Background:TheSCN5Agene encodes the α subunit of the cardiac voltage-gated sodium channel, NaV1.5. The missense mutation, D1275N, has been associated with a range of unusual phenotypes associated ...with reduced NaV1.5 function, including cardiac conduction disease and dilated cardiomyopathy. Curiously, the reported biophysical properties ofSCN5A-D1275N channels vary with experimental system.Methods and Results:First, using a human embryonic kidney (HEK) 293 cell-based heterologous expression system, theSCN5A-D1275N channels showed similar maximum sodium conductance but a significantly depolarizing shift of activation gate (+10 mV) compared to wild type. Second, we generated human-induced pluripotent stem cells (hiPSCs) from a 24-year-old female who carried heterozygousSCN5A-D1275N and analyzed the differentiated cardiomyocytes (CMs). AlthoughSCN5Atranscript levels were equivalent between D1275N and control hiPSC-CMs, both the total amount of NaV1.5 and the membrane fractions were reduced approximately half in the D1275N cells, which were rescued by the proteasome inhibitor MG132 treatment. Electrophysiological assays revealed that maximum sodium conductance was reduced to approximately half of that in control hiPSC-CMs in the D1275N cells, and maximum upstroke velocity of action potential was lower in D1275N, which was consistent with the reduced protein level of NaV1.5.Conclusions:This study successfully demonstrated diminished sodium currents resulting from lower NaV1.5 protein levels, which is dependent on proteasomal degradation, using a hiPSC-based model forSCN5A-D1275N-related sodium channelopathy.
Long QT syndrome type 3 (LQT3) is caused by gain-of-function mutations in the
gene, which encodes the α subunit of the cardiac voltage-gated sodium channel. LQT3 patients present bradycardia and ...lethal arrhythmias during rest or sleep. Further, the efficacy of β-blockers, the drug used for their treatment, is uncertain. Recently, a large multicenter LQT3 cohort study demonstrated that β-blocker therapy reduced the risk of life-threatening cardiac events in female patients; however, the detailed mechanism of action remains unclear.
This study aimed to establish LQT3-human induced pluripotent stem cells (hiPSCs) and to investigate the effect of propranolol in this model.
An hiPSCs cell line was established from peripheral blood mononuclear cells of a boy with LQT3 carrying the
-N1774D mutation. He had suffered from repetitive torsades de pointes (TdPs) with QT prolongation since birth (QTc 680 ms), which were effectively treated with propranolol, as it suppressed lethal arrhythmias. Furthermore, hiPSCs were differentiated into cardiomyocytes (CMs), on which electrophysiological functional assays were performed using the patch-clamp method.
N1774D-hiPSC-CMs exhibited significantly prolonged action potential durations (APDs) in comparison to those of the control cells (N1774D: 440 ± 37 ms vs. control: 272 ± 22 ms; at 1 Hz pacing;
< 0.01). Furthermore, N1774D-hiPSC-CMs presented gain-of-function features: a hyperpolarized shift of steady-state activation and increased late sodium current compared to those of the control cells. 5 μM propranolol shortened APDs and inhibited late sodium current in N1774D-hiPSC-CMs, but did not significantly affect in the control cells. In addition, even in the presence of intrapipette guanosine diphosphate βs (GDPβs), an inhibitor of G proteins, propranolol reduced late sodium current in N1774D cells. Therefore, these results suggested a unique inhibitory effect of propranolol on late sodium current unrelated to β-adrenergic receptor block in N1774D-hiPSC-CMs.
We successfully recapitulated the clinical phenotype of LQT3 using patient-derived hiPSC-CMs and determined that the mechanism, by which propranolol inhibited the late sodium current, was independent of β-adrenergic receptor signaling pathway.
Calmodulin is a ubiquitous Ca2+ sensor molecule encoded by three distinct calmodulin genes, CALM1-3. Recently, mutations in CALM1-3 have been reported to be associated with severe early-onset long-QT ...syndrome (LQTS). However, the underlying mechanism through which heterozygous calmodulin mutations lead to severe LQTS remains unknown, particularly in human cardiomyocytes. We aimed to establish an LQTS disease model associated with a CALM2 mutation (LQT15) using human induced pluripotent stem cells (hiPSCs) and to assess mutant allele-specific ablation by genome editing for the treatment of LQT15. We generated LQT15-hiPSCs from a 12-year-old boy with LQTS carrying a CALM2-N98S mutation and differentiated these hiPSCs into cardiomyocytes (LQT15-hiPSC-CMs). Action potentials (APs) and L-type Ca2+ channel (LTCC) currents in hiPSC-CMs were analyzed by the patch-clamp technique and compared with those of healthy controls. Furthermore, we performed mutant allele-specific knockout using a CRISPR-Cas9 system and analyzed electrophysiological properties. Electrophysiological analyses revealed that LQT15-hiPSC-CMs exhibited significantly lower beating rates, prolonged AP durations, and impaired inactivation of LTCC currents compared with control cells, consistent with clinical phenotypes. Notably, ablation of the mutant allele rescued the electrophysiological abnormalities of LQT15-hiPSC-CMs, indicating that the mutant allele caused dominant-negative suppression of LTCC inactivation, resulting in prolonged AP duration. We successfully recapitulated the disease phenotypes of LQT15 and revealed that inactivation of LTCC currents was impaired in CALM2-N98S hiPSC model. Additionally, allele-specific ablation using the latest genome-editing technology provided important insights into a promising therapeutic approach for inherited cardiac diseases.
Background
Nestin, which was originally described as a neural crest stem cell marker, is known to be expressed in bulge follicle cells of human, canine and murine anagen hairs. However, the capacity ...of nestin‐expressing cells to differentiate into the components of the hair follicle or the epidermis has been insufficiently investigated.
Hypothesis/objectives
To determine whether nestin‐expressing cells are capable of differentiating into keratinocytes.
Animals/materials
A double‐transgenic mouse line Nes‐Cre/CAG‐CAT‐EGFP, in which enhanced green fluorescent protein (EGFP) is expressed upon Cre‐based recombination driven by the nestin promoter.
Methods and materials
The tissue distribution of EGFP+ and nestin+ cells in the skin of the mouse line was analysed by immunofluorescence and immunohistochemical analyses.
Results
EGFP+ cells were recognized in the outer epithelial cell layers of anagen and telogen hair follicles, but rarely seen in the interfollicular epidermis. The EGFP+ cells in the outer layers of the hair follicles coexpressed keratin 14, a marker of the outer root sheath (ORS) keratinocytes, but not trichohyalin granules, an inner root sheath keratinocyte cell marker. Immunostaining for nestin failed to detect its expression in the majority of hair follicle epithelial cells, suggesting that the EGFP+ cells in the ORS were derived from nestin‐expressing progenitor cells that had become further committed along the epithelial cell lineage, where nestin is no longer expressed.
Conclusions and clinical importance
These results suggest that progenitor cells that differentiate into ORS keratinocytes are distinct from those for other hair follicle or epidermal components and provide implications for regenerative medicine and the molecular classification of hair follicle tumours.
Résumé
Contexte
La nestine, qui a été initialement décrite comme un marqueur des cellules souches des crêtes neurales, est connue pour être exprimée par les cellules folliculaires du bulge des poils anagènes chez l'homme, le chien et la souris. Cependant, la capacité des cellules à exprimer la nestine pour différencier les différents composants du follicule pileux ou de l’épiderme a été insuffisamment étudiée.
Hypothèses/Objectifs
Déterminer si les cellules exprimant la nestine sont capables de se différencier en kératinocytes.
Sujets/matériels
Une lignée de souris double‐transgénique Nes‐Cre/CAG‐CAT‐EGFP, pour laquelle une augmentation de protéine fluorescente verte (EGFP) s'exprime sur la recombinaison Cre‐based due au prompteur de nestine.
Matériel et méthode
La distribution tissulaire des cellules EGFP+ et nestine+ dans la peau de souris a été analysée par immunofluorescence et immunohistochimie.
Résultats
Les cellules EGFP+ ont été reconnues dans les couches cellulaires épithéliales externes des follicules pileux anagènes et télogènes mais ont été rarement vus dans l’épiderme interfolliculaire. Les cellules EGFP+ des couches externes des follicules pileux co‐exprimaient kératine 14, un marqueur des kératinocytes de ORS (outer root sheath) mais pas de granules de trichohyaline, un marqueur cellulaire des kératinocytes de la gaine épithéliale interne. L'immunomarquage pour la nestine n’ a pas permis de détecter son expression dans la majorité des cellules épithéliales des follicules pileux, suggérant que les cellules EGFP+ de l'ORS étaient dérivées des cellules pro‐génitrices exprimant la nestine qui se sont engagées dans la lignée cellulaire épithéliale, ou la nestine n'est plus exprimée.
Conclusions et importance clinique
Ces résultats suggèrent que les cellules pro‐génitrices qui se différencient en kératinocytes ORS sont distinctes de celles des autres follicules pileux ou composants épidermiques et fournissent des implications pour la médecine régénérative et la classification moléculaire des tumeurs folliculaires.
Resumen
Introducción
se sabe que la nestina, que se describió originalmente como un marcador de células madre de la cresta neural, se expresa en las células del promontorio folícular en los pelos anágenos humanos, caninos y murinos. Sin embargo, la capacidad de las células que expresan nestina para diferenciarse en los componentes del folículo piloso o la epidermis no se ha investigado lo suficiente.
Hipótesis/objetivos
determinar si las células que expresan nestina son capaces de diferenciarse en queratinocitos.
Animales/materiales
una línea de ratón transgénica doble Nes‐Cre/CAG‐CAT‐EGFP, en la que la proteína fluorescente verde mejorada (EGFP) se expresa en la recombinación basada en Cre impulsada por el promotor de nestina.
Métodos y materiales
se analizó la distribución tisular de células EGFP + y nestina + en la piel de la línea de ratón mediante análisis de inmunofluorescencia e inmunohistoquímica.
Resultados
las células EGFP + se reconocieron en las capas de células epiteliales externas de los folículos pilosos anágenos y telógenos, pero rara vez se observaron en la epidermis interfolicular. Las células EGFP + en las capas externas de los folículos pilosos coexpresaron la queratina 14, un marcador de los queratinocitos de la vaina radicular externa (ORS), pero no contenían gránulos de tricohialina, un marcador de queratinocitos de la vaina radicular interna. La inmunotinción para nestina no pudo detectar su expresión en la mayoría de las células epiteliales del folículo piloso, lo que sugiere que las células EGFP + en la ORS se derivaron de células progenitoras que expresan nestina más avanzadas a lo largo del linaje de células epiteliales, donde ya no se expresa la nestina.
Conclusiones e importancia clínica
estos resultados sugieren que las células progenitoras que se diferencian en queratinocitos de ORS son distintas de las de otros componentes del folículo piloso o de la epidermis y proporcionan implicaciones para la medicina regenerativa y la clasificación molecular de los tumores del folículo piloso.
Zusammenfassung
Hintergrund
Von Nestin, ursprünglich als Stammzellmarker der Neuralleiste beschrieben, ist bekannt dass es in den Zellen der Bulge Region in anagenen Haaren des Menschen, der Hunde und der Mäuse exprimiert wird. Jedoch ist die Fähigkeit von Nestin‐exprimierenden Zellen sich in die Komponenten der Haarfollikel oder der Epidermis zu differenzieren, noch ungenügend erforscht.
Hypothese/Ziele
Das Ziel war festzustellen, ob Nestin‐exprimierende Zellen imstande sind sich in Keratinozyten zu differenzieren.
Tiere/Materialien
Eine doppelt transgene Mauszelllinie Nes‐Cre/CAG‐CAT‐EGFP, in der verstärktes grünes Fluoreszenzprotein (EGFP) auf einer Cre‐basierten Rekombination exprimiert wird, welche durch den Nestin Promotor gesteuert wird.
Methoden und Materialien
Die Gewebsverteilung von EGFP+ und Nestin+ Zellen in der Haut der Mauslinie wurde mittels Immunfluoreszenz und Immunhistochemie analysiert.
Ergebnisse
EGFP+ Zellen wurden in den äußeren Epithelzellschichten der anagenen und telogenen Haarfollikel gefunden, während sie in der interfollikulären Epidermis selten gesehen wurden. Die EGFP+ Zellen in den äußeren Schichten der Haarfollikel exprimierten auch Keratin 14, welches einen Marker der äußeren Wurzelscheide (ORS) der Keratinozyten darstellt, aber keine Trichohyalin Granula, die einen Zellmarker der inneren Wurzelscheide darstellen. Die Immunfärbung von Nestin konnte keine Exprimierung in der Mehrheit der Epithelzellen der Haarfollikel nachweisen, was darauf hinweist, dass die EGFP+ Zellen in der ORS von Nestin‐exprimierenden Progenitorzellen abstammen, die entlang der Epithelzelllinie gebunden waren, wo Nestin nicht mehr länger exprimiert wird.
Schlussfolgerungen und klinische Bedeutung
Diese Ergebnisse weisen darauf hin, dass sich Progenitorzellen, die zu ORS Keratinozyten differenzieren, von jenen der anderen Haarfollikel‐ oder Epidermiskomponenten unterscheiden und folglich Implikationen für regenerative Medizin und die molekulare Klassifizierung von Haarfollikeltumoren darstellen.
要約
背景
ネスチンは元来、神経堤幹細胞マーカーとして説明されており、ヒト、犬およびマウスの成長期毛では毛包バルジ細胞で発現が認められることが知られている。しかしながら、ネスチンを発現する細胞が毛包または表皮を構成する細胞に分化する能力を有するかは十分に検証されていない。
仮説/目的
本研究の目的は、ネスチン発現細胞が角化細胞に分化することができるかどうかを解析することである。
被験動物/材料
ネスチンプロモーターの活性下でCreリコンビナーゼが発現した結果、遺伝子組換えにより増強された緑色蛍光タンパク質(EGFP)を発現するNes‐Cre / CAG‐CAT‐EGFP二重トランスジェニックマウス系統。
材料および方法
上述したマウス系統の皮膚におけるEGFP +およびネスチン+細胞の組織分布を蛍光抗体法および免疫組織化学的解析によって解析した。
結果
EGFP +細胞は、成長期および休止期の毛包上皮最外層に認められたが、毛包間表皮ではほとんど認められなかった。毛包最外層に存在するEGFP +細胞は、外毛根鞘(ORS)細胞のマーカーであるケラチン14を共発現していたが、内毛根鞘細胞マーカーであるトリコヒアリン顆粒は共発現していなかった。一方で抗ネスチン抗体を用いた免疫染色では、毛包上皮細胞の多くがネスチンを発現していなかった。このことは、外毛根鞘に認められたEGFP +細胞はネスチン発現前駆細胞に由来しており、これらの前駆細胞がネスチンを発現しない上皮細胞系へと分化したことを示唆している。
結論と臨床的重要性
本研究結果は、外毛根鞘細胞へと分化する前駆細胞は他の毛包上皮細胞や表皮角化細胞とは異なること、また本研究の成果は再生医療および毛包腫瘍の分子学的分類に応用しうることを示唆している。
摘要
背景
巢蛋白最初被定义为神经嵴干细胞标记物,其在人、犬和小鼠生长期毛发的隆凸毛囊细胞上表达。 然而,巢蛋白表达细胞分化成毛囊或表皮组分的能力尚未得到充分研究。
假设/目标
确定表达巢蛋白的细胞是否能够分化成角质细胞。
动物/材料
双转基因小鼠系Nes‐Cre / CAG‐CAT‐EGFP,其中增强绿荧光蛋白(EGFP)在由巢蛋白启动子驱动的Cre基重组中表达。
方法和材料
用免疫荧光和免疫组化分析小鼠皮肤中EGFP +和巢蛋白+细胞的组织分布。
结果
EGFP +细胞在生长期和终止期毛囊的外上皮细胞层中被发现,但在毛囊间表皮中很少见。EGFP +细胞在毛囊外层共表达角质14,后者是外根鞘(ORS)角质细胞的标记物,而没有角质透明颗粒这种内根鞘角质细胞标记物。经免疫染色未能检测到巢蛋白在大多数毛囊上皮细胞中的表达,这表明ORS中的EGFP +细胞由表达巢蛋白的祖细胞分化而来,其沿着不再表达巢蛋白的上皮细胞谱系进一步传代。
结论和临床意义
这些结果表明,分化ORS角质细胞的祖细胞不同于其他毛囊或表皮成分,并为再生医学和毛囊肿瘤的分子分类提供了价值。
Resumo
Contexto
Sabe‐se que a nestina, originalmente descrita como um marcador de célula‐tronco da crista neural, é expressada nas células do bulbo folicular de pelos em anágeno de humanos, caninos e camundongos. Entretanto, a capacidade das células expressadoras de nestina de se diferenciarem em componentes do folículo piloso ou da epiderme não foi suficientemente investigada.
Hipótese/Objetivos
Determinar se as células expressadoras de nestina são capazes de se diferenciarem em queratinócitos.
Animais/materiais
Uma linhagem de camundongos duplo transgênico Nes‐Cre/CAG‐CAT‐EGFP, em que a proteína verde fluorescente enriquecida (EGFP) é expressa na recombinação baseada em Cre conduzida pelo promotor de nestina.
Métodos e materiais
A distribuição tecidual das células EGFP+ e nestin+ na pele
Nucleotide excision repair (NER) is a versatile DNA repair pathway, which can remove an extremely broad range of base lesions from the genome. In mammalian global genomic NER, the XPC protein complex ...initiates the repair reaction by recognizing sites of DNA damage, and this depends on detection of disrupted/destabilized base pairs within the DNA duplex. A model has been proposed that XPC first interacts with unpaired bases and then the XPD ATPase/helicase in concert with XPA verifies the presence of a relevant lesion by scanning a DNA strand in 5'-3' direction. Such multi-step strategy for damage recognition would contribute to achieve both versatility and accuracy of the NER system at substantially high levels. In addition, recognition of ultraviolet light (UV)-induced DNA photolesions is facilitated by the UV-damaged DNA-binding protein complex (UV-DDB), which not only promotes recruitment of XPC to the damage sites, but also may contribute to remodeling of chromatin structures such that the DNA lesions gain access to XPC and the following repair proteins. Even in the absence of UV-DDB, however, certain types of histone modifications and/or chromatin remodeling could occur, which eventually enable XPC to find sites with DNA lesions. Exploration of novel factors involved in regulation of the DNA damage recognition process is now ongoing.
Abstract
Glutathione S-transferase omega 2 (GSTO2), which belongs to the superfamily of GST omega class, lacks any appreciable GST activity. Although GSTO2 exhibits thioltransferase and glutathione ...dehydrogenase activities, its precise expression and physiological functions are still unclear. In the present study, we found that GSTO2 is exclusively expressed in the basal cell layer in Ki67-negative non-proliferative cells in the human esophageal mucosa. GSTO2 overexpression in esophageal squamous cell carcinoma (ESCC) cell lines inhibited cell growth and colony formation, and GSTO2-transfected cells formed smaller tumors in nude mice compared with mock-transfected cells. Interestingly, GSTO2 induction suppressed the expressions of E-cadherin and β-catenin at the cell–cell contact site. We quantified the phosphorylation levels of key proteins of MAPK signaling pathway and identified phosphorylation of p38. Additionally, HSP27, a downstream molecule of p38, was accelerated in GSTO2-transfected cells, unlike in mock-transfected cells. When GSTO2-transfected cells were treated with a p38 inhibitor, the expression of β-catenin and the membrane localization of E-cadherin was recovered. We next examined GSTO2 expression in 61 ESCC tissues using quantitative reverse transcription polymerase chain reaction and immunostaining. The results showed that GSTO2 mRNA and protein were significantly reduced in ESCC compared with normal tissues. When human ESCC cell lines were treated with 5-aza-2′-deoxycytidine, a DNA-methyltransferase inhibitor, GSTO2 transcription was induced, suggesting that aberrant hypermethylation is the cause of the down-regulated expression. Our results indicate that GSTO2 expression inhibits the membrane localization of E-cadherin, probably by modulation of the p38 signaling pathway. Down-regulation of GSTO2 by DNA hypermethylation contributes to the growth and progression of ESCC.
The present study first revealed that glutathione S-transferase omega 2 regulates cell growth and the expression of E-cadherin in esophageal epithelial cells, and it is a significant marker of esophageal stem/progenitor cells.
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