derivation-characterization-transgene-free-human-induced-pluripotent

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic ...

University of California, Los Angeles (UCLA)

Commentary on human cloning.

Nuclear reprogramming by the transplantation of somatic cell nuclei to eggs (in second meiotic metaphase) is always followed by a phase of chromosome replication and cell division before new gene expression is seen. To help understand the mechanism of nuclear reprogramming, we have asked whether the nuclei of normal, nontransformed, nondividing, and terminally differentiated mammalian cells can be directly reprogrammed, without DNA replication, by Xenopus oocytes. We find that nuclei of adult mouse thymocytes and of adult human blood lymphocytes, injected into Xenopus oocytes, are induced to extinguish a differentiation marker and to strongly express oct-4, the most diagnostic mammalian stem cell/pluripotency marker. In the course of 2 days at 18 degrees C, the mammalian oct-4 transcripts are spliced to mature mRNA. We conclude that normal mammalian nuclei can be directly reprogrammed by the nucleus (germinal vesicle) of amphibian oocytes to express oct-4 at a rate comparable to that of oct-4 in mouse ES cells. To our knowledge, this is the first demonstration of a stem cell marker being induced in a differentiated adult human cell nucleus. This is an early step toward the long-term aim of developing a procedure for reprogramming readily accessible human adult cells for cell replacement therapy.

Nuclei of adult mammalian somatic cells are directly reprogrammed to oct-4 stem cell gene expression by amphibian oocytes.

Embryonic stem cells (ESCs) may be able to cure or alleviate the symptoms of various degenerative diseases. However, unresolved issues regarding survival, functionality, and tumor formation mean a prudent approach should be adopted towards advancing ESCs into human clinical trials. The rhesus monkey provides an ideal model organism for developing strategies to prevent immune rejection and test the feasibility, safety, and efficacy of ESC-based medical treatments. Transcriptional profiling of rhesus monkey ESCs provides a foundation for pre-clinical ESC research in this species. In the present study, we used microarray technology, immunocytochemistry, reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) to characterize and transcriptionally profile rhesus monkey ESCs. We identified 367 stemness gene candidates that were highly (>85%) conserved across five different ESC lines. Rhesus monkey ESC lines maintained a pluripotent undifferentiated state over a wide range of POU5F1 (also known as OCT4) expression levels, and comparisons between rhesus monkey, mouse, and human stemness genes revealed five mammalian stemness genes: CCNB1, GDF3, LEFTB, POU5F1, and NANOG. These five mammalian genes are strongly expressed in rhesus monkey, mouse, and human ESCs, albeit only in the undifferentiated state, and represent the core key mammalian stemness factors.

Transcriptional profiling of rhesus monkey embryonic stem cells.

Embryonic stem cells (ESCs) can proliferate indefinitely, maintain an undifferentiated pluripotent state and differentiate into any cell type. Differentiation of ESCs into various specific cell-types may be able to cure or alleviate the symptoms of various degenerative diseases. Unresolved issues regarding maintaining function, possible apoptosis and tumor formation in vivo mean a prudent approach should be taken towards advancing ESCs into human clinical trials. Rhesus macaques provide the ideal model organism for testing the feasibility, efficacy and safety of ESC based therapies and significant numbers of primate ESC lines are now available. In this review, we will summarize progress in evaluating the genetic and epigenetic integrity of primate ESCs, examine their current use in pre-clinical trials and discuss the potential of producing ESC-derived cell populations that are genetically identical (isogenic) to the host by somatic cell nuclear transfer.

Current progress with primate embryonic stem cells.

The ability to reprogram somatic cell nuclei back into a pluripotent epigenetic state provides exciting new possibilities for in vitro research and cell transplantation therapy. There has been a significant quantity of recent research studies demonstrating that this epigenetic reprogramming process is possible with human and non-human primate somatic cells. In this review, various methodologies for reprogramming primate somatic cells into pluripotent stem cells are examined, epigenetic reprogramming following somatic cell nuclear transfer and normal primate embryonic development is compared, and future potential methods to induce direct reprogramming without using genetic modification are discussed.

Generation of isogenic pluripotent stem cells.

The derivation of induced pluripotent stem cells (iPSCs) provides new possibilities for basic research and novel cell-based therapies. Limitations, however, include our current lack of understanding regarding the underlying mechanisms and the inefficiency of reprogramming.

Enhanced generation of induced pluripotent stem cells from a subpopulation of human fibroblasts.

Approximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI) procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis.

Parthenogenic blastocysts derived from cumulus-free in vitro matured human oocytes.

&#x6458;&#x8981;&#xFF1A; &#x7B80;&#x4ECB;&#xFF1A; OCT4 &#x8F6C;&#x5F55;&#x56E0;&#x5B50;&#x53C2;&#x4E0E;&#x4E86;&#x8BB8;&#x591A;&#x7EC6;&#x80DE;&#x7684;&#x8FC7;&#x7A0B;&#xFF0C;&#x5305;&#x62EC;&#x53D1;&#x5C55;&#x3001; &#x91CD;&#x7F16;&#x7A0B;&#x3001; &#x4FDD;&#x6301;&#x591A;&#x6F5C;&#x80FD;&#x548C;&#x5206;&#x5316;&#x3002;&#x5408;&#x6210; OCT4 mRNA &#x6700;&#x8FD1; &#xFF08;&#x5728;&#x4E0E;&#x5176;&#x4ED6;&#x7F16;&#x7A0B;&#x56E0;&#x7D20;&#x4E00;&#x8D77;&#xFF09; &#x7528;&#x4E8E;&#x751F;&#x6210;&#x4EBA;&#x7C7B;&#x8BF1;&#x5BFC;&#x591A;&#x80FD;&#x5E72;&#x7EC6;&#x80DE;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#x6E7E; 11-7082 (BAY11)&#xFF0C;&#x81F3;&#x5C11;&#x90E8;&#x5206;&#x5730;&#x901A;&#x8FC7;&#x4E00;&#x4E2A;&#x57FA;&#x4E8E; NF &#x6D3B;&#x5316;&#x6291;&#x5236;&#x673A;&#x5236;&#xFF0C;&#x80FD;&#x591F;&#x5927;&#x5927;&#x589E;&#x52A0; OCT4 &#x8868;&#x8FBE;&#x540E;&#x6210;&#x4EBA;&#x4EBA;&#x4F53;&#x76AE;&#x80A4;&#x7EC6;&#x80DE;&#x57FA;&#x56E0;&#x8F6C;&#x67D3;&#x7684;&#x5408;&#x6210; mRNA (synRNA)&#x3002;&#x65B9;&#x6CD5;&#xFF1A; &#x6211;&#x4EEC;&#x6D4B;&#x8BD5;&#x5404;&#x79CD;&#x5316;&#x5B66;&#x548C;&#x5206;&#x5B50;&#x7684;&#x5C0F;&#x5206;&#x5B50;&#xFF0C;&#x6291;&#x5236;&#x57FA;&#x56E0;&#x8F6C;&#x67D3; mRNA &#x5408;&#x6210;&#x540E;&#x770B;&#x5230;&#x7684;&#x975E;&#x7279;&#x5F02;&#x6027;&#x514D;&#x75AB;&#x53CD;&#x5E94;&#x7684;&#x80FD;&#x529B;&#x3002;&#x4E09;&#x5206;&#x5B50;-B18R&#x3001; BX795&#xFF0C;&#x548C; BAY11-&#x88AB;&#x7528;&#x5728;&#x514D;&#x75AB;&#x7EC6;&#x80DE;&#x5316;&#x5B66;&#x548C;&#x6269;&#x6563;&#x57FA;&#x4E8E;&#x68C0;&#x6D4B;&#x65B9;&#x6CD5;&#x3002;&#x6211;&#x4EEC;&#x8FD8;&#x5229;&#x7528;&#x5168;&#x7403;&#x8F6C;&#x5F55; meta &#x5206;&#x6790;&#x52A0;&#x4E0A;&#x8367;&#x5149;&#x5B9A;&#x91CF; PCR &#x6765;&#x6807;&#x8BC6; OCT4 &#x4E0B;&#x6E38;&#x76F8;&#x5173;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x3002;&#x7ED3;&#x679C;&#xFF1A; &#x6211;&#x4EEC;&#x53D1;&#x73B0; BAY11 &#x4E0B;&#x7684;&#x4EBA;&#x4F53;&#x76AE;&#x80A4;&#x7EC6;&#x80DE;&#x5BFC;&#x81F4;&#x4E0D;&#x4F1A;&#x59A8;&#x788D;&#x6B63;&#x5E38;&#x7684;&#x7EC6;&#x80DE;&#x589E;&#x6B96;&#x7684; OCT4 &#x91CD;&#x73B0;&#x8868;&#x8FBE;&#x589E;&#x52A0;&#x3002;OCT4 &#x7684;&#x589E;&#x52A0;&#x5BFC;&#x81F4;&#x5927;&#x5927;&#x589E;&#x52A0;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x7684;&#x4E0B;&#x6E38; OCT4&#xFF0C;&#x5305;&#x62EC;&#x4EE5;&#x524D;&#x53D1;&#x73B0;&#x7684; SPP1&#x3001; DUSP4 &#x548C; GADD45G&#xFF0C;&#x6697;&#x793A;&#x8868;&#x793A;&#x7684; OCT4 &#x529F;&#x80FD;&#x3002;&#x6211;&#x4EEC;&#x8FD8;&#x53D1;&#x73B0;&#x65B0;&#x578B; OCT4 &#x6307;&#x8BA4;&#x4E0B;&#x6E38;&#x9776;&#x57FA;&#x56E0; SLC16A9&#xFF0C;&#x8868;&#x73B0;&#x5927;&#x5927;&#x589E;&#x52A0;&#x7684;&#x8868;&#x8FBE;&#x5F0F;&#x4E4B;&#x540E; OCT4 &#x6C34;&#x5E73;&#x7684;&#x63D0;&#x5347;&#x3002;&#x7ED3;&#x8BBA;&#xFF1A; &#x7B2C;&#x4E00;&#x6211;&#x4EEC;&#x6709;&#x663E;&#x793A;&#x5408;&#x6210; mRNA &#x8868;&#x8FBE;&#x8FD9;&#x5C0F;&#x5206;&#x5B50;&#x57FA;&#x4E8E;&#x7A33;&#x5B9A;&#x7684;&#x65F6;&#x95F4;&#x53EF;&#x4EE5;&#x5B9E;&#x73B0;&#x4F7F;&#x7528;&#x7684; BAY11&#x3002;&#x8FD9;&#x79CD;&#x5C0F;&#x5206;&#x5B50;&#x57FA;&#x4E8E;&#x56FA;&#x6709;&#x514D;&#x75AB;&#x53CD;&#x5E94;&#x7684;&#x6291;&#x5236;&#x4F5C;&#x7528;&#xFF0C;&#x5E76;&#x4E14;&#x968F;&#x540E;&#x9C81;&#x68D2;&#x8868;&#x8FBE;&#x8F6C;&#x67D3;&#x7684;&#x5408;&#x6210;&#x5206;&#x6CCC;&#x7684;&#x53EF;&#x80FD;&#x672A;&#x6765;&#x57FA;&#x4E8E;&#x5355;&#x5143;&#x683C;&#x7684;&#x7814;&#x7A76;&#x548C;&#x6CBB;&#x7597;&#x7684;&#x591A;&#x4E2A;&#x5E94;&#x7528;&#x7A0B;&#x5E8F;&#x3002;

BAY11 &#x589E;&#x5F3A; OCT4 &#x6210;&#x4EBA;&#x4EBA;&#x4F53;&#x76AE;&#x80A4;&#x7EC6;&#x80DE;&#x4E2D;&#x7684;&#x5408;&#x6210; mRNA &#x8868;&#x8FBE;&#x3002;

ABSTRACT: INTRODUCTION: The OCT4 transcription factor is involved in many cellular processes, including development, reprogramming, maintaining pluripotency and differentiation. Synthetic OCT4 mRNA was recently used (in conjunction with other reprogramming factors) to generate human induced pluripotent stem cells. Here, we discovered that BAY 11-7082 (BAY11), at least partially through a NF-kappaB-inhibition based mechanism, could significantly increase the expression of OCT4 following transfection of synthetic mRNA (synRNA) into adult human skin cells. METHODS: We tested various chemical and molecular small molecules on their ability to suppress the innate immune response seen upon synthetic mRNA transfection. Three molecules-B18R, BX795, and BAY11- were used in immunocytochemical and proliferation based assays. We also utilized global transcriptional meta-analysis coupled with quantitative PCR to identify relative gene expression downstream of OCT4. RESULTS: We found that human skin cells cultured in the presence of BAY11 resulted in reproducible increased expression of OCT4 that did not inhibit normal cell proliferation. The increased levels of OCT4 resulted in significantly increased expression of genes downstream of OCT4, including the previously identified SPP1, DUSP4 and GADD45G, suggesting the expressed OCT4 was functional. We also discovered a novel OCT4 putative downstream target gene SLC16A9 which demonstrated significantly increased expression following elevation of OCT4 levels. CONCLUSIONS: For the first time we have shown that small molecule-based stabilization of synthetic mRNA expression can be achieved with use of BAY11. This small molecule based inhibition of innate immune responses and subsequent robust expression of transfected synthetic mRNAs may have multiple applications for future cell-based research and therapeutics.

BAY11 enhances OCT4 synthetic mRNA expression in adult human skin cells.

R&eacute;sum&eacute; : INTRODUCTION : facteur de transcription OCT4 The est impliqu&eacute; dans de nombreux processus cellulaires, y compris le d&eacute;veloppement, reprogrammation, maintien de pluripotence et diff&eacute;renciation. Synth&eacute;tique OCT4 ARNm a r&eacute;cemment &eacute;t&eacute; utilis&eacute;e (en conjonction avec d&#39;autres facteurs de reprogrammation) pour g&eacute;n&eacute;rer des cellules souches humaines pluripotentes induites. Ici, nous avons d&eacute;couvert que la baie 11-7082 (BAY11), au moins partiellement gr&acirc;ce &agrave; un m&eacute;canisme de NF-kappaB-inhibition bas&eacute;, pouvant accro&icirc;tre consid&eacute;rablement l&#39;expression de OCT4 apr&egrave;s transfection des ARNm synth&eacute;tique (synRNA) dans les cellules de peau humaine adulte. M&eacute;thodes : Nous avons test&eacute; diff&eacute;rents chimiques et mol&eacute;culaires petites mol&eacute;cules sur leur capacit&eacute; &agrave; supprimer la r&eacute;ponse immunitaire inn&eacute;e, vue &agrave; la transfection d&#39;ADN messag&egrave;re synth&eacute;tique. Trois mol&eacute;cules-B18R, BX795 et BAY11 - servaient immunocytochimiques et prolif&eacute;ration selon les dosages. Nous avons utilis&eacute; aussi meta-analyse transcriptionnelle globale associ&eacute;e &agrave; la PCR quantitative pour identifier l&#39;expression du g&egrave;ne relative en aval de OCT4. R&Eacute;SULTATS : Nous avons constat&eacute; que les cellules de peau humaine cultiv&eacute;es en pr&eacute;sence de BAY11 entra&icirc;n&eacute; reproductible expression accrue de OCT4 qui n&#39;a pas inhib&eacute; la prolif&eacute;ration des cellules normales. L&#39;augmentation des niveaux de OCT4 a significativement augmentation de l&#39;expression des g&egrave;nes en aval de OCT4, SPP1, DUSP4 et GADD45G, sugg&eacute;rant que l&#39;expresse OCT4 fonctionnait pr&eacute;c&eacute;demment identifi&eacute;s. Nous avons aussi d&eacute;couvert un nouveau g&egrave;ne cible en aval putatif OCT4 SLC16A9 qui d&eacute;montre l&#39;expression significativement accrue suite &agrave; l&#39;&eacute;l&eacute;vation des niveaux de OCT4. CONCLUSIONS : Pour la premi&egrave;re fois, que nous avons montr&eacute; que petite stabilisation ax&eacute;e sur la mol&eacute;cule d&#39;ARNm synth&eacute;tique peut &ecirc;tre r&eacute;alis&eacute; avec utilisation de BAY11. Cette petite mol&eacute;cule bas&eacute;e l&#39;inhibition des r&eacute;ponses immunitaires inn&eacute;es et ult&eacute;rieure expression robuste des ARNm synth&eacute;tique transfect&eacute;es peut-&ecirc;tre avoir plusieurs applications de recherche sur les cellules et produits th&eacute;rapeutiques.

BAY11 am&eacute;liore OCT4 synth&eacute;tique expression de l&#39;ARNm dans les cellules de peau humaine adulte.

Zusammenfassung: Einleitung: The OCT4 &Uuml;bertragungfaktor engagiert sich in viele zellul&auml;re Prozesse, einschlie&szlig;lich Entwicklung, Neuprogrammierung, Aufrechterhaltung der Pluripotenz und Differenzierung. Synthetische OCT4-mRNA wurde vor kurzem (in Verbindung mit anderen Faktoren Neuprogrammierung) genutzt, um menschliche induzierten pluripotenten Stammzellen zu generieren. Hier entdeckten wir, dass BAY 11-7082 (BAY11), zumindest teilweise durch einen NF-KappaB-Hemmung basiert-Mechanismus, den Ausdruck des OCT4 nach Transfektion von synthetischen mRNA (SynRNA) in Erwachsenen menschlichen Hautzellen deutlich erh&ouml;hen k&ouml;nnte. Methoden: Wir testeten verschiedene chemische und Molekulare kleine Molek&uuml;le auf ihrer F&auml;higkeit, der angeborenen Immunantwort auf synthetische mRNA Transfektion gesehen zu unterdr&uuml;cken. Drei Molek&uuml;le-B18R, BX795 und BAY11 - dienten des Prozesses Immunocytochemical und Vermehrung Proben basiert. Wir genutzt auch globale transkriptionelle Meta-Analyse gepaart mit quantitativen PCR relative Genexpression stromabw&auml;rts des OCT4 zu identifizieren. Ergebnis: Wir fanden, dass menschliche Hautzellen in Anwesenheit von BAY11 kultivierten reproduzierbare erh&ouml;hte Expression von OCT4 gef&uuml;hrt, die nicht normale Zellvermehrung hemmen,. Die erh&ouml;hten Konzentration von OCT4 ergab deutlich erh&ouml;hte Expression von Genen stromabw&auml;rts des OCT4, einschlie&szlig;lich der zuvor identifizierten SPP1, DUSP4 und GADD45G, vorgeschlagen, die die exprimierten OCT4 funktionsf&auml;hig war. Wir entdeckten auch ein neuartiger OCT4 vermeintliche nachgeschaltete Ziel gen SLC16A9, die deutlich erh&ouml;hte Expression nach H&ouml;he OCT4 Level demonstriert. Schlussfolgerungen: Erstmals kann die Zeit, die wir, dass kleine niedermolekulare Stabilisierung des synthetischen mRNA Ausdruck gezeigt haben mit Einsatz von BAY11 erreicht werden. Diese niedermolekularen basierte Hemmung der angeborenen Immunantwort und nachfolgende robuste Ausdruck transfizierte synthetische mRNAs m&ouml;glicherweise mehrere Anwendungen f&uuml;r zellbasierte Zukunftsforschung und Therapeutika.

BAY11 verbessert OCT4 synthetische mRNA Ausdruck im Erwachsenen menschlichen Hautzellen.

ABSTRACT: Introduzione: fattore di trascrizione OCT4 The &egrave; coinvolto in molti processi cellulari, tra cui sviluppo, riprogrammazione, mantenimento della pluripotenza e differenziazione. Sintetico OCT4 mRNA &egrave; stato recentemente utilizzato (in combinazione con altri fattori di riprogrammazione) per generare cellule staminali pluripotenti indotte umana. Qui, abbiamo scoperto che BAY (BAY11), 11-7082 almeno parzialmente attraverso un meccanismo di NF-kappaB-inibizione basata, potrebbe aumentare significativamente l&#39;espressione di OCT4 seguito di transfezione di mRNA sintetici (synRNA) nelle cellule di pelle umana adulta. Metodi: Abbiamo testato vari chimici e molecolari piccole molecole sulla loro capacit&agrave; di sopprimere la risposta immunitaria innata visto su transfezione mRNA sintetici. Tre molecole-B18R, BX795 e BAY11 - sono stati utilizzati in immunocitochimica e proliferazione based assays. Abbiamo anche utilizzato transcriptional meta-analisi globale accoppiato con PCR quantitativa per identificare l&#39;espressione del gene relativo a valle di OCT4. RISULTATI: Abbiamo trovato che le cellule di pelle umana colture in presenza di BAY11 ha portato a riproducibile aumentata espressione di OCT4 che non inibiscono la proliferazione delle cellule normali. L&#39;aumento dei livelli di OCT4 causato significativamente aumentata espressione dei geni a valle di OCT4, inclusi precedentemente identificati SPP1, DUSP4 e GADD45G, suggerendo che l&#39;espresso OCT4 &egrave; stato funzionale. Abbiamo anche scoperto un romanzo OCT4 putativo downstream target gene SLC16A9 dimostrato significativamente aumentata espressione seguendo elevazione dei livelli di OCT4. CONCLUSIONI: Per la prima volta che abbiamo dimostrato che piccole stabilizzazione basata sulla molecola di espressione del mRNA sintetici ottenibile con l&#39;uso di BAY11. Questa piccola molecola base di inibizione della risposta immunitaria innata e successive robusta espressione dei mRNAs sintetico transfected pu&ograve; avere molteplici applicazioni per la ricerca futura cell-based e terapie.

BAY11 migliora OCT4 sintetica espressione del mRNA nelle cellule della pelle umana adulta.

&#x8981;&#x7D04;&#xFF1A; &#x5C0E;&#x5165;:&#x3001;OCT4 &#x8EE2;&#x5199;&#x56E0;&#x5B50;&#x958B;&#x767A;&#x306A;&#x3069;&#x3001;&#x30EA;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30DF;&#x30F3;&#x30B0;&#x3001;&#x5206;&#x5316;&#x591A;&#x80FD;&#x6027;&#x3092;&#x7DAD;&#x6301;&#x591A;&#x304F;&#x306E;&#x7D30;&#x80DE;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306B;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x5408;&#x6210;&#x3055;&#x308C;&#x308B; OCT4 mRNA &#x306F;&#x3072;&#x3068;&#x8A98;&#x5C0E;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x3092;&#x751F;&#x6210;&#x3059;&#x308B; (&#x4ED6;&#x306E;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30E0;&#x3057;&#x76F4;&#x3059;&#x3053;&#x3068;&#x306E;&#x8981;&#x56E0;&#x3068;&#x7D44;&#x307F;&#x5408;&#x308F;&#x305B;&#x3066;) &#x3067;&#x6700;&#x8FD1;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x30D9;&#x30A4; 11-7082 (BAY11)&#x3001;NF-&#x30AB;&#x30C3;&#x30D1; b-&#x6291;&#x5236;&#x8A55;&#x4FA1;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306B;&#x3088;&#x3063;&#x3066;&#x5C11;&#x306A;&#x304F;&#x3068;&#x3082;&#x90E8;&#x5206;&#x7684;&#x306B;&#x5927;&#x5E45;&#x306B;&#x5927;&#x4EBA;&#x306E;&#x4EBA;&#x9593;&#x306E;&#x76AE;&#x819A;&#x7D30;&#x80DE;&#x306B;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30D5;&#x30A7;&#x30AF;&#x30B7;&#x30E7;&#x30F3;&#x5408;&#x6210; mRNA (synRNA) &#x306E;&#x5F8C;&#x306B;&#x7D9A;&#x304F;&#x5F0F; oct4 &#x306F;&#x5834;&#x5408;&#x3092;&#x9AD8;&#x3081;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x3092;&#x767A;&#x898B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x65B9;&#x6CD5;: &#x3055;&#x307E;&#x3056;&#x307E;&#x306A;&#x5316;&#x5B66;&#x30FB;&#x5206;&#x5B50;&#x5C0F;&#x5206;&#x5B50;&#x5408;&#x6210; mRNA &#x306E;&#x907A;&#x4F1D;&#x5B50;&#x306B;&#x898B;&#x3089;&#x308C;&#x308B;&#x81EA;&#x7136;&#x514D;&#x75AB;&#x5FDC;&#x7B54;&#x3092;&#x6291;&#x5236;&#x3059;&#x308B;&#x80FD;&#x529B;&#x3092;&#x30C6;&#x30B9;&#x30C8;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;3 &#x5206;&#x5B50;-B18R&#x3001;BX795&#x3001;&#x304A;&#x3088;&#x3073; BAY11 &#x306B;&#x306F;&#x514D;&#x75AB;&#x7D44;&#x7E54;&#x5316;&#x5B66;&#x3068;&#x5897;&#x6B96;&#x306B;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x305F;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x3057;&#x307E;&#x3059;&#x3002;&#x79C1;&#x305F;&#x3061;&#x3082;&#x30B0;&#x30ED;&#x30FC;&#x30D0;&#x30EB;&#x306E;&#x8EE2;&#x5199;&#x30E1;&#x30BF;&#x306E;&#x89E3;&#x6790; oct4 &#x306F;&#x5834;&#x5408;&#x306E;&#x76F8;&#x5BFE;&#x7684;&#x306A;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x3092;&#x4E0B;&#x6D41;&#x3092;&#x8B58;&#x5225;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x91CF;&#x7684;&#x306A; PCR &#x3068;&#x306E;&#x7D50;&#x5408;&#x3092;&#x5229;&#x7528;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x7D50;&#x679C;&#xFF1A; &#x79C1;&#x305F;&#x3061;&#x4EBA;&#x9593;&#x306E;&#x76AE;&#x819A;&#x7D30;&#x80DE; BAY11 &#x306E;&#x5B58;&#x5728;&#x4E0B;&#x3067;&#x57F9;&#x990A;&#x6B63;&#x5E38;&#x306A;&#x7D30;&#x80DE;&#x5897;&#x6B96;&#x6291;&#x5236;&#x4F5C;&#x7528;&#x3092;&#x793A;&#x3055;&#x306A;&#x304B;&#x3063;&#x305F; OCT4 &#x306E;&#x5897;&#x52A0;&#x8868;&#x73FE;&#x306E;&#x3053;&#x3068;&#x3092;&#x767A;&#x898B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;Oct4 &#x306F;&#x5834;&#x5408;&#x306E;&#x5897;&#x52A0;&#x30EC;&#x30D9;&#x30EB;&#x4EE5;&#x524D;&#x306B;&#x8B58;&#x5225;&#x3055;&#x308C;&#x305F; spp1 &#x7531;&#x6765;&#x3001;DUSP4&#x3001;GADD45G&#x3001;&#x8868;&#x73FE;&#x3055;&#x308C;&#x308B; OCT4 &#x6A5F;&#x80FD;&#x3057;&#x3066;&#x3044;&#x305F;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x306A;&#x3069;&#x3067;&#x5927;&#x5E45;&#x306B;&#x5897;&#x52A0;&#x3057;&#x305F;&#x767A;&#x73FE; oct4 &#x306F;&#x5834;&#x5408;&#x3001;&#x4E0B;&#x6D41;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x7D50;&#x679C;&#x3002;&#x307E;&#x305F;&#x3001;oct4 &#x306F;&#x5834;&#x5408;&#x5024;&#x306E;&#x4E0A;&#x6607;&#x306E;&#x5F8C;&#x5927;&#x5E45;&#x306B;&#x5897;&#x52A0;&#x3057;&#x305F;&#x5F0F;&#x3067;&#x793A;&#x3057;&#x305F; OCT4 &#x63A8;&#x5B9A;&#x6A19;&#x7684;&#x907A;&#x4F1D;&#x5B50; SLC16A9 &#x3092;&#x767A;&#x898B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x7D50;&#x8AD6;: &#x6700;&#x521D;&#x306E; BAY11 &#x3092;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x6211;&#x3005; &#x306F;&#x5C0F;&#x5206;&#x5B50;&#x306B;&#x3088;&#x308B;&#x5B89;&#x5B9A;&#x5316;&#x5408;&#x6210;&#x306E; mRNA &#x767A;&#x73FE;&#x306E;&#x793A;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x6642;&#x9593;&#x3092;&#x9054;&#x6210;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x5C0F;&#x3055;&#x306A;&#x5206;&#x5B50;&#x81EA;&#x7136;&#x514D;&#x75AB;&#x306E;&#x6291;&#x5236;&#x306B;&#x57FA;&#x3065;&#x3044;&#x3066;&#x3001;&#x5F8C;&#x7D9A;&#x306E;&#x5805;&#x7262;&#x306A;&#x5F0F; transfected &#x5408;&#x6210; Mrna &#x306E;&#x5C06;&#x6765;&#x306E;&#x7D30;&#x80DE;&#x306B;&#x57FA;&#x3065;&#x304F;&#x7814;&#x7A76;&#x3068;&#x6CBB;&#x7642;&#x306E;&#x305F;&#x3081;&#x306E;&#x8907;&#x6570;&#x306E;&#x30A2;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

BAY11 OCT4 &#x5408;&#x6210; mRNA &#x767A;&#x73FE;&#x5927;&#x4EBA;&#x306E;&#x4EBA;&#x9593;&#x306E;&#x76AE;&#x819A;&#x7D30;&#x80DE;&#x3092;&#x5F37;&#x5316;&#x3057;&#x307E;&#x3059;&#x3002;

&#xAC1C;&#xC694;: &#xC18C;&#xAC1C;: The OCT4 &#xB179;&#xC74C; &#xBC29;&#xC1A1; &#xC694;&#xC778; &#xB9CE;&#xC740; &#xC138;&#xD3EC;&#xC9C8; &#xD504;&#xB85C;&#xC138;&#xC2A4; &#xAC1C;&#xBC1C;&#xC744; &#xD3EC;&#xD568; &#xD558; &#xC5EC;, &#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D;, pluripotency&#xC640; &#xCC28;&#xBCC4;&#xD654;&#xB97C; &#xC720;&#xC9C0;&#xC5D0; &#xCC38;&#xC5EC; &#xD569;&#xB2C8;&#xB2E4;. &#xD569;&#xC131; OCT4 Mrna&#xB294; &#xCD5C;&#xADFC;&#xC5D0; &#xC778;&#xAC04; &#xC720;&#xB3C4; &#xB9CC;&#xB2A5; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xC0DD;&#xC131;&#xC744; (&#xB2E4;&#xB978; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC744; &#xB2E4;&#xC2DC; &#xB9CC;&#xB4E4; &#xC694;&#xC18C;&#xC640; &#xD568;&#xAED8;)&#xC5D0; &#xC0AC;&#xC6A9; &#xB429;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xAC00; &#xBCA0;&#xC774; 11-7082 (BAY11), NF-kappaB-&#xC5B5;&#xC81C; &#xAE30;&#xBC18; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xD1B5;&#xD574; &#xC801;&#xC5B4;&#xB3C4; &#xBD80;&#xBD84;&#xC801;&#xC73C;&#xB85C; &#xC131;&#xC778; &#xC778;&#xAC04;&#xC758; &#xD53C;&#xBD80; &#xC138;&#xD3EC;&#xC5D0; &#xD569;&#xC131; mRNA (synRNA)&#xC758; transfection &#xB2E4;&#xC74C; OCT4 &#xC2DD; &#xD574;&#xC9C8; &#xC218; &#xBC1C;&#xACAC; &#xD588;&#xB2E4;. &#xBC29;&#xBC95;: &#xD569;&#xC131; mRNA transfection &#xC2DC; &#xBCF8; &#xD0C0;&#xACE0; &#xB09C; &#xBA74;&#xC5ED; &#xBC18;&#xC751;&#xC744; &#xC5B5;&#xC81C; &#xD558;&#xB294; &#xB2A5;&#xB825;&#xC5D0; &#xB2E4;&#xC591; &#xD55C; &#xD654;&#xD559;&#xACFC; &#xBD84;&#xC790; &#xC791;&#xC740; &#xBD84;&#xC790; &#xD14C;&#xC2A4;&#xD2B8;. 3 &#xBD84;&#xC790;-B18R, BX795, &#xBC0F; BAY11-immunocytochemical &#xBC0F; &#xD655;&#xC0B0;&#xC5D0;&#xC11C; &#xC0AC;&#xC6A9;&#xD55C; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC744; &#xAE30;&#xBC18;&#xC73C;&#xB85C; &#xD569;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xB610;&#xD55C; &#xAE00;&#xB85C;&#xBC8C; transcriptional &#xBA54;&#xD0C0;-&#xBD84;&#xC11D; oct4&#xC758; &#xD558;&#xB958; &#xC0C1;&#xB300; &#xC720;&#xC804;&#xC790; &#xBC1C;&#xD604;&#xC744; &#xC2DD;&#xBCC4; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC815;&#xB7C9;&#xC801; PCR&#xC640; &#xACB0;&#xD569;&#xC744; &#xD65C;&#xC6A9;. &#xACB0;&#xACFC;&#xB97C;: BAY11 &#xC874;&#xC7AC; &#xAD50;&#xC591;&#xB41C; &#xC778;&#xAC04;&#xC758; &#xD53C;&#xBD80; &#xC138;&#xD3EC;&#xC758; &#xC815;&#xC0C1;&#xC801;&#xC778; &#xC138;&#xD3EC; &#xC99D;&#xC2DD;&#xC744; &#xC5B5;&#xC81C; &#xD558;&#xC9C0; &#xC54A;&#xC558;&#xB2E4; OCT4 &#xC7AC;&#xD604;&#xD560; &#xC218; &#xC99D;&#xAC00; &#xC2DD;&#xC5D0; &#xACB0;&#xACFC; &#xCC3E;&#xC558;&#xC2B5;&#xB2C8;&#xB2E4;. Oct4&#xC758; &#xC99D;&#xAC00; &#xC218;&#xC900;&#xC744; &#xD3EC;&#xD568; &#xD558; &#xC5EC; &#xC774;&#xC804;&#xC5D0; &#xD655;&#xC778; &#xB41C; SPP1, DUSP4 &#xBC0F; &#xD45C;&#xD604;&#xB41C; oct4&#xB294; &#xAE30;&#xB2A5; &#xC81C;&#xC548; GADD45G OCT4, &#xD558;&#xB958; &#xC720;&#xC804;&#xC790;&#xC758; &#xD06C;&#xAC8C; &#xC99D;&#xAC00; &#xC2DD; &#xADC0;&#xCC29;&#xB418; &#xC5C8; &#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xB610;&#xD55C; &#xC18C;&#xC124; OCT4 putative &#xB2E4;&#xC6B4;&#xC2A4;&#xD2B8;&#xB9BC; &#xB300;&#xC0C1; &#xC720;&#xC804;&#xC790; SLC16A9 OCT4 &#xB808;&#xBCA8; &#xC0C1;&#xC2B9;&#xC5D0; &#xB530;&#xB77C; &#xD06C;&#xAC8C; &#xC99D;&#xAC00; &#xC2DD; &#xC2DC;&#xC5F0; &#xD558;&#xB294; &#xBC1C;&#xACAC;. &#xACB0;&#xB860;: &#xCC98;&#xC74C; &#xC6B0;&#xB9AC;&#xAC00; &#xADF8; &#xC791;&#xC740; &#xBD84;&#xC790; &#xAE30;&#xBC18; &#xC548;&#xC815;&#xD654; &#xD569;&#xC131; mRNA &#xD45C;&#xD604;&#xC758; &#xD45C;&#xC2DC; &#xB418;&#xB294; &#xC2DC;&#xAC04;&#xC774; bay11&#xC758; &#xC0AC;&#xC6A9;&#xC73C;&#xB85C; &#xC5BB;&#xC744; &#xC218; &#xC788;&#xB2E4;. &#xC774; &#xC791;&#xC740; &#xBD84;&#xC790; &#xAE30;&#xBC18;&#xC73C;&#xB85C; &#xD0C0;&#xACE0; &#xB09C; &#xBA74;&#xC5ED; &#xBC18;&#xC751;&#xC758; &#xC800;&#xD574; &#xBC0F; transfected &#xD569;&#xC131; Mrnas&#xC758; &#xD6C4;&#xC18D; &#xAC15;&#xB825;&#xD55C; &#xC2DD; &#xBBF8;&#xB798;&#xC758; &#xC138;&#xD3EC; &#xAE30;&#xBC18; &#xC5F0;&#xAD6C; &#xBC0F; &#xCE58;&#xB8CC; &#xD559;&#xC744; &#xC704;&#xD574; &#xC5EC;&#xB7EC; &#xC751;&#xC6A9; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC774; &#xC788;&#xC744; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

BAY11 &#xC131;&#xC778; &#xC778;&#xAC04;&#xC758; &#xD53C;&#xBD80; &#xC138;&#xD3EC;&#xC5D0; OCT4 &#xD569;&#xC131; mRNA &#xC2DD;&#xC744; &#xD5A5;&#xC0C1; &#xC2DC;&#xD0B5;&#xB2C8;&#xB2E4;.

Resumen: Introducci&oacute;n: factor de trascripci&oacute;n OCT4 el participa en muchos procesos celulares, incluyendo el desarrollo, reprogramaci&oacute;n, manteniendo la pluripotencia y la diferenciaci&oacute;n. Sint&eacute;tico OCT4 mRNA fue utilizado recientemente (en conjunci&oacute;n con otros factores de reprogramaci&oacute;n) para generar c&eacute;lulas madre pluripotentes inducidas humanas. Aqu&iacute;, hemos descubierto que BAY 11-7082 (BAY11), al menos parcialmente mediante un mecanismo de NF-kappaB-inhibici&oacute;n de la base, podr&iacute;a aumentar significativamente la expresi&oacute;n de OCT4 siguiendo la transfecci&oacute;n del mRNA sint&eacute;tico (synRNA) en las c&eacute;lulas de piel humana para adultos. M&Eacute;TODOS: Probamos varias peque&ntilde;as mol&eacute;culas de qu&iacute;micas y moleculares sobre su capacidad para suprimir la respuesta inmunitaria innata vista sobre sint&eacute;tico transfecci&oacute;n de mRNA. Tres mol&eacute;culas-B18R, BX795 y BAY11 fueron utilizados en immunocytochemical y proliferaci&oacute;n basada en ensayos. Tambi&eacute;n utilizamos metan&aacute;lisis transcripcional global, juntado con la PCR cuantitativa para identificar genes relativa aguas abajo de OCT4. Resultados: Encontramos que las c&eacute;lulas de piel humana cultivadas en presencia de BAY11 dio lugar a reproducible expresi&oacute;n creciente de OCT4 que no inhiben la proliferaci&oacute;n celular normal. Los niveles crecientes de OCT4 produjo significativamente mayor expresi&oacute;n de genes corriente abajo de OCT4, previamente identificadas SPP1, DUSP4 y GADD45G, sugiriendo que el OCT4 expresada era funcional. Tambi&eacute;n descubrimos un novela gen putativo Diana OCT4 SLC16A9 que demostraron significativamente mayor expresi&oacute;n tras la elevaci&oacute;n de los niveles de OCT4. CONCLUSIONES: Por primera vez que hemos mostrado esa estabilizaci&oacute;n basadas en la mol&eacute;cula peque&ntilde;a de expresi&oacute;n de mRNA sint&eacute;tico puede lograrse con el uso de BAY11. Esta peque&ntilde;a mol&eacute;cula basada en la inhibici&oacute;n de la respuesta inmune innata y posterior robusta expresi&oacute;n de los mRNAs sint&eacute;tico transfected puede tener m&uacute;ltiples aplicaciones para futuras investigaciones basadas en c&eacute;lulas y terap&eacute;utica.

BAY11 aumenta la expresi&oacute;n del mRNA sint&eacute;tica OCT4 en c&eacute;lulas de piel humana para adultos.

&#x90A3;&#x91CC;&#x7684;&#x62BD;&#x8C61;&#x8D8A;&#x6765;&#x8D8A;&#x591A;&#x7684;&#x8BC1;&#x636E;&#x8868;&#x660E;&#x8868;&#x89C2;&#x9057;&#x4F20;&#x91CD;&#x7F16;&#x7A0B;&#x7684;&#x5375;&#x6BCD;&#x7EC6;&#x80DE;&#x7684;&#x80FD;&#x529B;&#x662F;&#x4F18;&#x4E8E;&#x5F53;&#x524D;&#x57FA;&#x4E8E;&#x56E0;&#x5B50;&#x7684;&#x7F16;&#x7A0B;&#x65B9;&#x6CD5;&#x548C;&#x4E00;&#x4E9B;&#x56E0;&#x7D20;&#x91CD;&#x65B0;&#x8BF1;&#x5BFC;&#x591A;&#x80FD;&#x5E72;&#x7EC6;&#x80DE; &#xFF08;&#x9B54;&#x9B3C;&#xFF09; &#x4FDD;&#x7559;&#x4E00;&#x5B9A;&#x7A0B;&#x5EA6;&#x7684;&#x8868;&#x89C2;&#x9057;&#x4F20;&#x7684;&#x5185;&#x5B58;&#xFF0C;&#x53EF;&#x4EE5;&#x5F71;&#x54CD;&#x5206;&#x5316;&#x80FD;&#x529B;&#x5E76;&#x53EF;&#x94FE;&#x63A5;&#x5230;&#x89C2;&#x5BDF;&#x5230;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x7684;&#x514D;&#x75AB;&#x539F;&#x6027;&#x673A;&#x4F53;&#x529F;&#x80FD;&#x884D;&#x751F;&#x54C1;&#x3002;&#x4E3A;&#x6B64;&#x91CD;&#x65B0;&#x7F16;&#x7A0B;&#x80FD;&#x529B;&#x7684;&#x5DEE;&#x5F02;&#x7684;&#x4E00;&#x4E2A;&#x5047;&#x8BBE;&#x662F;&quot;&#x67D3;&#x8272;&#x8D28;&#x677E;&#x52A8;/&#x5F3A;&#x5316;&#x91CD;&#x7F16;&#x7A0B;&quot;&#x6982;&#x5FF5;&#xFF0C;&#x5982;&#x524D;&#x6587;&#x6240;&#x8FF0;&#x7684;&#x7EA6;&#x7FF0; &middot; &#x683C;&#x767B;&#x548C;&#x4F0A;&#x6069; &middot; &#x5A01;&#x5C14;&#xFF0C;&#x4EE5;&#x53CA;&#x5176;&#x4ED6;&#x4EBA;&#xFF0C;&#x5176;&#x4E2D;&#x5047;&#x8BBE;&#x5375;&#x6BCD;&#x7EC6;&#x80DE;&#x62E5;&#x6709;&#x677E;&#x5F00;&#x7EC6;&#x80DE;&#x6838;&#x67D3;&#x8272;&#x8D28;&#x7ED3;&#x6784;&#xFF0C;&#x63D0;&#x4F9B;&#x66F4;&#x591A;&#x7684;&#x51C6;&#x5907;&#x83B7;&#x5F97;&#x538B;&#x6291;&#x57FA;&#x56E0;&#x7684;&#x8868;&#x89C2;&#x9057;&#x4F20;&#x5B66;&#x4E0E;&#x8F6C;&#x5F55;&#x8C03;&#x8282;&#x56E0;&#x5B50;&#xFF0C;&#x4ECE;&#x800C;&#x5927;&#x5927;&#x589E;&#x52A0;&#x8868;&#x89C2;&#x9057;&#x4F20;&#x91CD;&#x7F16;&#x7A0B;&#x7684;&#x56E0;&#x7D20;&#x3002;&#x4F46;&#x662F;&#xFF0C;&#x6839;&#x636E;&#x7ECF;&#x9A8C;&#x6D4B;&#x8BD5;&#x8FD9;&#x4E00;&#x5047;&#x8BBE;&#x7684;&#x5019;&#x9009;&#x4EBA;&#x5375;&#x6BCD;&#x7EC6;&#x80DE;&#x91CD;&#x7F16;&#x7A0B;&#x56E0;&#x7D20;&#x5217;&#x8868; (CORFs) &#x5FC5;&#x987B;&#x67E5;&#x660E;&#x6240;&#x5927;&#x5927;&#x8868;&#x793A;&#x5728;&#x4E2D;&#x665A;&#x671F;&#x4E8C;&#x5375;&#x6BCD;&#x7EC6;&#x80DE;&#x4E2D;&#x3002;&#x4EE5;&#x524D;&#x7684;&#x7814;&#x7A76;&#x4FA7;&#x91CD;&#x4E8E;&#x83CC;&#x79CD;&#x5185;&#x6216;&#x8DE8;&#x7269;&#x79CD;&#x7684;&#x4E24;&#x4E2A;&#x4E0D;&#x540C;&#x54C1;&#x79CD;&#x7684;&#x5375;&#x6BCD;&#x7EC6;&#x80DE;&#x7684;&#x8F6C;&#x5F55;&#x5206;&#x6790;&#x3002;&#x5728;&#x8FD9;&#x9879;&#x7814;&#x7A76;&#xFF0C;&#x6211;&#x4EEC;&#x786E;&#x5B9A;&#x4E86;&#x516B;&#x4E2A; CORFs (ARID2&#x3001; ASF1A&#x3001; ASF1B&#x3001; DPPA3&#x3001; ING3&#x3001; MSL3&#x3001; H1FOO &#x548C; KDM6B&#xFF09; &#x4ECE;&#x4E09;&#x4E2A;&#x4E0D;&#x540C;&#x7684;&#x7269;&#x79CD; &#xFF08;&#x4EBA;&#x6743;&#x3001; &#x6052;&#x6CB3;&#x7334;&#x548C;&#x9F20;&#x6807;&#xFF09; &#x7684;&#x5375;&#x6BCD;&#x7EC6;&#x80DE;&#x65E0;&#x504F;&#x5168;&#x7403;&#x8F6C;&#x5F55;&#x5206;&#x6790;&#x7684;&#x57FA;&#x7840;&#x4E24;&#x8005;&#x90FD;&#x8868;&#x660E;&#x91CD;&#x5927; (p &lt; 0.05&#xFF0C;FC &gt; 3&#xFF09; &#x6240;&#x6709;&#x4E09;&#x4E2A;&#x7269;&#x79CD;&#x5728;&#x5375;&#x6BCD;&#x7EC6;&#x80DE;&#x4E2D;&#x7684;&#x8868;&#x8FBE;&#x6709;&#x677E;&#x52A8;/&#x5F00;&#x653E;&#x67D3;&#x8272;&#x8D28;&#x7ED3;&#x6784;&#x4E2D;&#x7684;&#x65E2;&#x5B9A;&#x89D2;&#x8272;&#x3002;&#x6211;&#x4EEC;&#x8FD8;&#x786E;&#x5B9A;&#x9002;&#x5408;&#x7684;&#x989D;&#x5916; 15 CORFs &#x5185;&#x6211;&#x4EEC;&#x62DF;&#x8BAE;&quot;&#x67D3;&#x8272;&#x8D28;&#x5F00;&#x5E55;/&#x547D;&#x8FD0;&#x53D8;&#x9769;&quot;&#xFF08;COFT&#xFF09; &#x6A21;&#x578B;&#x3002;&#x8FD9;&#x4E9B; CORFs &#x4E5F;&#x8BB8;&#x80FD;&#x591F;&#x589E;&#x52A0;&#x5F25;&#x7684;&#x5148;&#x524D;&#x88AB;&#x91CD;&#x65B0;&#x7F16;&#x7A0B; &#xFF08;OCT4&#x3001; SOX2&#x3001; KLF4 &#x548C; cMYC&#xFF09; &#x7684;&#x56E0;&#x7D20;&#x548C;&#x53EF;&#x80FD;&#x65B9;&#x4FBF;&#x7684;&#x8868;&#x89C2;&#x9057;&#x4F20;&#x8BB0;&#x5FC6;&#x7684;&#x9B54;&#x9B3C;&#x642C;&#x8FC1;&#x6216;&#x51CF;&#x5C11;&#x673A;&#x4F53;&#x529F;&#x80FD;&#x884D;&#x751F;&#x7269;&#xFF0C;&#x514D;&#x75AB;&#x539F;&#x6027;&#x57FA;&#x56E0;&#x7684;&#x8868;&#x8FBE;&#x548C;&#x53EF;&#x80FD;&#x5DF2;&#x5E94;&#x7528;&#x5728;&#x672A;&#x6765;&#x4E2A;&#x6027;&#x5316;&#x591A;&#x80FD;&#x5E72;&#x57FA;&#x4E8E;&#x5E72;&#x7EC6;&#x80DE;&#x7597;&#x6CD5;&#x3002;

&#x786E;&#x5B9A;&#x5019;&#x9009;&#x5375;&#x6BCD;&#x7EC6;&#x80DE;&#x91CD;&#x7F16;&#x7A0B;&#x4F7F;&#x7528;&#x8DE8;&#x7269;&#x79CD;&#x5168;&#x7403;&#x8F6C;&#x5F55;&#x5206;&#x6790;&#x7684;&#x56E0;&#x7D20;&#x3002;

Abstract There is mounting evidence to suggest that the epigenetic reprogramming capacity of the oocyte is superior to that of the current factor-based reprogramming approaches and that some factor-reprogrammed induced pluripotent stem cells (iPSCs) retain a degree of epigenetic memory that can influence differentiation capacity and may be linked to the observed expression of immunogenicity genes in iPSC derivatives. One hypothesis for this differential reprogramming capacity is the "chromatin loosening/enhanced reprogramming" concept, as previously described by John Gurdon and Ian Wilmut, as well as others, which postulates that the oocyte possesses factors that loosen the somatic cell chromatin structure, providing the epigenetic and transcriptional regulatory factors more ready access to repressed genes and thereby significantly increasing epigenetic reprogramming. However, to empirically test this hypothesis a list of candidate oocyte reprogramming factors (CORFs) must be ascertained that are significantly expressed in metaphase II oocytes. Previous studies have focused on intraspecies or cross-species transcriptional analysis of up to two different species of oocytes. In this study, we have identified eight CORFs (ARID2, ASF1A, ASF1B, DPPA3, ING3, MSL3, H1FOO, and KDM6B) based on unbiased global transcriptional analysis of oocytes from three different species (human, rhesus monkey, and mouse) that both demonstrate significant (p<0.05, FC>3) expression in oocytes of all three species and have well-established roles in loosening/opening up chromatin structure. We also identified an additional 15 CORFs that fit within our proposed "chromatin opening/fate transformative" (COFT) model. These CORFs may be able to augment Shinya Yamanaka's previously identified reprogramming factors (OCT4, SOX2, KLF4, and cMYC) and potentially facilitate the removal of epigenetic memory in iPSCs and/or reduce the expression of immunogenicity genes in iPSC derivatives, and may have applications in future personalized pluripotent stem cell based therapeutics.

Identifying Candidate Oocyte Reprogramming Factors Using Cross-Species Global Transcriptional Analysis.

R&eacute;sum&eacute; il est montage &eacute;l&eacute;ments portant &agrave; croire que l&#39;&eacute;pig&eacute;n&eacute;tique reprogrammation des capacit&eacute;s de l&#39;ovocyte est sup&eacute;rieure &agrave; celle des approches reprogrammation actuelles ax&eacute;e sur le facteur et que certains facteur-reprogramm&eacute; induite par les cellules souches pluripotentes (CISP) conservent une certaine m&eacute;moire &eacute;pig&eacute;n&eacute;tique qui peut influencer la capacit&eacute; de diff&eacute;renciation et pourrait &ecirc;tre li&eacute;e &agrave; l&#39;expression observ&eacute;e des g&egrave;nes d&#39;immunog&eacute;nicit&eacute; dans les d&eacute;riv&eacute;s de l&#39;iPSC. Une hypoth&egrave;se pour cette prime de reprogrammation de capacit&eacute; est le &laquo; chromatine desserrant/renforc&eacute;es reprogrammation &raquo; concept, comme pr&eacute;c&eacute;demment d&eacute;crit par John Gurdon et Ian Wilmut, ainsi que d&#39;autres, qui postule que l&#39;ovocyte poss&egrave;de des facteurs qui desserrer la structure de la chromatine de cellules somatiques, fournissant les facteurs de r&eacute;gulation &eacute;pig&eacute;n&eacute;tiques et transcriptionnelles acc&eacute;der plus facilement aux g&egrave;nes r&eacute;prim&eacute;s et augmente donc consid&eacute;rablement la reprogrammation &eacute;pig&eacute;n&eacute;tique. Toutefois, pour v&eacute;rifier empiriquement cette hypoth&egrave;se une liste d&#39;ovocyte candidat facteurs de reprogrammation (Viviers) doivent &ecirc;tre &eacute;tablies qui sont nettement exprim&eacute;s dans des ovocytes de m&eacute;taphase II. Des &eacute;tudes ant&eacute;rieures ont port&eacute; sur l&#39;analyse transcriptionnelle intrasp&eacute;cifique ou intersp&eacute;cifique de jusqu&#39;&agrave; deux diff&eacute;rentes esp&egrave;ces d&#39;ovocytes. Dans cette &eacute;tude, nous avons identifi&eacute; huit viviers (ARID2, ASF1A, ASF1B, DPPA3, ING3, MSL3, H1FOO et KDM6B), bas&eacute;s sur l&#39;objective analyse transcriptionnelle globale des ovocytes provenant de trois diff&eacute;rentes esp&egrave;ces (homme, singe rh&eacute;sus et souris) que les deux d&eacute;montrent significatif (p &lt; 0,05, FC &gt; 3) expression dans les ovocytes des trois esp&egrave;ces et ont un r&ocirc;le bien &eacute;tabli en desserrant ou l&#39;ouverture vers le haut de la structure de la chromatine. Nous avons aussi identifi&eacute; un viviers 15 suppl&eacute;mentaires qui s&#39;adaptent au sein de notre projet &laquo; chromatine ouverture/destin transformatrice &raquo; mod&egrave;le (hi&eacute;rarchiques). Ces viviers peuvent &ecirc;tre en mesure d&#39;augmenter de Shinya Yamanaka pr&eacute;c&eacute;demment identifi&eacute; reprogrammation des facteurs (OCT4, SOX2, KLF4 et cMYC) et potentiellement faciliter le retrait de la m&eacute;moire &eacute;pig&eacute;n&eacute;tique chez CISP et/ou r&eacute;duire l&#39;expression des g&egrave;nes d&#39;immunog&eacute;nicit&eacute; dans les d&eacute;riv&eacute;s de l&#39;iPSC, et peuvent avoir demandes &agrave; l&#39;avenir personnalis&eacute; pluripotent stem cell bas&eacute; therapeutics.

Identification des ovocytes candidat reprogrammation des facteurs &agrave; l&#39;aide d&#39;analyse transcriptionnelle globale intersp&eacute;cifique.

Abstrakt es ist mehrenden Beweise verlangt, dass die epigenetische Neuprogrammierung der Kapazit&auml;t von der Eizelle, die die aktuellen Ans&auml;tze der Faktor-basierte Neuprogrammierung &uuml;berlegen ist, und dass einige Faktor umprogrammiert pluripotenten Stammzellen (iPSCs induzierten) ein Ma&szlig; an epigenetischen Speicher aufbewahren, die Differenzierung Kapazit&auml;t beeinflussen k&ouml;nnen und werden die beobachteten Ausdruck der Immunogenit&auml;t Gene in iPSC Derivate verkn&uuml;pft. Eine Hypothese f&uuml;r diese Differential Neuprogrammierung Kapazit&auml;t ist das &quot;Chromatin Lockerung/Neuprogrammierung&quot; Konzept, wie bereits beschrieben, durch John Gurdon und Ian Wilmut, sowie andere, die postuliert, dass die Eizelle Faktoren, die die somatische Chromatin Struktur verf&uuml;gt, die die epigenetischen und transkriptionelle regulatorischen Faktoren eher bereit Zugang zu verdr&auml;ngten Gene und somit deutlich zu erh&ouml;hen, epigenetischen Reprogrammierung zu lockern. Um diese Hypothese eine Liste der Kandidaten Eizelle Neuprogrammierung Faktoren empirisch zu testen m&uuml;ssen jedoch (Sadki) feststellbar ist, dass deutlich in Metaphase-II-Oozyten ausgedr&uuml;ckt werden. Fr&uuml;here Studien konzentrierten sich auf intraspecies oder Kreuz-Arten transkriptionelle Analyse bis zu zwei verschiedenen Arten der Eizellen. In dieser Studie haben wir acht Sadki (ARID2, ASF1A, ASF1B, DPPA3, ING3 MSL3, H1FOO und KDM6B) basierend auf unvoreingenommene transkriptionelle Gesamtanalyse der Eizellen aus drei verschiedenen Arten (Mensch, Rhesus-Affen und Maus) identifiziert, dass beide signifikante zeigen (p &lt; 0,05, FC &gt; 3) Ausdruck in Oozyten aller drei Arten und etablierte Rollen bei Lockerung/&Ouml;ffnung Chromatin Struktur aufweisen. Wir eine zus&auml;tzliche 15 Sadki, die passen auch innerhalb unserer vorgeschlagenen identifiziert &quot;Chromatin-Er&ouml;ffnung/Schicksal transformative&quot; (COFT) Modell. Diese Sadki k&ouml;nnen m&ouml;glicherweise erweitern Yamanakas die zuvor identifizierten Faktoren (OCT4, SOX2, KLF4 und cMYC) Neuprogrammierung und potenziell erleichtern das Entfernen von epigenetischen Speicher in iPSCs und/oder verringern die Expression von Genen der Immunogenit&auml;t iPSC-Derivate und m&ouml;glicherweise haben Anwendungen in Zukunft personalisierte pluripotenten Stammzellen basierte Therapeutika.

Kandidat Eizelle Neuprogrammierung mit Cross-Species transkriptionelle Gesamtanalyse Faktoren zu ermitteln.

Astratto c&#39;&egrave; montaggio prove per suggerire che l&#39;epigenetica riprogrammazione capacit&agrave; dell&#39;ovocita &egrave; superiore a quella degli attuali approcci riprogrammazione basata sul fattore e che alcuni fattore riprogrammato induced pluripotent stem cells (iPSCs) mantenere un certo grado di memoria epigenetica che pu&ograve; influire sulla capacit&agrave; di differenziazione e pu&ograve; essere collegato all&#39;espressione dei geni immunogenicit&agrave; in iPSC derivati osservato. Una delle ipotesi per questo differenziale riprogrammazione capacit&agrave; &egrave; &quot;cromatina allentamento/enhanced riprogrammazione&quot; concetto, come precedentemente descritto da John Gurdon e Ian Wilmut, cos&igrave; come gli altri, che postula che l&#39;ovocita possiede fattori che allentare la struttura della cromatina di cellule somatiche, fornendo i fattori epigenetici e transcriptional normativi pi&ugrave; pronto accesso ai geni repressi e aumentando cos&igrave; notevolmente la riprogrammazione epigenetica. Tuttavia, per verificare empiricamente questa ipotesi un elenco dell&#39;ovocita candidato riprogrammazione fattori (CORFs) deve accertare che sono significativamente espressi in ovociti di metafase II. Precedenti studi si sono concentrati sull&#39;analisi trascrizionale intraspecie o cross-specie di fino a due diverse specie di ovociti. In questo studio, abbiamo individuato otto CORFs (ARID2, ASF1A, ASF1B, DPPA3, ING3, MSL3, H1FOO e KDM6B) basata su analisi imparziale di transcriptional globale degli ovociti da tre diverse specie (uomo, scimmia rhesus e mouse) che entrambi dimostrano significative (p &lt; 0,05, FC &amp;gt; 3) espressione in ovociti di tutte e tre le specie e hanno ruoli ben consolidati in allentamento/apertura struttura della cromatina. Abbiamo anche individuato un CORFs 15 aggiuntivi che si adattano all&#39;interno della nostra proposta &quot;della cromatina apertura/destino trasformativo&quot; modello (COFT). Questi CORFs possono essere in grado di aumentare di Shinya Yamanaka precedentemente identificati riprogrammazione fattori (OCT4, SOX2, KLF4 e cMYC) e potenzialmente di facilitare la rimozione della memoria epigenetica in iPSCs e/o ridurre l&#39;espressione dei geni di immunogenicit&agrave; in iPSC derivati, e potrebbero avere applicazioni nel futuro personalizzate terapie basate sulle cellule staminali pluripotenti.

Identificazione dei candidati ovocita riprogrammazione fattori mediante analisi Transcriptional globale Cross-specie.

&#x8981;&#x7D04;&#x304C;&#x3001;&#x30A8;&#x30D4;&#x30B8;&#x30A7;&#x30CD;&#x30C6;&#x30A3;&#x30C3;&#x30AF;&#x306A;&#x5BB9;&#x91CF;&#x3001;&#x5375;&#x6BCD;&#x7D30;&#x80DE;&#x306E;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30E0;&#x3057;&#x76F4;&#x3059;&#x3053;&#x3068;&#x306F;&#x73FE;&#x5728;&#x306E;&#x56E0;&#x5B50;&#x306B;&#x57FA;&#x3065;&#x304F;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30E0;&#x3057;&#x76F4;&#x3059;&#x65B9;&#x6CD5;&#x3092;&#x3088;&#x308A;&#x3082;&#x512A;&#x308C;&#x3066;&#x3044;&#x308B;&#x3057;&#x3001;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x8981;&#x56E0;&#x304C;&#x518D;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30E0;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE; (ips &#x7D30;&#x80DE;) &#x3092;&#x8A98;&#x5C0E;&#x5206;&#x5316;&#x306B;&#x5F71;&#x97FF;&#x3092;&#x4E0E;&#x3048;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x306E;&#x514D;&#x75AB;&#x539F;&#x6027;&#x907A;&#x4F1D;&#x5B50; iPSC &#x8A98;&#x5C0E;&#x4F53;&#x306E;&#x89B3;&#x5BDF;&#x3055;&#x308C;&#x305F;&#x8868;&#x73FE;&#x306B;&#x30EA;&#x30F3;&#x30AF;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x30A8;&#x30D4;&#x30B8;&#x30A7;&#x30CD;&#x30C6;&#x30A3;&#x30C3;&#x30AF;&#x306A;&#x30E1;&#x30E2;&#x30EA;&#x306E;&#x7A0B;&#x5EA6;&#x4FDD;&#x6301;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x3059;&#x308B;&#x8A3C;&#x62E0;&#x306F;&#x53D6;&#x4ED8;&#x3051;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x5DEE;&#x52D5;&#x5BB9;&#x91CF;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30E0;&#x3057;&#x76F4;&#x3059;&#x3053;&#x3068;&#x306E; 1 &#x3064;&#x306E;&#x4EEE;&#x8AAC;&#x306F;&#x5375;&#x6BCD;&#x7D30;&#x80DE;&#x30A8;&#x30D4;&#x30B8;&#x30A7;&#x30CD;&#x30C6;&#x30A3;&#x30C3;&#x30AF;&#x306A;&#x3068;&#x8EE2;&#x5199;&#x8ABF;&#x7BC0;&#x56E0;&#x5B50;&#x3088;&#x308A;&#x6291;&#x5727;&#x3055;&#x308C;&#x305F;&#x907A;&#x4F1D;&#x5B50;&#x3078;&#x306E;&#x30A2;&#x30AF;&#x30BB;&#x30B9;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x3001;&#x30A8;&#x30D4;&#x30B8;&#x30A7;&#x30CD;&#x30C6;&#x30A3;&#x30C3;&#x30AF;&#x306A;&#x518D;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30E0;&#x5316;&#x3092;&#x5927;&#x5E45;&#x306B;&#x5897;&#x52A0;&#x306F;&#x3001;&#x4F53;&#x7D30;&#x80DE;&#x306E;&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3;&#x306E;&#x69CB;&#x9020;&#x3092;&#x3086;&#x308B;&#x3081;&#x308B;&#x8981;&#x56E0;&#x3092;&#x6301;&#x3063;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x3092;&#x4EEE;&#x5B9A;&#x300C;&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3;&#x3086;&#x308B;&#x307F;/&#x30EA;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30DF;&#x30F3;&#x30B0;&#x5F37;&#x5316;&#x300D;&#x6982;&#x5FF5;&#x3001;&#x30B8;&#x30E7;&#x30F3; &#x30FB; &#x30B4;&#x30FC;&#x30C9;&#x30F3;&#x3068;&#x30A4;&#x30A2;&#x30F3; &#x30FB; &#x30A6;&#x30A3;&#x30EB;&#x30DE;&#x30C3;&#x30C8;&#x3068;&#x4ED6;&#x306E;&#x4EBA;&#x306B;&#x3088;&#x3063;&#x3066;&#x524D;&#x8FF0;&#x306E;&#x3088;&#x3046;&#x3067;&#x3059;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x7D4C;&#x9A13;&#x7684;&#x521D;&#x671F;&#x5316;&#x56E0;&#x5B50;&#x306E;&#x5019;&#x88DC;&#x8005;&#x306E;&#x5375;&#x6BCD;&#x7D30;&#x80DE;&#x306E;&#x4E00;&#x89A7;&#x3053;&#x306E;&#x4EEE;&#x8AAC;&#x3092;&#x30C6;&#x30B9;&#x30C8;&#x3059;&#x308B;&#x306B;&#x306F; (CORFs) &#x306F;&#x5927;&#x5E45;&#x306B;&#x4E2D;&#x671F; II &#x306E;&#x5375;&#x6BCD;&#x7D30;&#x80DE;&#x3067;&#x8868;&#x73FE;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x78BA;&#x8A8D;&#x3059;&#x308B;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x4EE5;&#x524D;&#x306E;&#x7814;&#x7A76;&#x5375;&#x6BCD;&#x7D30;&#x80DE;&#x306E;&#x6700;&#x5927; 2 &#x3064;&#x306E;&#x7570;&#x306A;&#x308B;&#x7A2E;&#x306E;&#x7A2E;&#x5185;&#x3084;&#x30AF;&#x30ED;&#x30B9;&#x7A2E;&#x306E;&#x8EE2;&#x5199;&#x89E3;&#x6790;&#x306B;&#x7126;&#x70B9;&#x3092;&#x5F53;&#x3066;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;&#x6211;&#x3005; &#x306F;&#x5375;&#x6BCD;&#x7D30;&#x80DE; &#xFF08;&#x30D2;&#x30C8;&#x3001;&#x30A2;&#x30AB;&#x30B2;&#x30B6;&#x30EB;&#x3068;&#x30DE;&#x30A6;&#x30B9;&#xFF09; &#x306E; 3 &#x3064;&#x306E;&#x7570;&#x306A;&#x308B;&#x7A2E;&#x304B;&#x3089;&#x306E;&#x516C;&#x5E73;&#x306A;&#x30B0;&#x30ED;&#x30FC;&#x30D0;&#x30EB;&#x8EE2;&#x5199;&#x5206;&#x6790;&#x306B;&#x57FA;&#x3065;&#x3044;&#x3066; 8 &#x3064;&#x306E; CORFs &#xFF08;ARID2&#x3001;ASF1A&#x3001;ASF1B&#x3001;DPPA3&#x3001;ING3&#x3001;MSL3&#x3001;H1FOO&#x3001;&#x304A;&#x3088;&#x3073; KDM6B) &#x3092;&#x540C;&#x5B9A;&#x3057;&#x305F;&#x4E21;&#x65B9;&#x91CD;&#x8981;&#x306A;&#x3092;&#x793A;&#x3059; (p &lt; 0.05&#x3001;FC &#x76EE; 3) &#x3059;&#x3079;&#x3066; 3 &#x7A2E;&#x306E;&#x5375;&#x6BCD;&#x7D30;&#x80DE;&#x767A;&#x73FE;&#x3068;&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3;&#x69CB;&#x9020;&#x306E;&#x3086;&#x308B;&#x307F;&#x3068;&#x30AA;&#x30FC;&#x30D7;&#x30CB;&#x30F3;&#x30B0;&#x3067;&#x5B9A;&#x8A55;&#x306E;&#x3042;&#x308B;&#x5F79;&#x5272;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x79C1;&#x305F;&#x3061;&#x3082;&#x79C1;&#x305F;&#x3061;&#x63D0;&#x6848;&#x306E;&#x5185;&#x306B;&#x5408;&#x308F;&#x305B;&#x3066;&#x3001;&#x8FFD;&#x52A0;&#x306E; 15 CORFs &#x8B58;&#x5225;&#x3055;&#x308C;&#x308B;&#x300C;&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3; &#x30AA;&#x30FC;&#x30D7;&#x30CB;&#x30F3;&#x30B0;/&#x904B;&#x547D;&#x5909;&#x9769;&#x300D;&#xFF08;COFT&#xFF09; &#x30E2;&#x30C7;&#x30EB;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E; CORFs &#x306F;&#x3001;&#x4E0A;&#x8A18;&#x521D;&#x671F;&#x5316;&#x56E0;&#x5B50; (OCT4, SOX2&#x3001;KLF4&#x3001;&#x304A;&#x3088;&#x3073; cMYC) &#x5C71;&#x4E2D;&#x4F38;&#x5F25;&#x3092;&#x5F37;&#x5316;&#x6F5C;&#x5728;&#x7684; ips &#x7D30;&#x80DE;&#x306B;&#x304A;&#x3051;&#x308B;&#x30A8;&#x30D4;&#x30B8;&#x30A7;&#x30CD;&#x30C6;&#x30A3;&#x30C3;&#x30AF;&#x306A;&#x8A18;&#x61B6;&#x306E;&#x9664;&#x53BB;&#x3092;&#x4FC3;&#x9032;&#x3059;&#x308B;/&#x307E;&#x305F;&#x306F; iPSC &#x8A98;&#x5C0E;&#x4F53;&#x306E;&#x514D;&#x75AB;&#x539F;&#x6027;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x767A;&#x73FE;&#x3092;&#x6291;&#x5236;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x5834;&#x5408;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3001;&#x30A2;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x306F;&#x3001;&#x5C06;&#x6765;&#x306E;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x30D9;&#x30FC;&#x30B9;&#x6CBB;&#x7642;&#x30D1;&#x30FC;&#x30BD;&#x30CA;&#x30EB;&#x5316;&#x3057;&#x305F;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

&#x521D;&#x671F;&#x5316;&#x56E0;&#x5B50;&#x306E;&#x7570;&#x7A2E;&#x30B0;&#x30ED;&#x30FC;&#x30D0;&#x30EB;&#x8EE2;&#x5199;&#x89E3;&#x6790;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x5019;&#x88DC;&#x8005;&#x306E;&#x5375;&#x6BCD;&#x7D30;&#x80DE;&#x3092;&#x8B58;&#x5225;&#x3057;&#x307E;&#x3059;&#x3002;

&#xCD94;&#xC0C1; &#xAC70;&#xAE30; &#xADF8;&#xB294; epigenetic &#xC7AC;&#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D; &#xB41C; oocyte &#xC6A9;&#xB7C9; &#xC6B0;&#xC218; &#xD569;&#xB2C8;&#xB2E4; &#xD604;&#xC7AC; &#xC694;&#xC18C; &#xAE30;&#xBC18; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC744; &#xB2E4;&#xC2DC; &#xB9CC;&#xB4E4; &#xC811;&#xADFC; &#xD558; &#xACE0; &#xC77C;&#xBD80; &#xC694;&#xC18C; &#xC7AC;&#xC124;&#xC815; pluripotent &#xC904;&#xAE30; &#xC138;&#xD3EC; (iPSCs)&#xC744; &#xC720;&#xBC1C; &#xC720;&#xC9C0; &#xCC28;&#xBCC4;&#xD654; &#xC6A9;&#xB7C9;&#xC5D0; &#xC601;&#xD5A5;&#xC744; &#xBBF8;&#xCE60; &#xC218; immunogenicity &#xC720;&#xC804;&#xC790; iPSC &#xD30C;&#xC0DD; &#xC0C1;&#xD488;&#xC5D0;&#xC11C;&#xC758; &#xAD00;&#xCC30;&#xB41C; &#xD45C;&#xD604;&#xC5D0; &#xC5F0;&#xACB0; &#xB420; &#xC218; &#xC788;&#xB294; epigenetic &#xBA54;&#xBAA8;&#xB9AC; &#xC815;&#xB3C4; &#xC81C;&#xC548; &#xC99D;&#xAC70;&#xB97C; &#xC7A5;&#xCC29;. &#xC6A9;&#xB7C9;&#xC744; &#xC7AC;&#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D;&#xC774; &#xBBF8;&#xBD84;&#xC5D0; &#xB300; &#xD55C; &#xAC00;&#xC124; &quot;chromatin &#xC644;&#xD654;/&#xACE0;&#xAE09; &#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D;&quot; &#xAC1C;&#xB150; &#xC774;&#xB2E4;, John Gurdon&#xC640;&#xC774; &#xC548; &#xC70C; &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; &#xB2E4;&#xB978; &#xC0AC;&#xB78C;, &#xC55E;&#xC5D0;&#xC11C; &#xC124;&#xBA85;&#xD55C; &#xB300;&#xB85C; &#xC5EC;&#xAE34;&#xB2E4;&#xB294; oocyte epigenetic transcriptional &#xADDC;&#xC81C; &#xC694;&#xC778; &#xC5B5;&#xC555; &#xB41C; &#xC720;&#xC804;&#xC790;&#xB97C; &#xB354; &#xC900;&#xBE44; &#xC561;&#xC138;&#xC2A4;&#xB97C; &#xC81C;&#xACF5; &#xD558; &#xACE0; epigenetic &#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D; &#xD568;&#xC73C;&#xB85C;&#xC368; &#xD06C;&#xAC8C; &#xC99D;&#xAC00; &#xCCB4;&#xC138;&#xD3EC; chromatin &#xAD6C;&#xC870; &#xC644;&#xD654; &#xC694;&#xC778;&#xC744; &#xBCF4;&#xC720; &#xD558; &#xACE0; &#xC788;&#xB294;. &#xADF8;&#xB7EC;&#xB098;, &#xACBD;&#xD5D8;&#xC801;&#xC73C;&#xB85C;&#xC774; &#xAC00;&#xC124; &#xD6C4;&#xBCF4; oocyte &#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D; &#xC694;&#xC18C; &#xBAA9;&#xB85D; &#xD14C;&#xC2A4;&#xD2B8; (CORFs) &#xD574;&#xC57C; &#xD569;&#xB2C8;&#xB2E4; &#xC218; ascertained&#xB294; &#xD06C;&#xAC8C; &#xBD84;&#xC5F4; II oocytes&#xC5D0;&#xC11C; &#xD45C;&#xD604; &#xB418;&#xB294;. &#xC774;&#xC804; &#xC5F0;&#xAD6C;&#xB294; &#xCD5C;&#xB300; 2 &#xAC1C;&#xC758; &#xB2E4;&#xB978; &#xC885;&#xC758; oocytes &#xACF5;&#xB7B5; &#xB610;&#xB294; &#xAC04; &#xC885; transcriptional &#xBD84;&#xC11D;&#xC5D0; &#xC9D1;&#xC911; &#xD588;&#xB2E4;. &#xC774; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; &#xC6B0;&#xB9AC;&#xAC00; oocytes 3 &#xC885; (&#xC778;&#xAC04;, &#xBD89;&#xC740; &#xD138; &#xC6D0;&#xC22D;&#xC774; &#xB9C8;&#xC6B0;&#xC2A4;)&#xC758; &#xD3B8;&#xACAC;&#xB41C; &#xAE00;&#xB85C;&#xBC8C; transcriptional &#xBD84;&#xC11D;&#xC744; &#xAE30;&#xBC18;&#xC73C;&#xB85C; 8 &#xAC1C;&#xC758; CORFs (ARID2, ASF1A, ASF1B, DPPA3, ING3, MSL3, H1FOO, &#xBC0F; KDM6B)&#xC744; &#xBC1C;&#xACAC; &#xB458; &#xB2E4; &#xC911;&#xC694; &#xD55C; &#xC785;&#xC99D; (p &lt; 0.05, FC &amp;gt; 3) &#xBAA8;&#xB450; 3 &#xC885;&#xC758; oocytes&#xC5D0;&#xC11C; &#xC2DD; chromatin &#xAD6C;&#xC870; &#xC644;&#xD654;/&#xAC1C;&#xBC29;&#xC5D0;&#xC11C; &#xC5ED;&#xD560;&#xC744; &#xC798; &#xC124;&#xB9BD; &#xD558; &#xACE0;. &#xC6B0;&#xB9AC; &#xB610;&#xD55C; &#xC6B0;&#xB9AC;&#xC758; &#xC81C;&#xC548;&#xC5D0; &#xB9DE;&#xB294; &#xCD94;&#xAC00; 15 Corfs&#xB294; &#xC2DD;&#xBCC4; &quot;chromatin &#xC624;&#xD504;&#xB2DD;/&#xC6B4;&#xBA85; transformative&quot; (COFT) &#xBAA8;&#xB378;. &#xC774;&#xB7EC;&#xD55C; CORFs &#xC774;&#xC804;&#xC5D0; &#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D;&#xC744; &#xC694;&#xC778; (OCT4, SOX2, KLF4, &#xBC0F; cMYC)&#xC744; &#xBC1C;&#xACAC; &#xC2E0; &#xC57C; &#xC57C; &#xB9C8;&#xB098;&#xCE74;&#xC758; &#xC99D;&#xAC00; &#xC7A0;&#xC7AC;&#xC801;&#xC73C;&#xB85C; iPSCs epigenetic &#xBA54;&#xBAA8;&#xB9AC; &#xC81C;&#xAC70;&#xB97C; &#xC6A9;&#xC774; &#xD558; &#xAC8C; &#xB610;&#xB294; immunogenicity &#xC720;&#xC804;&#xC790; iPSC &#xD30C;&#xC0DD; &#xC0C1;&#xD488;&#xC5D0;&#xC11C;&#xC758; &#xC2DD;&#xC744; &#xC904;&#xC77C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xADF8;&#xB9AC;&#xACE0; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xC560;&#xD50C; &#xB9AC; &#xCF00;&#xC774; &#xC158; &#xBBF8;&#xB798;&#xC5D0; &#xAC1C;&#xC778; &#xC124;&#xC815;&#xD55C; pluripotent &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xAE30;&#xBC18; &#xCE58;&#xB8CC;&#xC81C;.

&#xD6C4;&#xBCF4; Oocyte &#xC694;&#xC778; &#xAC04; &#xC885; &#xAE00;&#xB85C;&#xBC8C; Transcriptional &#xBD84;&#xC11D;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D;&#xC744; &#xC2DD;&#xBCC4; &#xD569;&#xB2C8;&#xB2E4;.

Resumen existe creciente evidencia para sugerir que la epigen&eacute;tica reprogramaci&oacute;n capacidad del ovocito es superior a la de los actuales enfoques de reprogramaci&oacute;n basadas en el factor y que algunos factor-reprogramado inducida por las c&eacute;lulas de v&aacute;stago pluripotent (iPSCs) conservan un grado de memoria epigen&eacute;tica que puede influir en la capacidad de diferenciaci&oacute;n y podr&iacute;a relacionarse con la expresi&oacute;n observada de genes de inmunogenicidad en derivados de iPSC. Una hip&oacute;tesis para este diferencial capacidad de reprogramaci&oacute;n es el &quot;cromatina aflojamiento mejorada reprogramaci&oacute;n&quot; concepto, seg&uacute;n se describi&oacute; anteriormente por John Gurdon y Ian Wilmut, as&iacute; como otros, que postula que el ovocito posee factores que afloje la estructura de la cromatina de la c&eacute;lula som&aacute;tica, proporcionando los factores reguladores transcripcionales y epigen&eacute;ticos de m&aacute;s f&aacute;cil acceso a los genes reprimidos y el aumento de tal modo perceptiblemente reprogramaci&oacute;n epigen&eacute;tica. Sin embargo, para probar emp&iacute;ricamente esta hip&oacute;tesis una lista del ovocito candidato reprogramaci&oacute;n factores (CORFs) deben ser comprobados que significativamente se expresan en ovocitos de metafase II. Estudios anteriores se han centrado en el an&aacute;lisis transcripcional intraespec&iacute;ficas o especie de dos especies diferentes de ovocitos. En este estudio, hemos identificado ocho CORFs (ARID2, ASF1A, ASF1B, DPPA3, ING3, MSL3, H1FOO y KDM6B), basados en an&aacute;lisis transcripcional global imparcial de ovocitos de tres especies (humanos, mono rhesus y rat&oacute;n) que tanto demuestran significativos (p &lt; 0.05, FC &gt; 3) expresi&oacute;n en ovocitos de las tres especies y tienen funciones bien establecidas en aflojar/apertura estructura de la cromatina. Tambi&eacute;n identificamos un adicional CORFs 15 que encajan dentro de nuestra propuesta &quot;cromatina apertura/destino transformadora&quot; modelo (COFT). Estos CORFs pueden ser capaces de aumentar de Shinya Yamanaka previamente identificado reprogramaci&oacute;n (OCT4, SOX2, KLF4 y cMYC) y potencialmente facilitan la expulsi&oacute;n de memoria epigen&eacute;tico en iPSCs y/o reducir la expresi&oacute;n de genes de inmunogenicidad en derivados de iPSC, y pueden han aplicaciones en futuro personalizado pluripotentes c&eacute;lulas base terap&eacute;utica.

Identificaci&oacute;n de ovocitos candidato reprogramaci&oacute;n factores utilizando an&aacute;lisis transcripcional Global entre especies.

Stage-specific embryonic antigen 3 (SSEA3) is a glycosphingolipid that has previously been used to identify cells with stem cell-like, multipotent, and pluripotent characteristics. A rare subpopulation of SSEA3-expressing cells exists in the dermis of adult human skin. These SSEA3-expressing cells undergo a significant increase in cell number in response to injury, suggesting a possible role in regeneration. These SSEA3-expressing regeneration-associated (SERA) cells were derived through primary cell culture, purified by fluorescence-activated cell sorting (FACS), and characterized. Longer in vitro culture of the primary skin cells led to lower SSEA3 expression stability after FACS-based purification, suggesting that the current culture conditions may need to be optimized to permit the large-scale expansion of SERA cells. The SERA cells demonstrated a global transcriptional state that was most similar to bone marrow- and fat-derived mesenchymal stem cells (MSCs), and the highest expressing SSEA3-expressing cells co-expressed CD105 (clone 35). However, while a rare population of MSCs was observed in primary human skin cell cultures that could differentiate into adipocytes, osteoblasts, or chondrocytes, SERA cells did not possess this differentiation capacity, suggesting that there are at least two different rare subpopulations in adult human skin primary cultures. The identification, efficient purification, and large-scale expansion of these rare subpopulations (SERA cells and MSCs) from heterogeneous adult human skin primary cell cultures may have applications for future patient-specific cellular therapies.

Human skin cells that express stage-specific embryonic antigen 3 associate with dermal tissue regeneration.

The reprogramming of a patient's somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming, however, represents a safety concern that should be addressed prior to clinical applications. The polycistronic "stem cell cassette" (STEMCCA), an excisable lentiviral reprogramming vector, provides, in our hands, the most consistent reprogramming approach that addresses this safety concern. Nevertheless, most viral integrations occur in genes, and exactly how the integration, epigenetic reprogramming, and excision of the STEMCCA reprogramming cassette influences those genes and whether these cells still have clinical potential are not yet known.

Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status.

Human arginase deficiency is characterized by hyperargininemia and infrequent episodes of hyperammonemia, which lead to neurological impairment with spasticity, loss of ambulation, seizures, and severe mental and growth retardation; uncommonly, patients suffer early death from this disorder. In a murine targeted knockout model, onset of the phenotypic abnormality is heralded by weight loss at around day 15, and death occurs typically by postnatal day 17 with hyperargininemia and markedly elevated ammonia. This discrepancy between the more attenuated juvenile-onset human disease and the lethal neonatal murine model has remained suboptimal for studying and developing therapy for the more common presentation of arginase deficiency. These investigations aimed to address this issue by creating an adult conditional knockout mouse to determine whether later onset of arginase deficiency also resulted in lethality. Animal survival and ammonia levels, body weight, circulating amino acids, and tissue arginase levels were examined as outcome parameters after widespread Cre-recombinase activation in a conditional knockout model of arginase 1 deficiency. One hundred percent of adult female and 70% of adult male mice died an average of 21.0 and 21.6days, respectively, after the initiation of tamoxifen administration. Animals demonstrated elevated circulating ammonia and arginine at the onset of phenotypic abnormalities. In addition, brain and liver amino acids demonstrated abnormalities. These studies demonstrate that (a) the absence of arginase in adult animals results in a disease profile (leading to death) similar to that of the targeted knockout and (b) the phenotypic abnormalities seen in the juvenile-onset model are not exclusive to the age of the animal but instead to the biochemistry of the disorder. This adult model will be useful for developing gene- and cell-based therapies for this disorder that will not be limited by the small animal size of neonatal therapy and for developing a better understanding of the characteristics of hyperargininemia.

Lethal phenotype in conditional late-onset arginase 1 deficiency in the mouse.

Improved therapies for cancer and other conditions have resulted in a growing population of long-term survivors. Infertility is an unfortunate side effect of some cancer therapies that impacts the quality of life of survivors who are in their reproductive or prereproductive years. Some of these patients have the opportunity to preserve their fertility using standard technologies that include sperm, egg, or embryo banking, followed by IVF and/or ET. However, these options are not available to all patients, especially the prepubertal patients who are not yet producing mature gametes. For these patients, there are several stem cell technologies in the research pipeline that may give rise to new fertility options and allow infertile patients to have their own biological children. We will review the role of stem cells in normal spermatogenesis as well as experimental stem cell-based techniques that may have potential to generate or regenerate spermatogenesis and sperm. We will present these technologies in the context of the fertility preservation paradigm, but we anticipate that they will have broad implications for the assisted reproduction field.

Germline stem cells: toward the regeneration of spermatogenesis.

Owing to the aging of the population, our society now faces an impending wave of age-related neurodegenerative pathologies, the most significant of which is Alzheimer's disease. Currently, no effective therapies for Alzheimer's disease have been developed. However, recent advances in the fields of neural stem cells and human induced pluripotent stem cells now provide us with the first real hope for a cure. The recent discovery by Blurton-Jones and colleagues that neural stem cells can effectively deliver disease-modifying therapeutic proteins throughout the brains of our best rodent models of Alzheimer's disease, combined with recent advances in human nuclear reprogramming, stem cell research, and highly customized genetic engineering, may represent a potentially revolutionary personalized cellular therapeutic approach capable of effectively curing, ameliorating, and/or slowing the progression of Alzheimer's disease.

Article	Year
Commentary on human cloning. Differentiation; research in biological diversity\| PubMed ID: 11841469	2002
Nuclei of adult mammalian somatic cells are directly reprogrammed to oct-4 stem cell gene expression by amphibian oocytes. Current biology : CB\| PubMed ID: 12867031	2003
Transcriptional profiling of rhesus monkey embryonic stem cells. Biology of reproduction\| PubMed ID: 16943365	2006
Current progress with primate embryonic stem cells. Current stem cell research & therapy\| PubMed ID: 18220862	2006
Generation of isogenic pluripotent stem cells. Human molecular genetics\| PubMed ID: 18632695	2008
Enhanced generation of induced pluripotent stem cells from a subpopulation of human fibroblasts. PloS one\| PubMed ID: 19774082	2009
Parthenogenic blastocysts derived from cumulus-free in vitro matured human oocytes. PloS one\| PubMed ID: 20539753	2010
BAY11 enhances OCT4 synthetic mRNA expression in adult human skin cells. Stem cell research & therapy\| PubMed ID: 23388106	2013
Identifying Candidate Oocyte Reprogramming Factors Using Cross-Species Global Transcriptional Analysis. Cellular reprogramming\| PubMed ID: 23458164	2013
Human skin cells that express stage-specific embryonic antigen 3 associate with dermal tissue regeneration. BioResearch open access\| PubMed ID: 23514702	2012
Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status. Stem cell research & therapy\| PubMed ID: 23890092	2013
Lethal phenotype in conditional late-onset arginase 1 deficiency in the mouse. Molecular genetics and metabolism\| PubMed ID: 23920045	2013
Germline stem cells: toward the regeneration of spermatogenesis. Fertility and sterility\| PubMed ID: 24314923	2014
Developing neural stem cell-based treatments for neurodegenerative diseases. Stem cell research & therapy\| PubMed ID: 25157920	2014

James A. Byrne

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JoVE Journal Publications

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