co-transplantation-human-ovarian-tissue-with-engineered-endothelial

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. ...

Weill Cornell Medical College

To examine the results of a 3-year trial using blastocyst cryopreservation to limit multiple pregnancy and optimize overall pregnancy per cycle.

High pregnancy rates can be achieved after freezing and thawing human blastocysts.

&#x4EE5;&#x524D;&#x7684;&#x52AA;&#x529B;&#x5206;&#x5316;&#x4E3A;&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x7EC6;&#x80DE;&#x7684;&#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE; (hESCs) &#x5E76;&#x672A;&#x5B9E;&#x73B0;&#x6301;&#x7EED;&#x7684;&#x6269;&#x5F20;&#x548C;&#x8840;&#x7BA1;&#x7EC6;&#x80DE;&#x7684;&#x7A33;&#x5B9A;&#x6027;&#x3002;&#x82E5;&#x8981;&#x5B9A;&#x4E49;&#x8840;&#x7BA1;&#x6027;&#x53D1;&#x5C55;&#x8DEF;&#x5F84;&#x5E76;&#x52A0;&#x5F3A;&#x5206;&#x5316;&#xFF0C;&#x6211;&#x4EEC;&#x4F7F;&#x7528;&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x7EC6;&#x80DE;&#x7279;&#x5F02;&#x6027;&#x9ECF;&#x86CB;&#x767D;&#x542F;&#x52A8;&#x5B50;&#xFF0C;&#x9A71;&#x52A8;&#x7EFF;&#x8272;&#x8367;&#x5149;&#x86CB;&#x767D; (GFP) (hVPr-GFP) &#x7B5B;&#x67E5; &#xFF1B; &#x4FC3;&#x8FDB;&#x8840;&#x7BA1;&#x627F;&#x8BFA;&#x7684;&#x56E0;&#x7D20;&#x3002;&#x7B2C;&#x4E00;&#x9636;&#x6BB5;&#x7684;&#x6211;&#x4EEC;&#x7684;&#x65B9;&#x6CD5;&#xFF0C;&#x5728;&#x5206;&#x5316;&#x7684;&#x7B2C; 7 &#x5929;&#x8F6C;&#x5316;&#x751F;&#x957F;&#x56E0;&#x5B50; (TGF) &beta; &#x6291;&#x5236;&#x589E;&#x52A0; hVPr-GFP(+) &#x7EC6;&#x80DE;&#x7684; 10 &#x500D;&#x3002;&#x5728;&#x9636;&#x6BB5; 2&#xFF0C;&#x6291;&#x5236;&#x7EF4;&#x62A4;&#x7684;&#x6269;&#x6563;&#x548C;&#x7EAF;&#x51C0;&#x7684;&#x5185;&#x76AE;&#x7EC6;&#x80DE;&#xFF0C;&#x5BFC;&#x81F4;&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x7EC6;&#x80DE;&#x7684;&#x8D28;&#x819C;&#xFF0C;&#x51C0;&#x989D; 36-fold &#x6269;&#x5F20;&#x8840;&#x7BA1;&#x8EAB;&#x4EFD; TGFbeta &#x7684;&#x5C55;&#x51FA;&#x8F6C;&#x5F55; Id1(high)VEGFR2(high)VE-cadherin(+) ephrinB2(+) &#x7684;&#x914D;&#x7F6E;&#x6587;&#x4EF6;&#x3002;&#x6211;&#x4EEC;&#x4F7F;&#x7528; Id1 YFP hESC &#x8BB0;&#x8005;&#x884C;&#xFF0C;&#x663E;&#x793A; TGFbeta &#x6291;&#x5236;&#x652F;&#x6491; Id1 hESC &#x6D3E;&#x751F;&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x7EC6;&#x80DE;&#x4E2D;&#x7684;&#x8868;&#x8FBE;&#x548C; Id1 &#x662F;&#x589E;&#x52A0;&#x7684;&#x6269;&#x6563;&#x548C;&#x4FDD;&#x62A4;&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x7EC6;&#x80DE;&#x7684;&#x627F;&#x8BFA;&#x6240;&#x5FC5;&#x9700;&#x7684;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x65B9;&#x6CD5;&#x5206;&#x5316;&#x548C;&#x957F;&#x671F;&#x7EF4;&#x62A4; hESC &#x6D3E;&#x751F;&#x7684;&#x5185;&#x76AE;&#x7EC6;&#x80DE;&#x5728;&#x4E34;&#x5E8A;&#x7684;&#x5E94;&#x7528;&#x7A0B;&#x5E8F;&#x76F8;&#x5173;&#x7684;&#x89C4;&#x6A21;&#x63D0;&#x4F9B;&#x65E0;&#x8840;&#x6E05;&#x7684;&#x65B9;&#x6CD5;&#x3002;

&#x6269;&#x5927;&#x548C;&#x7EF4;&#x6301;&#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x6E90;&#x6027;&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x7EC6;&#x80DE;&#x7531; TGFbeta &#x6291;&#x5236;&#x662F; Id1 &#x4F9D;&#x8D56;&#x3002;

Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell-specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)beta at day 7 of differentiation increases hVPr-GFP(+) cells by tenfold. In phase 2, TGFbeta inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id1(high)VEGFR2(high)VE-cadherin(+) ephrinB2(+). Using an Id1-YFP hESC reporter line, we showed that TGFbeta inhibition sustains Id1 expression in hESC-derived endothelial cells and that Id1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application.

Expansion and maintenance of human embryonic stem cell-derived endothelial cells by TGFbeta inhibition is Id1 dependent.

Efforts ant&eacute;rieurs pour les cellules souches embryonnaires humaines (CSEh) se diff&eacute;rencient en cellules endoth&eacute;liales humaines n&#39;ont pas atteint une expansion soutenue et la stabilit&eacute; des cellules vasculaires. Pour d&eacute;finir des voies de d&eacute;veloppement vasculogenic et am&eacute;liorer la diff&eacute;renciation, nous avons utilis&eacute; un promoteur de VE-cadh&eacute;rine sp&eacute;cifiques des cellules endoth&eacute;lial prot&eacute;ine fluorescente verte (GFP) (hVPr-GFP) pour d&eacute;pister les facteurs qui favorisent l&#39;engagement vasculaire au volant. Dans la phase 1 de notre m&eacute;thode, inhibition de transforming growth factor (TGF) beta au septi&egrave;me jour de diff&eacute;renciation d&eacute;cuple les cellules hVPr-GFP(+) par. Dans la phase 2, TGF inhibition maintient la prolif&eacute;ration et l&#39;identit&eacute; vasculaire des purifi&eacute;e cellules endoth&eacute;liales, entra&icirc;nant une nette 36-fold expansion des cellules endoth&eacute;liales en monocouches homog&egrave;nes, qui pr&eacute;sentait un profil transcriptionnel des Id1(high)VEGFR2(high)VE-cadherin(+) ephrinB2(+). &Agrave; l&#39;aide d&#39;une ligne de journaliste de CSEh Id1-YFP, nous avons montr&eacute; que l&#39;inhibition TGF soutient l&#39;Id1 expression dans les cellules endoth&eacute;liales d&eacute;riv&eacute;es de CSEh et que Id1 est requise pour la prolif&eacute;ration accrue et de la pr&eacute;servation de l&#39;engagement des cellules endoth&eacute;liales. Notre approche fournit une m&eacute;thode sans s&eacute;rum pour la diff&eacute;renciation et de la maintenance &agrave; long terme des CSEh d&eacute;riv&eacute;es des cellules endoth&eacute;liales &agrave; l&#39;&eacute;chelle pertinente pour l&#39;application clinique.

Expansion et l&#39;entretien des cellules embryonnaires humaines d&eacute;riv&eacute;es de cellules souches endoth&eacute;liales par inhibition de TGF est Id1 d&eacute;pendants.

Fr&uuml;here Bem&uuml;hungen, menschlichen embryonalen Stammzellen (Zellkulturen) in Endothelzellen zu unterscheiden haben nicht nachhaltige Expansion und Stabilit&auml;t der vaskul&auml;ren Zellen erreicht. Um Vasculogenic entwicklungsbedingte Bahnen zu definieren und Differenzierung zu verbessern, haben wir eine endotheliale zellspezifische VE-Cadherin Projekttr&auml;ger fahren gr&uuml;nes fluoreszierendes Protein (GFP) (hVPr-GFP) Sie auf die Faktoren, die vaskul&auml;re Engagement f&ouml;rdern verwendet. In der ersten Phase unserer Methode erh&ouml;ht sich hVPr-GFP(+) Zellen durch Hemmung der transforming-Growth-Factor (TGF) Beta bei Tag 7 der Differenzierung verzehnfacht. In Phase 2, TGFbeta Hemmung der Proliferation und vaskul&auml;re Identit&auml;t des gereinigten Endothelzellen, wodurch eine net 36-fold Expansion der Endothelzellen in homogenen Monolayers unterh&auml;lt ausgestellt die transkriptionelle Profil von Id1(high)VEGFR2(high)VE-cadherin(+)-ephrinB2(+). &Uuml;ber eine Id1-YFP hESC-Reporter-Leitung, zeigten wir, dass TGFbeta Hemmung Id1 Ausdruck in Endothelzellen hESC abgeleitete zugezogen und Id1 f&uuml;r die Verbreitung und Erhaltung der endothelial Zelle Engagement erforderlich ist. Unser Ansatz stellt eine serumfreie Methode f&uuml;r Differenzierung und langfristige Erhaltung der hESC-abgeleitet von Endothelzellen im Ma&szlig;stab f&uuml;r klinische Anwendung.

Erweiterung und Wartung von menschlichen embryonalen Stammzellen Cell-derived Endothelzellen durch TGFbeta-Hemmung ist Id1 abh&auml;ngig.

Precedenti tentativi di differenziare cellule staminali embrionali umane (hESC) in cellule endoteliali non hanno raggiunto espansione sostenuta e la stabilit&agrave; delle cellule vascolari. Per definire percorsi di sviluppo vasculogenic e migliorare la differenziazione, abbiamo utilizzato un promotore di VE-caderina specifici delle cellule endotelio guida proteina fluorescente verde (GFP) (hVPr-GFP) a schermo per fattori che promuovono l&#39;impegno vascolare. Nella prima fase del nostro metodo, inibizione della trasformazione di fattore di crescita (TGF) beta al 7 giorno di differenziazione aumenta le cellule hVPr-GFP(+) di dieci volte. Nella fase 2, TGFbeta inibizione mantiene la proliferazione e l&#39;identit&agrave; vascolare delle cellule endoteliali purificate, risultante in un netto 36-fold espansione delle cellule endoteliali in monostrati omogenee, che espone un profilo trascrizionale di Id1(high)VEGFR2(high)VE-cadherin(+) ephrinB2(+). Utilizzando una linea di reporter hESC Id1-YFP, abbiamo dimostrato che l&#39;inibizione TGFbeta sostiene Id1 espressione in cellule endoteliali hESC-derivato e che Id1 &egrave; necessaria per aumentata proliferazione e conservazione dell&#39;impegno delle cellule endoteliali. Il nostro approccio fornisce un metodo privo di siero per la differenziazione e la manutenzione a lungo termine delle cellule endoteliali derivati hESC a scala pertinente all&#39;applicazione clinica.

Ampliamento e manutenzione di cellule embrionali umane derivate da cellule staminali endoteliali tramite inibizione TGFbeta &egrave; Id1 dipendente.

&#x30D2;&#x30C8;&#x80DA;&#x6027;&#x5E79;&#x7D30;&#x80DE; &#xFF08;hESCs&#xFF09; &#x5185;&#x76AE;&#x306E;&#x7D30;&#x80DE;&#x306B;&#x5206;&#x5316;&#x3059;&#x308B;&#x4EE5;&#x524D;&#x306E;&#x52AA;&#x529B;&#x306F;&#x3001;&#x6301;&#x7D9A;&#x7684;&#x306A;&#x62E1;&#x5927;&#x3068;&#x8840;&#x7BA1;&#x7D30;&#x80DE;&#x306E;&#x5B89;&#x5B9A;&#x6027;&#x3092;&#x9054;&#x6210;&#x3057;&#x3066;&#x3044;&#x307E;&#x305B;&#x3093;&#x3002;Vasculogenic &#x767A;&#x9054;&#x7D4C;&#x8DEF;&#x3092;&#x5B9A;&#x7FA9;&#x304A;&#x3088;&#x3073;&#x5206;&#x5316;&#x3092;&#x5F37;&#x5316;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x7DD1;&#x8272;&#x86CD;&#x5149;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA; (GFP &#x8840;&#x7BA1;&#x306E;&#x30B3;&#x30DF;&#x30C3;&#x30C8;&#x30E1;&#x30F3;&#x30C8;&#x3092;&#x4FC3;&#x9032;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x8981;&#x56E0;&#x3092;&#x753B;&#x9762;&#x306B;) (hVPr-GFP) &#x904B;&#x8EE2;&#x3001;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x7279;&#x7570; VE &#x30AB;&#x30C9;&#x30D8;&#x30EA;&#x30F3;&#x306E;&#x30D7;&#x30ED;&#x30E2;&#x30FC;&#x30BF;&#x30FC;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x672C;&#x624B;&#x6CD5;&#x306E;&#x7B2C; 1 &#x6BB5;&#x968E;&#x3067;&#x306F;&#x3001;&#x5206;&#x5316;&#x306F; 7 &#x65E5;&#x76EE;&#x306E;&#x6210;&#x9577;&#x56E0;&#x5B50; (TGF) &#x30D9;&#x30FC;&#x30BF;&#x7248;&#x306E;&#x5909;&#x63DB;&#x306E;&#x6291;&#x5236;&#x306B;&#x3088;&#x308B; hVPr-GFP(+) &#x7D30;&#x80DE;&#x306F; 10 &#x500D;&#x306B;&#x5897;&#x52A0;&#x3057;&#x307E;&#x3059;&#x3002;&#x30D5;&#x30A7;&#x30FC;&#x30BA; 2&#x3001;&#x5897;&#x6B96;&#x3068;&#x8840;&#x7BA1;&#x306E;&#x30A2;&#x30A4;&#x30C7;&#x30F3;&#x30C6;&#x30A3;&#x30C6;&#x30A3;&#x5747;&#x8CEA;&#x306A;&#x5358;&#x5206;&#x5B50;&#x819C;&#x306B;&#x304A;&#x3051;&#x308B;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x306E;&#x7D14; 36-fold &#x62E1;&#x5927;&#x306E;&#x7D50;&#x679C;&#x3001;&#x7CBE;&#x88FD;&#x306E;&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x306E;&#x6291;&#x5236;&#x3092;&#x7DAD;&#x6301; tgf &beta; &#x306B;&#x5BFE;&#x3059;&#x308B;&#x8EE2;&#x5199;&#x30D7;&#x30ED;&#x30D5;&#x30A1;&#x30A4;&#x30EB; Id1(high)VEGFR2(high)VE-cadherin(+) ephrinB2(+) &#x306E;&#x5C55;&#x793A;&#x3002;&#x300C;Id1 YFP hESC &#x8A18;&#x8005;&#x56DE;&#x7DDA;&#x3092;&#x4F7F;&#x3063;&#x3066;&#x3001;tgf &beta; &#x306B;&#x5BFE;&#x3059;&#x308B;&#x6291;&#x5236;&#x304C; Id1 &#x5F0F;&#x306E;&#x305F;&#x3081;&#x306B;&#x60AA;&#x540D;&#x9AD8;&#x304F;&#x7531;&#x6765;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x3092;&#x7DAD;&#x6301;&#x3057;&#x3001;&#x3001;Id1 &#x304C;&#x5897;&#x52A0;&#x5897;&#x6B96;&#x3068;&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x30B3;&#x30DF;&#x30C3;&#x30C8;&#x30E1;&#x30F3;&#x30C8;&#x4FDD;&#x5B58;&#x306E;&#x305F;&#x3081;&#x5FC5;&#x8981;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002;&#x6211;&#x3005; &#x306E;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x306F;&#x5206;&#x5316;&#x3068;&#x9577;&#x671F;&#x7684;&#x306A;&#x4FDD;&#x5B88;&#x306E;&#x305F;&#x3081;&#x306B;&#x60AA;&#x540D;&#x9AD8;&#x304F;&#x7531;&#x6765;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x81E8;&#x5E8A;&#x30A2;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x306B;&#x95A2;&#x9023;&#x3059;&#x308B;&#x30B9;&#x30B1;&#x30FC;&#x30EB;&#x3067;&#x306E;&#x8840;&#x6E05;&#x7121;&#x6599;&#x306E;&#x30E1;&#x30BD;&#x30C3;&#x30C9;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;

&#x62E1;&#x5F35;&#x3068;&#x30D2;&#x30C8;&#x80DA;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x7531;&#x6765;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x306B;&#x3088;&#x308B; tgf &beta; &#x963B;&#x5BB3;&#x306E;&#x30E1;&#x30F3;&#x30C6;&#x30CA;&#x30F3;&#x30B9;&#x306F;&#x3001;Id1 &#x4F9D;&#x5B58;&#x3067;&#x3059;&#x3002;

&#xB178;&#xB825; &#xC774;&#xC804;&#xC5D0; &#xB0B4; &#xD53C; &#xC138;&#xD3EC;&#xB85C; &#xC778;&#xAC04; &#xBC30;&#xC544; &#xC904;&#xAE30; &#xC138;&#xD3EC; (hESCs)&#xB97C; &#xCC28;&#xBCC4;&#xD654; &#xD558;&#xB294; &#xB370; &#xC9C0;&#xC18D;&#xC801;&#xC778;&#xB41C; &#xD655;&#xC7A5; &#xBC0F; &#xD608;&#xAD00; &#xC138;&#xD3EC;&#xC758; &#xC548;&#xC815;&#xC131;&#xC744; &#xB2EC;&#xC131; &#xD558;&#xC9C0; &#xC54A;&#xC558;&#xC2B5;&#xB2C8;&#xB2E4;. Vasculogenic &#xBC1C;&#xB2EC; &#xACBD;&#xB85C; &#xC815;&#xC758; &#xD558; &#xACE0; &#xCC28;&#xBCC4;&#xD654;&#xB97C; &#xAC15;&#xD654;, &#xB179;&#xC0C9; &#xD615;&#xAD11; &#xB2E8;&#xBC31;&#xC9C8; (GFP) (hVPr-GFP) &#xD608;&#xAD00; &#xC57D;&#xC18D;&#xC744; &#xCD09;&#xC9C4; &#xD558;&#xB294; &#xC694;&#xC778;&#xC5D0; &#xB300; &#xD55C; &#xD654;&#xBA74;&#xC744; &#xC6B4;&#xC804; &#xB0B4; &#xD53C; &#xC138;&#xD3EC; &#xAD00;&#xB828; VE cadherin &#xBC1C;&#xAE30;&#xC778;&#xC744; &#xC0AC;&#xC6A9; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xBC29;&#xBC95;&#xC758; 1 &#xB2E8;&#xACC4;&#xC5D0;&#xC11C; &#xC5B5;&#xC81C; &#xCC28;&#xBCC4;&#xD654;&#xC758; &#xD558;&#xB8E8; 7&#xC5D0;&#xC11C; &#xC131;&#xC7A5; &#xC778;&#xC790; (TGF) &#xBCA0;&#xD0C0; &#xBCC0;&#xD658;&#xC758; &#xC5F4; &#xBC30; hVPr-GFP(+) &#xC140;&#xC5D0; &#xC758;&#xD574; &#xC99D;&#xAC00; &#xD569;&#xB2C8;&#xB2E4;. &#xB2E8;&#xACC4; 2, TGFbeta &#xC800;&#xD574; &#xD655;&#xC0B0; &#xBC0F; &#xADE0;&#xC9C8; &#xC131; monolayers &#xB0B4; &#xD53C; &#xC138;&#xD3EC;&#xC758; net 36-fold &#xD655;&#xC7A5;&#xC758; &#xACB0;&#xACFC; &#xC21C;&#xD654; &#xB41C; &#xB0B4; &#xD53C; &#xC138;&#xD3EC;&#xC758; &#xD608;&#xAD00; &#xC815;&#xCCB4;&#xC131;&#xC744; &#xC720;&#xC9C0; &#xD558;&#xB294; Id1(high)VEGFR2(high)VE-cadherin(+) Ephrinb2(+)&#xC758; transcriptional &#xD504;&#xB85C;&#xD544; &#xC804;&#xC2DC;. Id1 YFP hESC &#xAE30;&#xC790; &#xB77C;&#xC778;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC0B4;&#xD3B4;&#xBCF8; TGFbeta &#xC800;&#xD574; hESC &#xC720;&#xB798; &#xB0B4; &#xD53C; &#xC138;&#xD3EC;&#xC758; Id1 &#xC2DD; &#xACA9;&#xB824; &#xD558;&#xBA70; Id1 &#xC99D;&#xAC00; &#xD655;&#xC0B0;&#xACFC; &#xB0B4; &#xD53C; &#xC138;&#xD3EC;&#xC758; &#xC9C0;&#xC758; &#xBCF4;&#xC874; &#xD544;&#xC694;. &#xC6B0;&#xB9AC;&#xC758; &#xC811;&#xADFC; &#xBC29;&#xC2DD;&#xC744; &#xCC28;&#xBCC4;&#xD654;&#xC640; &#xADDC;&#xBAA8; &#xC784;&#xC0C1; &#xC751;&#xC6A9; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC5D0; &#xAD00;&#xB828; &#xB41C; hESC &#xC720;&#xB798; &#xB0B4; &#xD53C; &#xC138;&#xD3EC;&#xC758; &#xC7A5;&#xAE30;&#xC801;&#xC778; &#xC720;&#xC9C0; &#xAD00;&#xB9AC;&#xC5D0; &#xB300; &#xD55C; &#xD608; &#xCCAD; &#xBB34;&#xB8CC; &#xBA54;&#xC11C;&#xB4DC;&#xB97C; &#xC81C;&#xACF5;&#xD569;&#xB2C8;&#xB2E4;.

&#xD655;&#xC7A5; &#xBC0F; &#xC778;&#xAC04; &#xBC30;&#xC544; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xC720;&#xB798; &#xB0B4; &#xD53C; &#xC138;&#xD3EC;&#xC758; TGFbeta &#xC800;&#xD574; &#xD558; &#xC5EC; &#xC720;&#xC9C0; &#xBCF4;&#xC218; Id1 &#xC885;&#xC18D; &#xC774;&#xB2E4;.

Los esfuerzos anteriores para las c&eacute;lulas madre embrionarias humanas (hESCs) se diferencian en c&eacute;lulas endoteliales humanas no han logrado expansi&oacute;n sostenido y la estabilidad de las c&eacute;lulas vasculares. Para definir las v&iacute;as del desarrollo vasculog&eacute;nica y aumentar la diferenciaci&oacute;n, utilizamos un promotor de VE-cadherina espec&iacute;ficas de la c&eacute;lula endotelial conducir la prote&iacute;na fluorescente verde (GFP) (hVPr-GFP) para detectar factores que promueven el compromiso vascular. En la fase 1 de nuestro m&eacute;todo, inhibici&oacute;n de transformar el factor de crecimiento (TGF) beta en el d&iacute;a 7 de diferenciaci&oacute;n aumenta las c&eacute;lulas hVPr-GFP(+) por diez veces. En la fase 2, TGFbeta inhibici&oacute;n mantiene la proliferaci&oacute;n e identidad vascular de las c&eacute;lulas endoteliales purificadas, resultando en una red 36-fold expansi&oacute;n de c&eacute;lulas endoteliales en monocapas homog&eacute;neas, que exhibi&oacute; un perfil transcripcional de Id1(high)VEGFR2(high)VE-cadherin(+) ephrinB2(+). Usando una l&iacute;nea de reportero de hESC Id1-YFP, demostramos que la inhibici&oacute;n de TGFbeta sostiene Id1 expresi&oacute;n en c&eacute;lulas derivadas de c&eacute;lulas endoteliales y que Id1 es necesaria para el aumento de la proliferaci&oacute;n y preservaci&oacute;n de compromiso de la c&eacute;lula endotelial. Nuestro enfoque proporciona un m&eacute;todo libre de suero para la diferenciaci&oacute;n y mantenimiento a largo plazo de las c&eacute;lulas endoteliales derivados de c&eacute;lulas a una escala pertinente para la aplicaci&oacute;n cl&iacute;nica.

Expansi&oacute;n y mantenimiento de c&eacute;lulas endoteliales derivadas de c&eacute;lulas madre embrionarias humanas por la inhibici&oacute;n de TGFbeta es Id1 dependiente.

Generation of vascular endothelial cells (EC) from human embryonic stem cells (hESC) is a vital component of cell-based strategies for treatment of cardiovascular disease. Before hESC-derived ECs can be administered in therapeutic modalities, however, chemically defined culture conditions must be developed that reproducibly and robustly induce vascular differentiation. One approach to screening for culture conditions that support differentiation of hESCs to any cell type is their genetic modification with exogenous DNA sequence comprising a tissue-specific gene promoter driving reporters such as fluorescent protein or antibiotic drug resistance. The protocols herein provide instructions for the generation of clonal hESC lines containing a reporter transgene that is specifically expressed in vascular endothelial derivatives. Additionally, they demonstrate the methodology employed to assess vascular differentiation from clonal lines. Together, these protocols provide a solid foundation for study of vascular differentiation, and may also be applied, in principle, to studies of other specialized cell types derived from hESCs.

Lentiviral transduction and clonal selection of hESCs with endothelial-specific transgenic reporters.

To report the chance of sperm discovery in the laboratory when sperm were not identified in the operating room (OR).

Role of tissue digestion and extensive sperm search after microdissection testicular sperm extraction.

Several studies have demonstrated that hematopoietic cells originate from endotheliumin early development; however, the phenotypic progression of progenitor cells during human embryonic hemogenesis is not well described. Here, we define the developmental hierarchy among intermediate populations of hematopoietic progenitor cells (HPCs) derived from human embryonic stem cells (hESCs). We genetically modified hESCs to specifically demarcate acquisition of vascular (VE-cadherin) and hematopoietic (CD41a) cell fate and used this dual-reporting transgenic hESC line to observe endothelial to hematopoietic transition by real-time confocal microscopy. Live imaging and clonal analyses revealed a temporal bias in commitment of HPCs that recapitulates discrete waves of lineage differentiation noted during mammalian hemogenesis. Specifically, HPCs isolated at later time points showed reduced capacity to form erythroid/ megakaryocytic cells and exhibited a tendency toward myeloid fate that was enabled by expression of the Notch ligand Dll4 on hESC-derived vascular feeder cells. These data provide a framework for defining HPC lineage potential, elucidate a molecular contribution from the vascular niche in promoting hematopoietic lineage progression, and distinguish unique subpopulations of hemogenic endothelium during hESC differentiation.

Human ESC-derived hemogenic endothelial cells undergo distinct waves of endothelial to hematopoietic transition.

Does follicular flushing during oocyte retrieval improve the number of oocytes retrieved in the poorest responders?

Follicular flushing and in vitro fertilization outcomes in the poorest responders: a randomized controlled trial.

Fragile X syndrome (FXS) is caused by a CGG repeat expansion in the FMR1 gene that appears to occur during oogenesis and during early embryogenesis. One model proposes that repeat instability depends on the replication fork direction through the repeats such that (CNG)n hairpin-like structures form, causing DNA polymerase to stall and slip. Examining DNA replication fork progression on single DNA molecules at the endogenous FMR1 locus revealed that replication forks stall at CGG repeats in human cells. Furthermore, replication profiles of FXS human embryonic stem cells (hESCs) compared to nonaffected hESCs showed that fork direction through the repeats is altered at the FMR1 locus in FXS hESCs, such that predominantly the CCG strand serves as the lagging-strand template. This is due to the absence of replication initiation that would typically occur upstream of FMR1, suggesting that altered replication origin usage combined with fork stalling promotes repeat instability during early embryonic development. 

The DNA replication program is altered at the FMR1 locus in fragile X embryonic stem cells.

Epigenetic gene silencing is seen in several repeat-expansion diseases. In fragile X syndrome, the most common genetic form of mental retardation, a CGG trinucleotide-repeat expansion adjacent to the fragile X mental retardation 1 (FMR1) gene promoter results in its epigenetic silencing. Here, we show that FMR1 silencing is mediated by the FMR1 mRNA. The FMR1 mRNA contains the transcribed CGG-repeat tract as part of the 5' untranslated region, which hybridizes to the complementary CGG-repeat portion of the FMR1 gene to form an RNA·DNA duplex. Disrupting the interaction of the mRNA with the CGG-repeat portion of the FMR1 gene prevents promoter silencing. Thus, our data link trinucleotide-repeat expansion to a form of RNA-directed gene silencing mediated by direct interactions of the trinucleotide-repeat RNA and DNA. 

Promoter-bound trinucleotide repeat mRNA drives epigenetic silencing in fragile X syndrome.

Stem cells are characterized by their absolute or relative lack of specialization their ability for self-renewal, as well as their ability to generate differentiated progeny through cellular lineages with one or more branches. The increased availability of embryonic tissue and greatly improved derivation methods have led to a large increase in the number of hESC lines. 

Derivation of human embryonic stem cells (hESC).

Fragile X syndrome (FXS) is caused by CGG repeat expansion that leads to FMR1 silencing. Women with a premutation allele are at risk of having a full mutation child with FXS. To investigate the mechanism of repeat expansion, we examined the relationship between a single-nucleotide polymorphism (SNP) variant that is linked to repeat expansion in haplogroup D and a replication origin located ∼53 kb upstream of the repeats. This origin is absent in FXS human embryonic stem cells (hESCs), which have the SNP variant C, but present in the nonaffected hESCs, which have a T variant. The SNP maps directly within the replication origin. Interestingly, premutation hESCs have a replication origin and the T variant similar to nonaffected hESCs. These results suggest that a T/C SNP located at a replication origin could contribute to the inactivation of this replication origin in FXS hESCs, leading to altered replication fork progression through the repeats, which could result in repeat expansion to the FXS full mutation. 

Cis-acting DNA sequence at a replication origin promotes repeat expansion to fragile X full mutation.

To study the outcomes of microdissection testicular sperm extraction (microTESE) among men with pure Sertoli cell-only histology on diagnostic testicular biopsy.

Microdissection testicular sperm extraction in men with Sertoli cell-only testicular histology.

Despite major advances in tissue cryopreservation and auto-transplantation, reperfusion ischemia and hypoxia have been reported as major obstacles to successful recovery of the follicular pool within grafted ovarian tissue. We demonstrate a benefit to follicular survival and function in human ovarian tissue that is co-transplanted with exogenous endothelial cells (ExEC). ExECs were capable of forming functionally perfused vessels at the host/graft interface and increased both viability and follicular volume in ExEC-assisted grafts with resumption of antral follicle development in long-term grafts. ExECs that were engineered to constitutively express anti-mullerian hormone (AMH) induced a greater proportion of quiescent primordial follicles than control ExECs, indicating suppression of premature mobilization that has been noted in the context of ovarian tissue transplantation. These findings present a cell-based strategy that combines accelerated perfusion with direct paracrine delivery of a bioactive payload to transplanted ovarian tissue.

Engineered endothelium provides angiogenic and paracrine stimulus to grafted human ovarian tissue.

Time-lapse microscopy (TLM) is an exciting novel technology with great potential for enhancing embryo selection in the embryology laboratory. This non-invasive objective assessment of embryos has provided a new tool for predicting embryo development and implantation potential. TLM detects several morphological phenomena that are often missed with static observations using conventional incubators, such as irregular divisions, blastocyst collapse and re-expansion, timing of blastocoel appearance, and timing of formation and internalization of fragments. Nevertheless, it should be recognized that conventional morphological assessment has been widely accepted as the gold standard by most embryologists. TLM can enhance conventional morphological assessments to improve embryo selection and subsequent reproductive outcomes. Furthermore, morphokinetic parameters can aid in differentiating between euploid and aneuploid embryos, although they are not sufficiently accurate to replace preimplantation genetic testing for aneuploidy. Morphokinetic assessment together with chromosomal screening may ultimately help identify euploid embryos with the highest developmental potential.

Article	Year
High pregnancy rates can be achieved after freezing and thawing human blastocysts. Fertility and sterility\| PubMed ID: 15533370	2004
Expansion and maintenance of human embryonic stem cell-derived endothelial cells by TGFbeta inhibition is Id1 dependent. Nature biotechnology\| PubMed ID: 20081865	2010
Lentiviral transduction and clonal selection of hESCs with endothelial-specific transgenic reporters. Current protocols in stem cell biology\| PubMed ID: 21509935	2011
Role of tissue digestion and extensive sperm search after microdissection testicular sperm extraction. Fertility and sterility\| PubMed ID: 21669413	2011
Human ESC-derived hemogenic endothelial cells undergo distinct waves of endothelial to hematopoietic transition. Blood\| PubMed ID: 23169780	2013
Follicular flushing and in vitro fertilization outcomes in the poorest responders: a randomized controlled trial. Human reproduction (Oxford, England)\| PubMed ID: 24014603	2013
The DNA replication program is altered at the FMR1 locus in fragile X embryonic stem cells. Molecular cell\| PubMed ID: 24289922	2014
Promoter-bound trinucleotide repeat mRNA drives epigenetic silencing in fragile X syndrome. Science (New York, N.Y.)\| PubMed ID: 24578575	2014
Derivation of human embryonic stem cells (hESC). Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 24782008	2014
Cis-acting DNA sequence at a replication origin promotes repeat expansion to fragile X full mutation. The Journal of cell biology\| PubMed ID: 25179629	2014
Microdissection testicular sperm extraction in men with Sertoli cell-only testicular histology. Fertility and sterility\| PubMed ID: 25441063	2014
Engineered endothelium provides angiogenic and paracrine stimulus to grafted human ovarian tissue. Scientific reports\| PubMed ID: 28811567	2017
Assessment of embryo morphology and developmental dynamics by time-lapse microscopy: is there a relation to implantation and ploidy? Fertility and sterility\| PubMed ID: 29101997	2017
Time-lapse imaging: clearly useful to both laboratory personnel and patient outcomes versus just because we can doesn't mean we should. Fertility and sterility\| PubMed ID: 29653705	2018

Nikica Zaninovic

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