efficient-gene-transfer-chick-retinas-for-primary-cell-culture

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An <em>Ex-ovo</em> Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

The Johns Hopkins University School of Medicine

&#x6211;&#x4EEC;&#x786E;&#x5B9A;&#x4E86;&#x4E00;&#x79CD;&#x673A;&#x5236;&#x4F7F;&#x89C6;&#x7F51;&#x819C;&#x518D;&#x751F;&#x7684;&#x80DA;&#x80CE;&#x5C0F;&#x9E21;&#x53EF;&#x4EE5;&#x8BF1;&#x5BFC;&#x5E72;/&#x7956;&#x7EC6;&#x80DE;&#x7684;&#x8D21;&#x732E;&#x3002;&#x6211;&#x4EEC;&#x663E;&#x793A;&#x9AA8;&#x5F62;&#x6001;&#x53D1;&#x751F;&#x86CB;&#x767D; (BMP) &#x4FE1;&#x53F7;&#x662F;&#x5145;&#x5206;&#x548C;&#x5FC5;&#x8981;&#x8BF1;&#x4F7F;&#x89C6;&#x7F51;&#x819C;&#x518D;&#x751F;&#x548C;&#x5176;&#x884C;&#x52A8;&#x53EF;&#x4EE5;&#x5206;&#x4E3A;&#x4E24;&#x4E2A;&#x9636;&#x6BB5;&#x3002;Postretinectomy (d PR) &#x540E;&#x7684; 3 &#x5929;&#xFF0C;BMP &#x901A;&#x8DEF;&#x5C06;&#x5B9A;&#x5411;&#x6269;&#x6563;&#x548C;&#x901A;&#x8FC7;&#x6FC0;&#x6D3B; Smad (&#x5178;&#x578B; BMP &#x901A;&#x8DEF;) &#x4E0E; FGF &#x4FE1;&#x53F7;&#x7531; mapk &#x4FE1;&#x53F7;&#x8F6C;&#x5BFC;&#x901A;&#x8DEF;&#x7684;&#x8C03;&#x63A7;&#x518D;&#x751F;&#x3002;7 D &#x516C;&#x5173;&#xFF0C;&#x7531;&#x5B83;&#x8BF1;&#x5BFC;&#x51CB;&#x4EA1;&#x901A;&#x8FC7;&#x6FC0;&#x6D3B; p38 &#xFF08;&#x975E;&#x89C4;&#x8303;&#x5316;&#x7684; BMP &#x901A;&#x8DEF;&#xFF09; &#x548C;&#x5411;&#x4E0B;&#x8C03;&#x8282; FGF &#x4FE1;&#x53F7; &#xFF08;&#x901A;&#x8FC7; MAPK &#x548C; AKT &#x9014;&#x5F84;&#xFF09;&#x3002;&#x5728;&#x6B64;&#x4EE5;&#x540E;&#x9636;&#x6BB5;&#x7684;&#x7EC6;&#x80DE;&#x51CB;&#x4EA1;&#x662F;&#x53EF;&#x4EE5;&#x9884;&#x9632;&#x7684;&#x548C; BMP &#x8BF1;&#x5BFC;&#x518D;&#x751F;&#x53EF;&#x4EE5;&#x8FDB;&#x4E00;&#x6B65;&#x8BF1;&#x5BFC; p38 &#x7684;&#x6291;&#x5236;&#x4F5C;&#x7528;&#x3002;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x89E3;&#x5F00;&#x5E72;/&#x7956;&#x7EC6;&#x80DE;&#x4ECB;&#x5BFC;&#x7684;&#x89C6;&#x7F51;&#x819C;&#x518D;&#x751F;&#xFF0C;&#x51E1; BMP &#x6FC0;&#x6D3B;&#x786E;&#x7ACB;&#x4E86;&#x76F8;&#x58F0;&#x4E0E; FGF &#x901A;&#x8DEF;&#x548C;&#x6709;&#x9009;&#x62E9;&#x5730;&#x6FC0;&#x6D3B;&#x7684;&#x89C4;&#x8303;&#x5316;&#x4E0E;&#x975E;&#x89C4;&#x8303;&#x5316;&#x7684; BMP &#x901A;&#x8DEF;&#x7684;&#x673A;&#x5236;&#x3002;&#x89C6;&#x7F51;&#x819C;&#x5E72;/&#x7956;&#x7EC6;&#x80DE;&#x5B58;&#x5728;&#x4E8E;&#x5305;&#x62EC;&#x4EBA;&#x7C7B;&#x5728;&#x5185;&#x7684;&#x5176;&#x4ED6;&#x7269;&#x79CD;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x63D0;&#x4F9B;&#x53EF;&#x80FD;&#x7684;&#x518D;&#x751F;&#x7597;&#x6CD5;&#x53EF;&#x4EE5;&#x6FC0;&#x6D3B;&#x5982;&#x4F55;&#x89C6;&#x7F51;&#x819C;&#x5E72;&#x7EC6;&#x80DE;&#x7684;&#x89C1;&#x89E3;&#x3002;

&#x9AA8;&#x5F62;&#x6001;&#x53D1;&#x751F;&#x86CB;&#x767D;&#x4FE1;&#x53F7;&#x8F6C;&#x5BFC;&#x4ECB;&#x5BFC;&#x5E72;/&#x7956;&#x7EC6;&#x80DE;&#x8BF1;&#x5BFC;&#x89C6;&#x7F51;&#x819C;&#x518D;&#x751F;&#x3002;

We identified a mechanism whereby retina regeneration in the embryonic chick can be induced by the contribution of stem/progenitor cells. We show that bone morphogenetic protein (BMP) signaling is sufficient and necessary to induce retina regeneration and that its action can be divided into two phases. By 3 days after postretinectomy (d PR), the BMP pathway directs proliferation and regeneration through the activation of Smad (canonical BMP pathway) and the up-regulation of FGF signaling by the MAPK pathway. By 7d PR, it induces apoptosis by activating p38 (a noncanonical BMP pathway) and down-regulating FGF signaling (by both MAPK and AKT pathways). Apoptosis at this later stage can be prevented, and BMP-induced regeneration can be further induced by inhibition of p38. These results unravel a mechanism for stem/progenitor cell-mediated retina regeneration, where BMP activation establishes a cross-talk with the FGF pathway and selectively activates the canonical and noncanonical BMP pathways. Retina stem/progenitor cells exist in other species, including humans. Thus, our findings provide insights on how retinal stem cells can be activated for possible regenerative therapies.

BMP signaling mediates stem/progenitor cell-induced retina regeneration.

Nous avons identifi&#xE9; un m&#xE9;canisme par lequel la r&#xE9;g&#xE9;n&#xE9;ration r&#xE9;tine chez le poulet embryonnaire peut &#xEA;tre induite par la contribution des cellules souches / prog&#xE9;nitrices. Nous montrons que la prot&#xE9;ine morphog&#xE9;n&#xE9;tique osseuse (BMP) de signalisation est n&#xE9;cessaire et suffisant pour induire la r&#xE9;g&#xE9;n&#xE9;ration r&#xE9;tine et que son action peut &#xEA;tre divis&#xE9; en deux phases. En 3 jours apr&#xE8;s postretinectomy (d PR), la voie BMP dirige la prolif&#xE9;ration et la r&#xE9;g&#xE9;n&#xE9;ration &#xE0; travers l&#x26;#39;activation des Smad (canonique BMP voie) et la r&#xE9;gulation &#xE0; la hausse du FGF de signalisation par la voie MAPK. Par 7d PR, il induit l&#x26;#39;apoptose en activant p38 (une voie non canonique BMP) et vers le bas de r&#xE9;gulation FGF de signalisation (&#xE0; la fois par les voies MAPK et AKT). L&#x26;#39;apoptose &#xE0; ce stade ult&#xE9;rieur peut &#xEA;tre emp&#xEA;ch&#xE9;, et BMP-induite de r&#xE9;g&#xE9;n&#xE9;ration peut &#xEA;tre en outre induite par l&#x26;#39;inhibition de la p38. Ces r&#xE9;sultats d&#xE9;m&#xEA;ler un m&#xE9;canisme de tige / prog&#xE9;nitrices &#xE0; m&#xE9;diation cellulaire de r&#xE9;g&#xE9;n&#xE9;ration r&#xE9;tine, o&#xF9; BMP activation &#xE9;tablit une diaphonie avec la voie FGF et active s&#xE9;lectivement les voies canoniques et non canonique BMP. Retina cellules souches / prog&#xE9;nitrices existent dans d&#x26;#39;autres esp&#xE8;ces, y compris les humains. Ainsi, nos r&#xE9;sultats donnent un aper&#xE7;u sur la fa&#xE7;on dont les cellules souches r&#xE9;tiniennes peuvent &#xEA;tre activ&#xE9;s pour d&#x26;#39;&#xE9;ventuelles th&#xE9;rapies r&#xE9;g&#xE9;n&#xE9;ratives.

BMP m&#xE9;diateur de signalisation souches / prog&#xE9;nitrices de cellules induite par la r&#xE9;g&#xE9;n&#xE9;ration Retina

Wir identifizieren einen Mechanismus, wobei Netzhaut Erneuerung in der embryonalen K&uuml;ken durch den Beitrag von Stammzellen und Vorl&auml;uferzellen induziert werden kann. Wir zeigen, dass Bone morphogenetic Proteins (BMP) Signalisierung ausreichend und notwendig, um die Netzhaut Regenerierung zu induzieren und, dass ihre Klage in zwei Phasen unterteilt werden kann. Von 3 Tagen nach Postretinectomy (d PR) leitet die BMP-Pathway Verbreitung und Regeneration durch die Aktivierung der Smad (kanonische BMP Pathway) und oben-Regulierung der FGF Signalisierung durch die MAPK-Pathway. Von 7D PR, induziert es Apoptosis durch Aktivierung p38 (ein Mitgliedattribut BMP-Pathway) und Down-Regulierung FGF Signalisierung (durch MAPK und AKT Pfade). Apoptose zu diesem sp&auml;teren Zeitpunkt vermieden werden k&ouml;nnen, und BMP-induzierte Regenerierung kann weiter durch Hemmung der p38 induziert werden. Diese Ergebnisse zu entwirren einen Mechanismus f&uuml;r die Vorbau/Stammvater zellvermittelte Netzhaut Regeneration, wo BMP Aktivierung stellt ein Nebensprechen mit der FGF-Pathway und selektiv aktiviert die kanonischen und Mitgliedattribut BMP-Bahnen. Netzhaut Vorbau/Vorl&auml;uferzellen gibt es bei anderen Arten, einschlie&szlig;lich des Menschen. Somit bieten unsere Ergebnisse, dass Einblicke, wie retinale Stammzellen f&uuml;r regenerative Therapien m&ouml;glich aktiviert werden k&ouml;nnen.

BMP Signalisierung vermittelt Vorbau/Stammvater Zelle induziert Netzhaut Regenerierung.

Abbiamo individuato un meccanismo per cui la rigenerazione della retina nel pulcino embrionale pu&ograve; essere indotta dal contributo delle cellule staminali/progenitrici. Mostriamo che bone morphogenetic protein (BMP) segnalazione &egrave; sufficiente e necessario per indurre la rigenerazione della retina e che la sua azione pu&ograve; essere diviso in due fasi. Da 3 giorni dopo postretinectomy (d. PR), il pathway BMP dirige la proliferazione e la rigenerazione attraverso l&#39;attivazione di Smad (pathway canonico BMP) e la up-regolazione di FGF segnalazione dalla via MAPK. Da 7 d PR, induce apoptosi attivando p38 (un percorso BMP canonica) e gi&ugrave;-regolazione FGF segnalando (da percorsi entrambi MAPK e AKT). Apoptosis in questa fase successiva pu&ograve; essere prevenuto e rigenerazione indotta da BMP pu&ograve; essere ulteriormente indotta dall&#39;inibizione della p38. Questi risultati svelare un meccanismo per la rigenerazione di retina cellulo-mediata di staminali/progenitrici, dove BMP attivazione stabilisce un cross-talk con il percorso FGF e attiva selettivamente i percorsi BMP canonici e canonica. Cellule staminali/progenitrici retina esistono in altre specie, inclusi gli esseri umani. Cos&igrave;, i nostri risultati forniscono approfondimenti su cellule staminali come retiniche possono essere attivati per possibili terapie rigenerative.

Segnalazione BMP da intermediario rigenerazione retina indotta da cellule staminali/progenitrici.

&#x30CB;&#x30EF;&#x30C8;&#x30EA;&#x80DA;&#x306E;&#x7DB2;&#x819C;&#x518D;&#x751F;&#x304C;&#x5E79;&#x7D30;&#x80DE;/&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x306E;&#x5BC4;&#x4E0E;&#x306B;&#x3088;&#x308A;&#x8A98;&#x767A;&#x3055;&#x308C;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x6211;&#x3005;&#x306F;&#x3001;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x540C;&#x5B9A;&#x3057;&#x305F;&#x3002;&#x6211;&#x3005;&#x306F;&#x3001;&#x9AA8;&#x5F62;&#x6210;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#xFF08;BMP&#xFF09;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x306F;&#x7DB2;&#x819C;&#x306E;&#x518D;&#x751F;&#x3092;&#x8A98;&#x5C0E;&#x3059;&#x308B;&#x306E;&#x306B;&#x5341;&#x5206;&#x3068;&#x304C;&#x5FC5;&#x8981;&#x3067;&#x3042;&#x308B;&#x3068;&#x3001;&#x305D;&#x306E;&#x30A2;&#x30AF;&#x30B7;&#x30E7;&#x30F3;&#x306F;&#x3001;2&#x3064;&#x306E;&#x30D5;&#x30A7;&#x30FC;&#x30BA;&#x306B;&#x5206;&#x3051;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002; postretinectomy 3&#x65E5;&#x5F8C;&#xFF08;D PR&#xFF09;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x308A;&#x3001;BMP&#x7D4C;&#x8DEF;&#x306F;&#x3001;&#x5897;&#x6B96;&#x3068;&#x518D;&#x751F;&#x306E;Smad&#x306E;&#x6D3B;&#x6027;&#x5316;&#xFF08;&#x6A19;&#x6E96;&#x7684;&#x306A;BMP&#x7D4C;&#x8DEF;&#xFF09;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x3068;MAPK&#x7D4C;&#x8DEF;&#x306B;&#x3088;&#x308B;&#x30B7;&#x30B0;&#x200B;&#x200B;&#x30CA;&#x30EB;&#x4F1D;&#x9054;FGF&#x306E;&#x30A2;&#x30C3;&#x30D7;&#x30EC;&#x30AE;&#x30E5;&#x30EC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3092;&#x6307;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002; 7D PR&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x3001;&#x305D;&#x308C;&#x306F;&#x3001;p38&#xFF08;&#x975E;&#x6A19;&#x6E96;BMP&#x7D4C;&#x8DEF;&#xFF09;&#x3068;&#x30C0;&#x30A6;&#x30F3;&#x30EC;&#x30AE;&#x30E5;&#x30EC;&#x30FC;&#x30C8;FGF&#x30B7;&#x30B0;&#x30CA;&#x30EA;&#x30F3;&#x30B0;&#xFF08;MAPK&#x53CA;&#x3073;AKT&#x7D4C;&#x8DEF;&#x306E;&#x4E21;&#x65B9;&#x306B;&#x3088;&#x3063;&#x3066;&#xFF09;&#x6D3B;&#x6027;&#x5316;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x308A;&#x3001;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x3092;&#x8A98;&#x5C0E;&#x3059;&#x308B;&#x3002;&#x3053;&#x306E;&#x5F8C;&#x306E;&#x6BB5;&#x968E;&#x3067;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x3092;&#x9632;&#x6B62;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x3001;BMP&#x8A98;&#x5C0E;&#x518D;&#x751F;&#x306F;&#x3055;&#x3089;&#x306B;&#x3001;p38&#x306E;&#x963B;&#x5BB3;&#x306B;&#x3088;&#x308A;&#x8A98;&#x5C0E;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D50;&#x679C;&#x306F;&#x3001;BMP&#x306E;&#x6D3B;&#x6027;&#x5316;&#x306F;FGF&#x7D4C;&#x8DEF;&#x3068;&#x306E;&#x30AF;&#x30ED;&#x30B9;&#x30C8;&#x30FC;&#x30AF;&#x3092;&#x78BA;&#x7ACB;&#x3057;&#x3001;&#x9078;&#x629E;&#x7684;&#x6B63;&#x898F;&#x5316;&#x3068;&#x975E;&#x6A19;&#x6E96;&#x306E;BMP&#x7D4C;&#x8DEF;&#x3092;&#x6D3B;&#x6027;&#x5316;&#x5E79;&#x7D30;&#x80DE;/&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x5A92;&#x4ECB;&#x6027;&#x7DB2;&#x819C;&#x518D;&#x751F;&#x306E;&#x305F;&#x3081;&#x306E;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x89E3;&#x660E;&#x3059;&#x308B;&#x3002;&#x7DB2;&#x819C;&#x5E79;&#x7D30;&#x80DE;/&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x306F;&#x3001;&#x30D2;&#x30C8;&#x3092;&#x542B;&#x3080;&#x4ED6;&#x306E;&#x7A2E;&#x306B;&#x5B58;&#x5728;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x6211;&#x3005;&#x306E;&#x6240;&#x898B;&#x306F;&#x3001;&#x7DB2;&#x819C;&#x5E79;&#x7D30;&#x80DE;&#x304C;&#x518D;&#x751F;&#x53EF;&#x80FD;&#x306A;&#x6CBB;&#x7642;&#x306E;&#x305F;&#x3081;&#x306B;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306B;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x65B9;&#x6CD5;&#x306B;&#x3064;&#x3044;&#x3066;&#x306E;&#x6D1E;&#x5BDF;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

BMP&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x3092;&#x4EF2;&#x4ECB;&#x3059;&#x308B;&#x5E79;&#x7D30;&#x80DE;/&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x8A98;&#x5C0E;&#x7DB2;&#x819C;&#x518D;&#x751F;

&#xC6B0;&#xB9AC;&#xB294; &#xBC30;&#xC544; &#xBCD1;&#xC544;&#xB9AC;&#xC5D0;&#xC11C; &#xB9DD;&#xB9C9; &#xC7AC;&#xC0DD; &#xC904;&#xAE30;/&#xC2DC; &#xC870; &#xC138;&#xD3EC;&#xC758; &#xAE30;&#xC5EC;&#xC5D0; &#xC758;&#xD574; &#xC720;&#xB3C4; &#xB420; &#xC218; &#xC788;&#xB2E4; &#xADF8;&#xAC83;&#xC5D0; &#xC758;&#xD558;&#xC5EC; &#xBA54;&#xCEE4;&#xB2C8;&#xC998; &#xBC1C;&#xACAC;. &#xC6B0;&#xB9AC;&#xB294; &#xADF8; &#xBF08; &#xC804;&#xCCB4;&#xC801; &#xB2E8;&#xBC31;&#xC9C8; (BMP) &#xC2E0;&#xD638;&#xB294; &#xCDA9;&#xBD84; &#xD558; &#xACE0; &#xB9DD;&#xB9C9; &#xC7AC;&#xC0DD;&#xC131;&#xC744; &#xC720;&#xB3C4; &#xD558;&#xB294; &#xB370; &#xD544;&#xC694;&#xD55C; &#xD45C;&#xC2DC;&#xB294; &#xB450; &#xB2E8;&#xACC4;&#xB85C; &#xB098;&#xB20C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. Postretinectomy (d &#xD64D;&#xBCF4;) &#xD6C4; 3 &#xC77C;&#xC73C;&#xB85C; BMP &#xD1B5;&#xB85C; &#xD655;&#xC0B0; &#xBC0F; &#xC7AC;&#xC0DD;&#xC131; Smad (&#xC815;&#xC2DD; BMP &#xD1B5;&#xB85C;)&#xC758; &#xD65C;&#xC131;&#xD654;&#xC640; MAPK &#xACBD;&#xB85C; &#xC758;&#xD574; &#xC2E0;&#xD638; FGF&#xC758; &#xC5C5; &#xADDC;&#xC81C;&#xB97C; &#xD1B5;&#xD574; &#xC548;&#xB0B4; &#xD569;&#xB2C8;&#xB2E4;. 7 D &#xD64D;&#xBCF4;, &#xADF8;&#xAC83; p38 &#xD65C;&#xC131;&#xD654; &#xD558; &#xC5EC; apoptosis &#xC720;&#xB3C4; (&#xBE44;&#xC815;&#xADDC; BMP &#xD1B5;&#xB85C;) &#xBC0F; &#xB2E4;&#xC6B4; &#xADDC;&#xC81C; FGF &#xC2E0;&#xD638; (AKT&#xC640; MAPK &#xACBD;&#xB85C;)&#xC5D0; &#xC758;&#xD574;. &#xC774; &#xB098;&#xC911; &#xB2E8;&#xACC4;&#xC5D0;&#xC11C; Apoptosis&#xB97C; &#xB9C9;&#xC744; &#xC218; &#xC788;&#xB2E4; &#xACE0; BMP &#xC720;&#xB3C4; &#xC7AC;&#xC0DD; &#xB354; p38&#xC758; &#xC800;&#xD574;&#xC5D0; &#xC758;&#xD574; &#xC720;&#xB3C4; &#xB420; &#xC218; &#xC788;&#xB2E4;. &#xC774; &#xACB0;&#xACFC; &#xC904;&#xAE30;/&#xBFCC;&#xB9AC; &#xC140; &#xC911;&#xC7AC; &#xB9DD;&#xB9C9; &#xC911;&#xC0DD;, BMP &#xD65C;&#xC131;&#xD654; FGF &#xD1B5;&#xB85C;&#xC640; &#xC7A1;&#xB2F4;&#xC744; &#xC124;&#xC815; &#xD558; &#xACE0; &#xC120;&#xD0DD;&#xC801;&#xC73C;&#xB85C; &#xC815;&#xADDC; &#xBC0F; &#xBE44;&#xC815;&#xADDC; BMP &#xACBD;&#xB85C; &#xD65C;&#xC131;&#xD654; &#xD558;&#xB294; &#xBA54;&#xCEE4;&#xB2C8;&#xC998; &#xD574;&#xBA85;. &#xB9DD;&#xB9C9; &#xC904;&#xAE30;/&#xC2DC; &#xC870; &#xC138;&#xD3EC; &#xC778;&#xAC04;&#xC744; &#xD3EC;&#xD568; &#xD55C; &#xB2E4;&#xB978; &#xC885;&#xC5D0; &#xC874;&#xC7AC; &#xD569;&#xB2C8;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, &#xC6B0;&#xB9AC;&#xC758; &#xC5F0;&#xAD6C; &#xACB0;&#xACFC; &#xC81C;&#xACF5; &#xAC00;&#xB2A5;&#xD55C; &#xC7AC;&#xC0DD; &#xC694;&#xBC95;&#xC5D0; &#xB300; &#xD55C; &#xC5B4;&#xB5BB;&#xAC8C; &#xB9DD;&#xB9C9; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC5D0; &#xB300; &#xD55C; &#xD1B5;&#xCC30;&#xB825;&#xC744; &#xD65C;&#xC131;&#xD654;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

BMP &#xC2E0;&#xD638; &#xC904;&#xAE30;/&#xC2DC; &#xC870; &#xC138;&#xD3EC; &#xC720;&#xB3C4; &#xB41C; &#xB9DD;&#xB9C9; &#xC7AC;&#xC0DD;&#xC131; &#xC911;&#xC7AC;.

Hemos identificado un mecanismo que permita la regeneraci&#xF3;n de la retina en el embri&#xF3;n de pollo puede ser inducida por la contribuci&#xF3;n de las c&#xE9;lulas madre / progenitoras. Se demuestra que la prote&#xED;na morfogen&#xE9;tica &#xF3;sea (BMP) de se&#xF1;alizaci&#xF3;n es suficiente y necesaria para inducir la regeneraci&#xF3;n retina y que su acci&#xF3;n se puede dividir en dos fases. A los 3 d&#xED;as despu&#xE9;s de postretinectomy (d PR), la v&#xED;a BMP dirige la proliferaci&#xF3;n y regeneraci&#xF3;n a trav&#xE9;s de la activaci&#xF3;n de Smad (BMP v&#xED;a can&#xF3;nica) y la regulaci&#xF3;n de la FGF se&#xF1;alizaci&#xF3;n de la v&#xED;a MAPK. Por 7d PR, que induce la apoptosis mediante la activaci&#xF3;n de p38 (una v&#xED;a no can&#xF3;nica BMP) y abajo de la regulaci&#xF3;n de se&#xF1;alizaci&#xF3;n FGF (tanto por las v&#xED;as MAPK y AKT). La apoptosis en esta etapa posterior se puede prevenir, y BMP-inducida por la regeneraci&#xF3;n puede ser m&#xE1;s inducida por la inhibici&#xF3;n de p38. Estos resultados desentra&#xF1;ar un mecanismo para la madre / progenitoras de c&#xE9;lulas mediada por la regeneraci&#xF3;n de la retina, donde la activaci&#xF3;n de BMP establece una estrecha relaci&#xF3;n con la v&#xED;a FGF y activa selectivamente las v&#xED;as BMP can&#xF3;nicos y no can&#xF3;nicos. Retina c&#xE9;lulas madre / progenitoras existentes en otras especies, incluyendo seres humanos. Por lo tanto, nuestros resultados proporcionan ideas sobre c&#xF3;mo las c&#xE9;lulas madre de la retina puede ser activado para posibles terapias regenerativas.

Regula la se&#xF1;alizaci&#xF3;n BMP madre / progenitoras de la c&#xE9;lula inducida por la regeneraci&#xF3;n de Retina

Among 397 homeless participants studied, the overall West Nile virus (WNV) seroprevalence was 6.8%. Risk factors for WNV infection included being homeless >1 year, spending >6 hours outside daily, regularly taking mosquito precautions, and current marijuana use. Public health interventions need to be directed toward this high-risk population.

West Nile virus infection among the homeless, Houston, Texas.

Advanced devices capable of selective delivery of compounds to targeted tissues are lacking, especially in small animal research. Biomedical microelectromechanical systems (bioMEMS) are uniquely suited to this application through the combination of scalability and precise control of fluid handling. Polymer-based drug delivery components and pumps for acute and chronic delivery in small animals are discussed.

Implantable MEMS drug delivery pumps for small animal research.

A new class of transducers based on a Parylene C encapsulated fluid element are introduced. These versatile units have thus far been explored as impedance-based contact sensors and electrolysis-based actuators for fine positioning of neural recording electrodes. These sensors may be fabricated on thin, flexible substrates which permits application on non-planar or three dimensional surfaces. Another interesting modality is the coupling of individual mechanically-responsive, impedance-based sensing elements distributed over a surface but interconnected through microfluidic channel networks in a manner that emulates mechanotransduction in natural biological systems. These fluidic elements offer interesting new possibilities in neural prosthetics.

Parylene-based encapsulated fluid MEMS sensors.

We present the first electrochemical actuator with Parylene bellows for large-deflection operation. The bellows diaphragm was fabricated using a polyethylene-glycol-based sacrificial molding technique followed by coating in Parylene C. Bellows were mechanically characterized and integrated with a pair of interdigitated electrodes to form an electrochemical actuator that is suitable for low-power pumping of fluids. Pump performance (gas generation rate and pump efficiency) was optimized through a careful examination of geometrical factors. Overall, a maximum pump efficiency of 90% was achieved in the case of electroplated electrodes, and a deflection of over 1.5 mm was demonstrated. Real-time wireless operation was achieved. The complete fabrication process and the materials used in this actuator are bio-compatible, which makes it suitable for biological and medical applications.

A Parylene Bellows Electrochemical Actuator.

Relapsing polychondritis-associated meningitis and encephalitis: response to infliximab.

Understanding the mechanisms governing cell fate specification remains one of the main challenges in the study of retinal development. In this context, molecular markers that identify specific cell types become crucial tools for the analysis and interpretation of these phenomena. In studies using the developing chick retina, expression of the mid-size neurofilament (NF-M) and a chick-specific microtubule associated protein recognized by the RA4 antibody (MAP(RA4)), have been broadly used to selectively identify ganglion cells and their committed precursors. However, observations in our laboratory suggested that the expression of these proteins may not be restricted to cells of the ganglion cell lineage. Because of its potential significance in the field, we pursued a detailed analysis of the expression of these two molecules in combination with an array of proteins that allowed precise identification of all retinal cell-type precursors throughout the development of the chick retina.

Cytoskeleton proteins previously considered exclusive to ganglion cells are transiently expressed by all retinal neuronal precursors.

A Parylene C neural probe with a three dimensional sheath structure was designed, fabricated, and characterized. Multiple platinum (Pt) electrodes for recording neural signals were fabricated on both inner and outer surfaces of the sheath structure. Thermoforming of Parylene was used to create the three dimensional sheath structures from flat surface micromachined microchannels using solid microwires as molds. Benchtop electrochemical characterization was performed on the thin film Pt electrodes using cyclic voltammetry and electrochemical impedance spectroscopy and showed that electrodes possessed low impedances suitable for neuronal recordings. A procedure for implantation of the neural probe was developed and successfully demonstrated in vitro into an agarose brain tissue model. The electrode-lined sheath will be decorated with eluting neurotrophic factors to promote in vivo neural tissue ingrowth post-implantation. These features will enhance tissue integration and improve recording quality towards realizing reliable chronic neural interfaces.

Novel flexible Parylene neural probe with 3D sheath structure for enhancing tissue integration.

We present the preliminary electrochemical characterization of 3D Parylene C sheath microelectrode array probes towards realizing reliable chronic neuroprosthetic recordings. Electrochemical techniques were used to verify electrode integrity after our novel post-fabrication thermoforming process was applied to flat surface micromachined structures to achieve a hollow sheath probe shape. Characterization of subsequent neurotrophic coatings was performed and accelerated life testing was used to simulate six months in vivo. Prior to probe implantation, crosstalk was measured and electrode surface properties were evaluated through the use of electrochemical impedance spectroscopy.

Pre-implantation electrochemical characterization of a Parylene C sheath microelectrode array probe.

Primary retinal cultures constitute valuable tools not only for basic research on retinal cell development and physiology, but also for the identification of factors or drugs that promote cell survival and differentiation. In order to take full advantage of the benefits of this system it is imperative to develop efficient and reliable techniques for the manipulation of gene expression. However, achieving appropriate transfection efficiencies in these cultures has remained challenging. The purpose of this work was to develop and optimize a technique that would allow the transfection of chick retinal cells with high efficiency and reproducibility for multiple applications. We developed an ex vivo electroporation method applied to dissociated retinal cell cultures that offers a significant improvement over other currently available transfection techniques, increasing efficiency by five-fold. In this method, eyes were enucleated, devoid of RPE, and electroporated with GFP-encoding plasmids using custom-made electrodes. Electroporated retinas were then dissociated into single cells and plated in low density conditions, to be analyzed after 4 days of incubation. Parameters such as voltage and number of electric pulses, as well as plasmid concentration and developmental stage of the animal were optimized for efficiency. The characteristics of the cultures were assessed by morphology and immunocytochemistry, and cell viability was determined by ethidium homodimer staining. Cell imaging and counting was performed using an automated high-throughput system. This procedure resulted in transfection efficiencies in the order of 22-25% of cultured cells, encompassing both photoreceptors and non-photoreceptor neurons, and without affecting normal cell survival and differentiation. Finally, the feasibility of the technique for cell-autonomous studies of gene function in a biologically relevant context was tested by carrying out gain and loss-of-function experiments for the transcription factor PAX6. Electroporation of a plasmid construct expressing PAX6 resulted in a marked upregulation in the expression levels of this protein that could be measured in the whole culture as well as cell-intrinsically. This was accompanied by a significant decrease in the percentage of cells differentiating as photoreceptors among the transfected population. Conversely, electroporation of an RNAi construct targeting PAX6 resulted in a significant decrease in the levels of this protein, with a concomitant increase in the proportion of photoreceptors. Taken together these results provide strong proof-of-principle of the suitability of this technique for genetic studies in retinal cultures. The combination of the high transfection efficiency obtained by this method with automated high-throughput cell analysis supplies the scientific community with a powerful system for performing functional studies in a cell-autonomous manner.

Ex vivo electroporation of retinal cells: a novel, high efficiency method for functional studies in primary retinal cultures.

Many forms of blindness result from the dysfunction or loss of retinal photoreceptors. Induced pluripotent stem cells (iPSCs) hold great potential for the modelling of these diseases or as potential therapeutic agents. However, to fulfill this promise, a remaining challenge is to induce human iPSC to recreate in vitro key structural and functional features of the native retina, in particular the presence of photoreceptors with outer-segment discs and light sensitivity. Here we report that hiPSC can, in a highly autonomous manner, recapitulate spatiotemporally each of the main steps of retinal development observed in vivo and form three-dimensional retinal cups that contain all major retinal cell types arranged in their proper layers. Moreover, the photoreceptors in our hiPSC-derived retinal tissue achieve advanced maturation, showing the beginning of outer-segment disc formation and photosensitivity. This success brings us one step closer to the anticipated use of hiPSC for disease modelling and open possibilities for future therapies.

Christian Gutierrez

Wilmer Eye Institute

The Johns Hopkins University School of Medicine

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Christian Gutierrez

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The Johns Hopkins University School of Medicine

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Wilmer Eye Institute