metabolismo , Animais , Edição de Genes/métodos , Terapia Genética/métodos , Humanos , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/terapia , Medicina de Precisão/métodosRESUMO
Induction of pluripotency with defined factors (octamer-binding transcription factor 4 [Oct4], SRY (sex determining region Y)-box 2 [Sox2], Kruppel-like factor 4 [Klf4], c-Myc) raises hopes for successful clinical trials. Despite considerable efforts, the molecular mechanism of reprogramming remains poorly understood. The aim of the present study was to identify the role of calcineurin/nuclear factor of activated T cells (NFAT) in reprogramming. Our results demonstrated a biphasic role for calcineurin/NFAT signaling during reprogramming. In the early phase of reprogramming, calcineurin activity is required to maintain proper cell cycle division and for mesenchymal-epithelial transition. In the late phase, calcineurin exerts a negative effect that is mediated by NFATc2. NFATc2 interacts with Hdac3, Ezh2, and Suv39h1 to increase H3K9me3 and H3K27me3 over the Sox2 enhancer and Klf2 promoter, respectively, resulting in the downregulation of their expression. Moreover, Gαq was identified as a positive upstream regulator for calcineurin. The Gαq/calcineurin/NFATc2 axis negatively regulates the late step of reprogramming. By inhibiting NFATc2 or calcineurin, induced pluripotent stem cells could be established without exogenous Sox2. Thus, the present study revealed another regulatory level of reprogramming, and proposes a biological axis that could be useful for cancer therapy. Stem Cells 2017;35:1162-1175.
Assuntos
Calcineurina/metabolismo , Reprogramação Celular , Fatores de Transcrição NFATC/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Animais , Biomarcadores/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Técnicas de Silenciamento de Genes , Heterocromatina/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Niemann-Pick disease type C (NPC) is an autosomal recessive lipid storage disorder, and the majority of cases are caused by mutations in the NPC1 gene. In this study, we clarified how a single gene mutation in the NPC1 gene impacts the cellular glycome by analyzing the total glycomic expression profile of Chinese hamster ovary cell mutants defective in the Npc1 gene (Npc1 KO CHO cells). A number of glycomic alterations were identified, including increased expression of lactosylceramide, GM1, GM2, GD1, various neolacto-series glycosphingolipids, and sialyl-T (O-glycan), which was found to be the major sialylated protein-bound glycan, as well as various N-glycans, which were commonly both fucosylated and sialylated. We also observed significant increases in the total amounts of free oligosaccharides (fOSs), especially in the unique complex- and hybrid-type fOSs. Treatment of Npc1 KO CHO cells with 2-hydroxypropyl-ß-cyclodextrin (HPBCD), which can reduce cholesterol and glycosphingolipid (GSL) storage, did not affect the glycomic alterations observed in the GSL-, N-, and O-glycans of Npc1 KO CHO cells. However, HPBCD treatment corrected the glycomic alterations observed in fOSs to levels observed in wild-type cells.
Assuntos
Glicômica , Mutação , Doença de Niemann-Pick Tipo C/genética , Animais , Antígenos CD/metabolismo , Células CHO , Cricetulus , Glicoesfingolipídeos/metabolismo , Lactosilceramidas/metabolismo , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/genética , Polissacarídeos/análise , beta-Ciclodextrinas/farmacologiaRESUMO
Niemann-Pick type C (NPC) disease, characterized by intracellular accumulation of unesterified cholesterol and other lipids owing to defects in two proteins NPC1 and NPC2, causes neurodegeneration and other fatal neurovisceral symptoms. Currently, treatment of NPC involves the use of 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD). HP-ß-CD is effective in the treatment of hepatosplenomegaly in NPC disease, albeit at a very high dose. One of the methods to reduce the required dose of HP-ß-CD for treatment of NPC is to actively targeting hepatocytes with ß-cyclodextrin (ß-CD). The aim of the present study was to synthesize a novel multi-lactose-appended ß-CD (multi-Lac-ß-CD) and to evaluate its cholesterol-lowering effect in U18666A-treated HepG2 (NPC-like HepG2) cells. Further, the study aimed at delivering ß-CD to hepatocytes via cholesterol-accumulated HepG2 cells, and indicated that the newly synthesized multi-Lac-ß-CD had an average degree of substitution of lactose (DSL) of 5.6. This newly synthesized multi-Lac-ß-CD was found to significantly decrease the concentration of intracellular cholesterol with negligible cytotoxicity as compared to HP-ß-CD. An increased internalization of TRITC-multi-Lac-ß-CD (DSL 5.6) as compared to TRITC-HP-ß-CD was observed in NPC-like HepG2 cells. Further, the dissociation constant of peanut lectin with multi-Lac-ß-CD (DSL5.6) was found to be extremely low (2.5 × 10-8 M). These results indicate that multi-Lac-ß-CD (DSL5.6) diminished intracellular cholesterol levels in NPC-like HepG2 cells via asialoglycoprotein receptor (ASGPR)-mediated endocytosis.
RESUMO
Niemann-Pick disease type C (NPC) is a lysosomal storage disease characterized by abnormal accumulation of free cholesterol and glycolipids. Here, we established induced pluripotent stem cell (iPSC) lines from NPC patients. Hepatocyte-like cells (HLCs) and neural progenitors derived from the iPSC lines accumulated cholesterol and displayed impaired autophagy and ATP production. A molecular signature related to lipid metabolism was also impaired in the NPC-iPSC-derived HLCs. These findings indicate that iPSC-derived cells can phenocopy human NPC. We also newly found that 2-hydroxypropyl-γ-cyclodextrin (HPGCD) could reduce the cholesterol accumulation and restore the functional and molecular abnormalities in the NPC patient-derived cells, and do so more effectively than 2-hydroxypropyl-ß-cyclodextrin treatment. In addition, NPC model mice showed an improved liver status and prolonged survival with HPGCDs. Thus, iPSC lines derived from patient cells are powerful tools to study cellular models of NPC, and HPGCD is a potential new drug candidate for future treatment of this disease.
Assuntos
Células-Tronco Pluripotentes Induzidas/patologia , Doença de Niemann-Pick Tipo C/patologia , beta-Ciclodextrinas/farmacologia , gama-Ciclodextrinas/farmacologia , 2-Hidroxipropil-beta-Ciclodextrina , Animais , Técnicas de Cocultura , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/metabolismo , Resultado do Tratamento , beta-Ciclodextrinas/uso terapêutico , gama-Ciclodextrinas/uso terapêuticoRESUMO
Niemann-Pick disease type C (NPC) is an autosomal recessive lysosomal storage disorder, which is an inherited disease characterized by the accumulation of unesterified cholesterol in endolysosomes. Recently, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CyD) has been used for the treatment of NPC, and ameliorated a hepatosplenomegaly in the patients. However, to obtain the treatment efficacy, a high dose of HP-ß-CyD was necessary. Therefore, the decrease in dose by using active intracellular delivery system of ß-CyD to NPC cells is expected. In this study, to efficiently deliver ß-CyD to NPC-like cells, we newly synthesized octaarginine (R8)-appended ß-CyD with a spacer of γ-aminobutyric acid (R8-ß-CyD) and evaluated its cytotoxicity, intracellular distribution, endocytosis pathway and cholesterol-lowering effect in Npc1-trap-Chinese hamster ovary (CHO) cells, cholesterol-accumulated cells through the impairment of NPC1 function. R8-ß-CyD did not show cytotoxicity in the cells. In addition, Alexa568-labeled R8-ß-CyD was actively internalized into Npc1-trap-CHO cells, possibly through micropinocytosis. Notably, R8-ß-CyD significantly decreased intracellular cholesterol content compared with HP-ß-CyD. These results suggest that R8-ß-CyD may be a promising therapeutic agent for ameliorating cholesterol accumulation in NPC.
Assuntos
Colesterol/metabolismo , Oligopeptídeos/farmacologia , beta-Ciclodextrinas/farmacologia , Ácido gama-Aminobutírico/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , Endocitose , Doença de Niemann-Pick Tipo C , Oligopeptídeos/química , beta-Ciclodextrinas/química , Ácido gama-Aminobutírico/químicaRESUMO
Platelet-derived growth factor (PDGF) and its receptor play an important role in embryogenesis. PDGF receptor α (PDGFRα) is expressed specifically in the embryonic day 7.5 (E7.5) mesoderm and in the E9.5 neural crest among other tissues. PDGFRα-expressing cells and their descendants are involved in the formation of various tissues. To trace PDGFRα-expressing cells in vivo, we generated a knock-in mouse line that expressed a fusion protein of green fluorescent protein (GFP), Cre recombinase (Cre), and mutated estrogen receptor ligand-binding domain (ERT2) under the control of the PDGFRα promoter. In these mice, Cre activity in PDGFRα-expressing cells could be induced by tamoxifen treatment. Taken together, our results suggest that the knock-in mouse line generated here could be useful for studying PDGFRα-expressing cells and their descendants in vivo at various stages of development.
Assuntos
Proteínas de Fluorescência Verde/genética , Integrases/genética , Domínios e Motivos de Interação entre Proteínas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptores de Estrogênio/genética , Animais , Imunofluorescência , Expressão Gênica , Técnicas de Introdução de Genes , Ordem dos Genes , Marcação de Genes , Genes Reporter , Loci Gênicos , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Transgênicos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Estrogênio/química , TransgenesRESUMO
Rest (RE1-silencing transcription factor, also called Nrsf) is involved in the maintenance of the undifferentiated state of neuronal stem/progenitor cells in vitro by preventing precocious expression of neuronal genes. However, the function of Rest during neurogenesis in vivo remains to be elucidated because of the early embryonic lethal phenotype of conventional Rest knockout mice. In the present study, we have generated Rest conditional knockout mice, which allow the effect of genetic ablation of Rest during embryonic neurogenesis to be examined in vivo. We show that Rest plays a role in suppressing the expression of neuronal genes in cultured neuronal cells in vitro, as well as in non-neuronal cells outside of the central nervous system, but that it is dispensable for embryonic neurogenesis in vivo. Our findings highlight the significance of extrinsic signals for the proper intrinsic regulation of neuronal gene expression levels in the specification of cell fate during embryonic neurogenesis in vivo.
Assuntos
Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/fisiologia , Neurônios/fisiologia , Proteínas Repressoras/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Camundongos , Camundongos Knockout , Neurônios/citologia , Proteínas Repressoras/genética , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
The Niemann-Pick type C disease (NPC) is one of inherited lysosomal storage disorders, emerges the accumulation of unesterified cholesterol in endolysosomes. Currently, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CyD) has been applied for the treatment of NPC. HP-ß-CyD improved hepatosplenomegaly in NPC patients, however, a high dose of HP-ß-CyD was necessary. Therefore, the decrease in dose by actively targeted-ß-CyD to hepatocytes is expected. In the present study, to deliver ß-CyD selectively to hepatocytes, we newly fabricated mono-lactose-appended ß-CyD (Lac-ß-CyD) and evaluated its cholesterol lowering effects in NPC-like HepG2 cells, cholesterol accumulated HepG2 cells induced by treatment with U18666A. Lac-ß-CyD (degree of substitution of lactose (DSL) 1) significantly decreased the intracellular cholesterol content in a concentration-dependent manner. TRITC-Lac-ß-CyD was associated with NPC-like HepG2 cells higher than TRITC-ß-CyD. In addition, TRITC-Lac-ß-CyD was partially localized with endolysosomes after endocytosis. Thus, Lac-ß-CyD entered NPC-like HepG2 cells via asialoglycoprotein receptor (ASGPR)-mediated endocytosis and decreased the accumulation of intracellular cholesterol in NPC-like HepG2 cells. These results suggest that Lac-ß-CyD may have the potential as a drug for the treatment of hepatosplenomegaly in NPC disease.
RESUMO
Hereditary transthyretin (TTR) amyloidosis (ATTRv amyloidosis) is autosomal dominant and caused by mutation of TTR gene. Heterozygous ATTR Tyr114Cys (p.Tyr134Cys) amyloidosis is a lethal disease with a life expectancy of about 10 years after onset of the disease. However, the molecular pathogenesis of ATTR Tyr114Cys amyloidosis is still largely unknown. In this study, we took advantage of disease-specific induced pluripotent stem (iPS) cells and generated & characterized the heterozygous ATTR Tyr114Cys amyloidosis-specific iPS cells (Y114C iPS cells), to determine whether Y114C iPS cells could be useful for elucidating the pathogenesis of ATTR Tyr114Cys amyloidosis. We successfully differentiated heterozygous Y114C iPS cells into hepatocyte like cells (HLCs) mainly producing TTR protein. On day 27 after differentiation, the expression of hepatocyte maker albumin was detected, and TTR expression was significantly increased in HLCs differentiated from Y114C iPS cells. LC-MS/MS analysis showed that both WT TTR & ATTR Y114C protein were indeed expressed in the HLCs differentiated from Y114C iPS cells. Notably, the number of detected peptides derived from ATTR Y114C protein was lower than that of WT TTR protein, indeed indicating the clinical phenotype of ATTR Tyr114Cys amyloidosis. Taken together, we first reported the heterozygous Y114C iPS cells generated from patient with ATTR Tyr114Cys amyloidosis, and suggested that Y114C iPS cells could be a potential pathological tool, which may contribute to elucidating the molecular pathogenesis of heterozygous ATTR Tyr114Cys amyloidosis.
RESUMO
Niemann-Pick disease type C (NPC) is a lysosomal lipid storage disorder characterized by progressive neurodegeneration and hepatic dysfunction. A cyclic heptasaccharide, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), is currently under clinical investigation for NPC, but its adverse events remain problematic. We previously identified that a cyclic octasaccharide, 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD), also ameliorated NPC manifestations with higher biocompatibility than HP-ß-CD. However, preclinical studies describing the associations between the biodistribution and pharmacodynamics of these compounds, which are essential for clinical application, are still lacking. Here, we investigated these properties of HP-γ-CD by measuring its organ biodistribution and therapeutic effect after systemic and central administration. The effect of HP-γ-CD on disturbed cholesterol homeostasis appeared within several hours after exposure and persisted for several days in NPC model cells and mice. Tissue distribution indicated that only a small fraction of subcutaneously administered HP-γ-CD rapidly distributed to peripheral organs and contributed to disease amelioration. We found that a subcutaneous dose of HP-γ-CD negligibly ameliorated neurological characteristics because it has limited penetration of the blood-brain barrier; however, an intracerebroventricular microdose unexpectedly attenuated hepatic dysfunction without the detection of HP-γ-CD in the liver. These results demonstrate that central administration of HP-γ-CD can indirectly attenuate peripheral manifestations of NPC.
Assuntos
Modelos Animais de Doenças , Fígado , Doença de Niemann-Pick Tipo C , gama-Ciclodextrinas , Animais , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/patologia , Camundongos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , gama-Ciclodextrinas/farmacologia , Distribuição Tecidual , Colesterol/metabolismo , Masculino , Camundongos Endogâmicos BALB CRESUMO
The regulation of mesenchymal cell growth by signaling molecules plays an important role in maintaining tissue functions. Aberrant mesenchymal cell proliferation caused by disruption of this regulatory process leads to pathogenetic events such as fibrosis. In the current study we have identified a novel nuclear factor, Phf14, which controls the proliferation of mesenchymal cells by regulating PDGFRα expression. Phf14-null mice died just after birth due to respiratory failure. Histological analyses of the lungs of these mice showed interstitial hyperplasia with an increased number of PDGFRα(+) mesenchymal cells. PDGFRα expression was elevated in Phf14-null mesenchymal fibroblasts, resulting in increased proliferation. We demonstrated that Phf14 acts as a transcription factor that directly represses PDGFRα expression. Based on these results, we used an antibody against PDGFRα to successfully treat mouse lung fibrosis. This study shows that Phf14 acts as a negative regulator of PDGFRα expression in mesenchymal cells undergoing normal and abnormal proliferation, and is a potential target for new treatments of lung fibrosis.
Assuntos
Proliferação de Células , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Mesoderma/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Fibroblastos/patologia , Proteínas de Homeodomínio/genética , Mesoderma/patologia , Camundongos , Camundongos Knockout , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Fibrose Pulmonar/terapia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genéticaRESUMO
Melanocytes are pigment-producing cells generated from neural crest cells (NCCs) that delaminate from the dorsal neural tube. The widely accepted premise that NCCs migrating along the dorsolateral pathway are the main source of melanocytes in the skin was recently challenged by the finding that Schwann cell precursors are the major cellular source of melanocytes in the skin. Still, in a wide variety of vertebrate embryos, melanocytes are exclusively derived from NCCs. In this study, we show that a NCC population that is not derived from Sox1(+) dorsal neuroepithelial cells but are derived from Sox1(-) cells differentiate into a significant population of melanocytes in the skin of mice. Later, these Sox1(-) cells clearly segregate from cells that originated from Sox1(+) dorsal neuroepithelial cell-derived NCCs. The possible derivation of Sox1(-) cells from epidermal cells also strengthens their non-neuroepithelial origin.
Assuntos
Melanócitos/metabolismo , Crista Neural/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Células de Schwann/metabolismo , Pele/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Movimento Celular/genética , Células Cultivadas , Células Epidérmicas , Epiderme/embriologia , Epiderme/metabolismo , Imuno-Histoquímica , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Melanócitos/citologia , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Crista Neural/citologia , Crista Neural/embriologia , Células Neuroepiteliais/citologia , Células Neuroepiteliais/metabolismo , Fatores de Transcrição SOXB1/genética , Células de Schwann/citologia , Pele/citologia , Pele/embriologiaRESUMO
Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder characterized by progressive ossification of soft tissues. FOP is caused by mutations in activin receptor-like kinase 2 (ALK2) that cause its constitutive activation and result in dysregulation of BMP signaling. Here, we show that generation of induced pluripotent stem cells (iPSCs) from FOP-derived skin fibroblasts is repressed because of incomplete reprogramming and inhibition of iPSC maintenance. This repression was mostly overcome by specific suppression of ALK2 expression and treatment with an ALK2 inhibitor, indicating that the inhibition of iPSC generation and maintenance observed in FOP-derived skin fibroblasts results from constitutive activation of ALK2. Using this system, we identified an ALK2 inhibitor as a potential candidate for future drug development. This study highlights the potential of the inhibited production and maintenance of iPSCs seen in diseases as a useful phenotype not only for studying the molecular mechanisms underlying iPS reprogramming but also for identifying drug candidates for future therapies.
Assuntos
Receptores de Ativinas Tipo I/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Miosite Ossificante/patologia , Receptores de Ativinas Tipo I/antagonistas & inibidores , Receptores de Ativinas Tipo I/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Técnicas de Cocultura , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Mutação de Sentido Incorreto , Miosite Ossificante/genética , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais , Pele/patologia , TranscriptomaRESUMO
Musculocontractural Ehlers-Danlos syndrome caused by mutations in the carbohydrate sulfotransferase 14 gene (mcEDS-CHST14) is a heritable connective tissue disorder characterized by multiple congenital malformations and progressive connective tissue fragility-related manifestations in the cutaneous, skeletal, cardiovascular, visceral, and ocular systems. Progressive skeletal deformities are among the most frequent and serious complications affecting the quality of life and activities of daily living in patients. After establishing induced pluripotent stem cells (iPSCs) from cultured skin fibroblasts of three patients with mcEDS-CHST14, we generated a patient iPSC-based human osteogenesis model and performed an in vitro assessment of the phenotype and pathophysiology of skeletal deformities. Patient-derived iPSCs presented with remarkable downregulation of osteogenic-specific gene expression, less alizarin red staining, and reduced calcium deposition compared with wild-type iPSCs at each stage of osteogenic differentiation, including osteoprogenitor cells, osteoblasts, and osteocytes. These findings indicated that osteogenesis was impaired in mcEDS-CHST14 iPSCs. Moreover, the decrease in decorin (DCN) expression and increase in collagen (COL12A1) expression in patient-derived iPSCs elucidated the contribution of CHST14 dysfunction to skeletal deformities in mcEDS-CHST14. In conclusion, this disease-in-a-dish model provides new insight into the pathophysiology of EDS and may have the potential for personalized gene or drug therapy.