Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

Institute of Pathology, University of Bonn Medical School

&#x5728;&#x54FA;&#x4E73;&#x52A8;&#x7269;&#x4E2D;&#x7684;&#x7B2C;&#x4E00;&#x4E2A;&#x5355;&#x5143;&#x683C;&#x7684;&#x547D;&#x8FD0;&#x51B3;&#x5B9A;&#x7531; 8-16 &#x7EC6;&#x80DE;&#x9636;&#x6BB5;&#x7684;&#x80DA;&#x80CE;&#x690D;&#x5165;&#x524D;&#x80DA;&#x80CE;&#x7EC6;&#x80DE;&#x6781;&#x5316;&#x521D;&#x59CB;&#x5316;&#x3002;&#x5728;&#x6B64;&#x9636;&#x6BB5;&#xFF0C;&#x5916;&#x90E8;&#x5355;&#x5143;&#x683C;&#x91C7;&#x7528; trophectoderm &#xFF08;TE&#xFF09; &#x7684;&#x547D;&#x8FD0;&#xFF0C;&#x800C;&#x5185;&#x90E8;&#x7EC6;&#x80DE;&#x4EBA;&#x53E3;&#x5185;&#x7EC6;&#x80DE;&#x56E2; &#xFF08;ICM&#xFF09; &#x5F15;&#x8D77;&#x3002;&#x5728;&#x690D;&#x5165;&#x672F;&#x3001; &#x8F6C;&#x5F55;&#x4E92;&#x52A8;&#x7F51;&#x7EDC;&#x548C;&#x8868;&#x89C2;&#x9057;&#x4F20;&#x4FEE;&#x6539;&#x4E4B;&#x524D;&#x4E0D;&#x53EF;&#x633D;&#x56DE;&#x5730;&#x5212;&#x5206;&#x7684;&#x80DA;&#x5916;&#x548C;&#x80DA;&#x80CE;&#x7684;&#x547D;&#x8FD0;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x62A5;&#x544A;&#x80DA;&#x5916;&#x6ECB;&#x517B;&#x7EC6;&#x80DE;&#x5E72;&#x7EC6;&#x80DE; (TSC) &#x7CFB;&#x7531;&#x8D85;&#x91CF;&#x8868;&#x8FBE; Oct4&#x3001; Sox2&#x3001; Klf4&#x3001; cMyc &#x8F6C;&#x6362;&#x4E3A;&#x8BF1;&#x5BFC;&#x7684;&#x591A;&#x529F;&#x80FD;&#x5E72;&#x7EC6;&#x80DE; (TSC &#x9B54;&#x9B3C;)&#x3002;&#x7532;&#x57FA;&#x5316;&#x7684;&#x7814;&#x7A76;&#x548C;&#x57FA;&#x56E0;&#x9635;&#x5217;&#x5206;&#x6790;&#x8868;&#x793A; TSC &#x9B54;&#x9B3C;&#x5DF2;&#x7ECF;&#x901A;&#x8FC7;&#x4E86;&#x6F5C;&#x5728;&#x591A;&#x80FD;&#x5E72;&#x3002;&#x8F6C;&#x6362;&#x7387;&#x4F4E;&#x4E8E;&#x4F53;&#x7EC6;&#x80DE;&#x7684;&#x7F16;&#x7A0B;&#x5B9E;&#x9A8C;&#xFF0C;&#x53EF;&#x80FD;&#x662F;&#x7531;&#x4E8E;&#x63A7;&#x5236;&#x80DA;&#x5916;&#x6CBF;&#x88AD;&#x56FA;&#x5B9A;&#x7684;&#x72EC;&#x7279;&#x7684;&#x9057;&#x4F20;&#x7F51;&#x7EDC;&#x3002;&#x4F53;&#x5916;&#x548C;&#x4F53;&#x5185;&#xFF0C;TSC &#x9B54;&#x9B3C;&#x5206;&#x5316;&#x4E3A;&#x7EC4;&#x7EC7;&#x4EE3;&#x8868;&#x6240;&#x6709;&#x4E09;&#x4E2A;&#x80DA;&#x80CE;&#x80DA;&#x5C42;&#xFF0C;&#x6307;&#x793A;&#x53EF;&#x4EE5;&#x8BF1;&#x5BFC;&#x4F53;&#x7EC6;&#x80DE;&#x547D;&#x8FD0;&#x3002;&#x6700;&#x540E;&#xFF0C;TSC &#x9B54;&#x9B3C;&#x7684; chimerized &#x80DA;&#x80CE;&#x9002;&#x5F53;&#x548C;&#x5BF9;&#x5C0F;&#x9F20;&#xFF0C;&#x8868;&#x660E;&#x8FD9;&#x4E9B;&#x7EC6;&#x80DE;&#x83B7;&#x5F97;&#x4E86;&#x5145;&#x5206;&#x4F53;&#x7EC6;&#x80DE;&#x5206;&#x5316;&#x6F5C;&#x80FD;&#x7684;&#x80DA;&#x82BD;&#x884C;&#x4F5C;&#x51FA;&#x8D21;&#x732E;&#x3002;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x5BFC;&#x81F4;&#x7684;&#x5206;&#x5B50;&#x7684;&#x8FC7;&#x7A0B;&#xFF0C;&#x6307;&#x5BFC;&#x5728;&#x54FA;&#x4E73;&#x52A8;&#x7269;&#x4E2D;&#x7684;&#x7B2C;&#x4E00;&#x4E2A;&#x6CBF;&#x88AD;&#x51B3;&#x5B9A;&#x66F4;&#x597D;&#x5730;&#x7406;&#x89E3;&#x3002;

&#x6CBF;&#x88AD;&#x8F6C;&#x6362;&#x4E3A;&#x591A;&#x80FD;&#x5E72;&#x7EC6;&#x80DE;&#x5C0F;&#x9F20;&#x80DA;&#x5916;&#x6ECB;&#x517B;&#x7EC6;&#x80DE;&#x5E72;&#x7EC6;&#x80DE;&#x3002;

In mammals, the first cell fate decision is initialized by cell polarization at the 8- to 16-cell stage of the preimplantation embryo. At this stage, outside cells adopt a trophectoderm (TE) fate, whereas the inside cell population gives rise to the inner cell mass (ICM). Prior to implantation, transcriptional interaction networks and epigenetic modifications divide the extraembryonic and embryonic fate irrevocably. Here, we report that extraembryonic trophoblast stem cell (TSC) lines are converted to induced pluripotent stem cells (TSC-iPSCs) by overexpressing Oct4, Sox2, Klf4, and cMyc. Methylation studies and gene array analyses indicated that TSC-iPSCs had adopted a pluripotent potential. The rate of conversion was lower than those of somatic reprogramming experiments, probably due to the unique genetic network controlling extraembryonic lineage fixation. Both in vitro and in vivo, TSC-iPSCs differentiated into tissues representing all three embryonic germ layers, indicating that somatic cell fate could be induced. Finally, TSC-iPSCs chimerized the embryo proper and contributed to the germ line of mice, indicating that these cells had acquired full somatic differentiation potential. These results lead to a better understanding of the molecular processes that govern the first lineage decision in mammals.

Lineage conversion of murine extraembryonic trophoblast stem cells to pluripotent stem cells.

Chez les mammif&egrave;res, la premi&egrave;re d&eacute;cision de destin cellulaire est initialis&eacute;e par la polarisation de la cellule au stade 8 &agrave; 16 cellules de l&#39;embryon pr&eacute;implantatoire. &Agrave; ce stade, les cellules externes adoptent un destin trophectoderme (TE), alors que la population de cellules de l&#39;int&eacute;rieur donne lieu &agrave; la masse cellulaire interne (MCI). Avant l&#39;implantation, r&eacute;seaux d&#39;interaction transcriptionnelle et les modifications &eacute;pig&eacute;n&eacute;tiques divisent le sort extra-embryonnaires et embryonnaire irr&eacute;vocablement. Nous rapportons ici que des lign&eacute;es de cellules souches (TSC) extra-embryonnaires trophoblastiques sont converties en des cellules souches pluripotentes induites (TSC-CISP) par surexprimant Oct4, Sox2, Klf4 et cMyc. &Eacute;tudes de m&eacute;thylation et g&egrave;ne tableau a indiqu&eacute; que TSC-CISP avait adopt&eacute; un pluripotentes potentiels. Le taux de conversion &eacute;tait inf&eacute;rieur &agrave; celui des exp&eacute;riences de reprogrammation somatiques, probablement en raison de l&#39;unique r&eacute;seau g&eacute;n&eacute;tique contr&ocirc;lant la fixation de la lign&eacute;e extra-embryonnaires. In vitro et in vivo, TSC-CISP se diff&eacute;rencie en tissus qui repr&eacute;sente tous les trois couches germinales embryonnaires, indiquant que le destin des cellules somatiques peut &ecirc;tre induit. Enfin, chimerized TSC-CISP l&#39;embryon appropri&eacute;e et a contribu&eacute; &agrave; la lign&eacute;e germinale des souris, ce qui indique que ces cellules avaient acquis la diff&eacute;renciation somatique complet potentielle. Ces r&eacute;sultats conduisent &agrave; une meilleure compr&eacute;hension des processus mol&eacute;culaires qui r&eacute;gissent la premi&egrave;re d&eacute;cision de lign&eacute;e chez les mammif&egrave;res.

Conversion de la lign&eacute;e de cellules souches trophoblastiques extra-embryonnaires murin de cellules souches pluripotentes.

Bei S&auml;ugetieren ist die erste Zelle Schicksal Entscheidung von Zelle Polarisierung in der 8 - 16-Zellen-Stadium des Embryos Pr&auml;implantationsdiagnostik initialisiert. Zu diesem Zeitpunkt erlassen au&szlig;en Zellen ein Trophectoderm (TE)-Schicksal, w&auml;hrend innen Zelle Bev&ouml;lkerung gibt Grund zu der inneren Zelle Masse (ICM). Vor der Implantation Teilen transkriptionelle Interaktion Netze und epigenetischen Modifikationen das extraembryonic und embryonale Schicksal unwiderruflich. Hier berichten wir, dass extraembryonic Trophoblast (TSC) Stammzelllinien von &uuml;berexprimierenden Oct4, Sox2, Klf4 und cMyc induzierten pluripotenten Stammzellen (TSC-iPSCs) konvertiert werden. Methylierung-Studien und Analysen der Gen-Array angegeben, dass die TSC-iPSCs einer pluripotenten potentiellen angenommen hatte. Umrechnung war niedriger als die der somatischen Neuprogrammierung Experimente, wahrscheinlich aufgrund der einzigartigen genetischen Netzwerk steuern extraembryonic &Uuml;bertragungslinie Fixierung. Sowohl in Vitro und in Vivo differenzierte TSC-iPSCs in das Gewebe, die alle drei embryonalen Keim Schichten repr&auml;sentieren, dass dieses Schicksal somatischen Zellen induziert werden konnte. Schlie&szlig;lich TSC-iPSCs Chimerized der Embryo ordnungsgem&auml;&szlig;e und trug zum die Keimbahn von M&auml;usen, die darauf hinweist, dass diese Zellen potenzielle volle somatische Differenzierung erworben hatte. Diese Ergebnisse f&uuml;hren zu einem besseren Verst&auml;ndnis der molekularen Prozesse, die die erste Linie-Entscheidung bei S&auml;ugetieren zu Regeln.

Linie Umwandlung von murinen extraembryonic Trophoblast Stammzellen zu pluripotenten Stammzellen.

Nei mammiferi, la prima decisione del destino delle cellule viene inizializzata da polarizzazione delle cellule nella fase 8-16 cellule dell&#39;embrione preimpianto. In questa fase, le cellule esterne adottano un destino trophectoderm (TE), mentre all&#39;interno delle cellule popolazione d&agrave; luogo alla massa cellulare interna (ICM). Prima dell&#39;impianto, reti di transcriptional interazione e modifiche epigenetiche dividono il destino extraembryonic ed embrionale irrevocabilmente. Qui, segnaliamo che linee di cellule staminali (TSC) extraembryonic trofoblasto vengono convertiti in cellule staminali pluripotenti indotte (TSC-iPSCs) da con sovraesprimono relativa Oct4, Sox2, Klf4 e cMyc. Studi di metilazione e gene array analisi indicato che TSC-iPSCs aveva adottato un potenziale pluripotenti. Il tasso di conversione &egrave; stato inferiore a quelli degli esperimenti di riprogrammazione somatici, probabilmente a causa della rete genetico unico controllo della fissazione del lignaggio extraembryonic. Sia in vitro che in vivo, TSC-iPSCs differenziato in tessuti che rappresenta tutti i tre strati germinali embrionali, che indica che il destino delle cellule somatiche potrebbe essere indotta. Infine, TSC-iPSCs chimerized l&#39;embrione corretta e contribuito alla linea germinale dei topi, che indica che queste cellule avevano acquisito la differenziazione somatica pieno potenziale. Questi risultati portano a una migliore comprensione dei processi molecolari che regolano la prima decisione di lignaggio in mammiferi.

Conversione di lignaggio di murino extraembryonic trophoblast cellule staminali in cellule staminali pluripotenti.

&#x54FA;&#x4E73;&#x985E;&#x3067;&#x3001;&#x6700;&#x521D;&#x306E;&#x7D30;&#x80DE;&#x904B;&#x547D;&#x6C7A;&#x5B9A;&#x7740;&#x5E8A;&#x524D;&#x671F;&#x80DA;&#x306E; 8-16 &#x7D30;&#x80DE;&#x671F;&#x3067;&#x7D30;&#x80DE;&#x5206;&#x6975;&#x306B;&#x3088;&#x3063;&#x3066;&#x521D;&#x671F;&#x5316;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x6BB5;&#x968E;&#x3067;&#x306F;&#x3001;&#x7D30;&#x80DE;&#x5916;&#x306F;&#x3001;&#x6804;&#x990A;&#x5916;&#x80DA;&#x8449; &#xFF08;TE&#xFF09; &#x306E;&#x904B;&#x547D;&#x3092;&#x63A1;&#x7528;&#x3059;&#x308B;&#x4EBA;&#x53E3;&#x306E;&#x5185;&#x5074;&#x306E;&#x30BB;&#x30EB;&#x306B;&#x5BFE;&#x3057;&#x5185;&#x90E8;&#x7D30;&#x80DE;&#x584A; &#xFF08;ICM&#xFF09; &#x3092;&#x751F;&#x3080;&#x3002;&#x6CE8;&#x5165;&#x3001;&#x8EE2;&#x5199;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x30CD;&#x30C3;&#x30C8;&#x30EF;&#x30FC;&#x30AF;&#x3001;&#x30A8;&#x30D4;&#x30B8;&#x30A7;&#x30CD;&#x30C6;&#x30A3;&#x30C3;&#x30AF;&#x4FEE;&#x98FE;&#x524D;&#x521D;&#x671F;&#x80DA;&#x3068;&#x80DA;&#x306E;&#x904B;&#x547D;&#x306F;&#x53D6;&#x6D88;&#x4E0D;&#x80FD;&#x306E;&#x5F62;&#x3001;&#x306A;&#x3044;&#x5206;&#x5272;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x975E;&#x80DA;&#x6027;&#x5E79;&#x7D30;&#x80DE; (TSC) &#x682A;&#x8A98;&#x5C0E;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE; (TSC ips &#x7D30;&#x80DE;) Oct4, Sox2, Klf4, &#x304A;&#x3088;&#x3073; cMyc &#x306E;&#x904E;&#x5270;&#x306B;&#x3088;&#x3063;&#x3066;&#x5909;&#x63DB;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x3092;&#x5831;&#x544A;&#x3057;&#x307E;&#x3059;&#x3002;&#x30E1;&#x30C1;&#x30EB;&#x5316;&#x7814;&#x7A76;&#x3001;&#x907A;&#x4F1D;&#x5B50;&#x914D;&#x5217;&#x89E3;&#x6790; TSC ips &#x7D30;&#x80DE;&#x591A;&#x80FD;&#x6027;&#x306E;&#x6F5C;&#x5728;&#x7684;&#x306A;&#x63A1;&#x7528;&#x3057;&#x3066;&#x3044;&#x305F;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002;&#x63DB;&#x7B97;&#x7387;&#x306F;&#x304A;&#x305D;&#x3089;&#x304F;&#x30E6;&#x30CB;&#x30FC;&#x30AF;&#x306A;&#x907A;&#x4F1D;&#x7684;&#x30CD;&#x30C3;&#x30C8;&#x30EF;&#x30FC;&#x30AF;&#x521D;&#x671F;&#x80DA;&#x7CFB;&#x7D71;&#x56FA;&#x5B9A;&#x5236;&#x5FA1;&#x306B;&#x3088;&#x308B;&#x4F53;&#x7D30;&#x80DE;&#x306E;&#x30EA;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30DF;&#x30F3;&#x30B0;&#x5B9F;&#x9A13;&#x306E;&#x305D;&#x308C;&#x3089;&#x3088;&#x308A;&#x4F4E;&#x304B;&#x3063;&#x305F;&#x3002;&#x4E21;&#x65B9; in vitro &#x304A;&#x3088;&#x3073; in vivo &#x3067;&#x3001;TSC ips &#x7D30;&#x80DE;&#x306F;&#x4F53;&#x7D30;&#x80DE;&#x904B;&#x547D;&#x304C;&#x8A98;&#x767A;&#x3055;&#x308C;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3092;&#x793A;&#x3059;&#x3059;&#x3079;&#x3066;&#x306E;&#x4E09;&#x80DA;&#x6027;&#x751F;&#x6B96;&#x5C64;&#x3092;&#x8868;&#x3059;&#x30C6;&#x30A3;&#x30C3;&#x30B7;&#x30E5;&#x306B;&#x533A;&#x5225;&#x3002;&#x6700;&#x5F8C;&#x306B;&#x3001;TSC ips &#x7D30;&#x80DE; &#xFF05; &#x80DA;&#x9069;&#x5207;&#x3067;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x30BB;&#x30EB;&#x5B8C;&#x5168;&#x4F53;&#x7D30;&#x80DE;&#x306E;&#x5206;&#x5316;&#x306E;&#x80FD;&#x529B;&#x3092;&#x53D6;&#x5F97;&#x3057;&#x305F;&#x3053;&#x3068;&#x3092;&#x793A;&#x3059;&#x30DE;&#x30A6;&#x30B9;&#x306E;&#x751F;&#x6B96;&#x7CFB;&#x5217;&#x306B;&#x5BC4;&#x4E0E;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D50;&#x679C;&#x306F;&#x3001;&#x54FA;&#x4E73;&#x985E;&#x306E;&#x6700;&#x521D;&#x306E;&#x7CFB;&#x5217;&#x6C7A;&#x5B9A;&#x3092;&#x652F;&#x914D;&#x3059;&#x308B;&#x5206;&#x5B50;&#x306E;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306E;&#x7406;&#x89E3;&#x3092;&#x6DF1;&#x3081;&#x308B;&#x306B; &#x3057;&#x307E;&#x3059;&#x3002;

&#x30EA;&#x30CD;&#x30FC;&#x30B8;&#x30E5; &#x30DE;&#x30A6;&#x30B9;&#x521D;&#x671F;&#x80DA;&#x7D68;&#x6BDB;&#x7D30;&#x80DE;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x3078;&#x306E;&#x5909;&#x63DB;&#x3002;

&#xD3EC;&#xC720;&#xB958;, &#xCCAB; &#xBC88;&#xC9F8; &#xC138;&#xD3EC; &#xC6B4;&#xBA85; &#xACB0;&#xC815; preimplantation &#xBC30;&#xC544;&#xC758; 8-16 &#xC140; &#xB2E8;&#xACC4;&#xC5D0;&#xC11C; &#xC140; &#xC591;&#xADF9; &#xD654;&#xC5D0; &#xC758;&#xD574; &#xCD08;&#xAE30;&#xD654; &#xB429;&#xB2C8;&#xB2E4;. &#xC774; &#xB2E8;&#xACC4;&#xC5D0;&#xC11C; &#xC678;&#xBD80; &#xC140; &#xB0B4;&#xBD80; &#xC138;&#xD3EC; &#xC778;&#xAD6C; &#xBC18;&#xBA74; trophectoderm (&#xD14C;) &#xC6B4;&#xBA85;&#xC744; &#xCC44;&#xD0DD; &#xB0B4;&#xBD80; &#xC140; &#xC9C8;&#xB7C9; (ICM)&#xC5D0; &#xC0C1;&#xC2B9; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;. &#xC774;&#xC2DD;, transcriptional &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xB124;&#xD2B8;&#xC6CC;&#xD06C;&#xC640; epigenetic &#xC218;&#xC815; &#xC804;&#xC5D0; &#xBD84;&#xD560; &#xD558;&#xC9C0; extraembryonic &#xBC0F; &#xBC30;&#xC544; &#xC6B4;&#xBA85; &#xC601;&#xAD6C;&#xC801;&#xC73C;&#xB85C; &#xC81C;&#xAC70;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xAC00; &#xBCF4;&#xACE0; extraembryonic trophoblast &#xC904;&#xAE30; &#xC138;&#xD3EC; (TSC) &#xB77C;&#xC778; Oct4, Sox2, Klf4, &#xBC0F; cMyc overexpressing&#xC5D0; &#xC758;&#xD574; &#xC720;&#xB3C4; &#xB9CC;&#xB2A5; &#xC904;&#xAE30; &#xC138;&#xD3EC; (tsc&#xAC00;-iPSCs)&#xC73C;&#xB85C; &#xBCC0;&#xD658; &#xB429;&#xB2C8;&#xB2E4;. &#xBA54; &#xD2F8; &#xD654; &#xC5F0;&#xAD6C;&#xC640; &#xC720;&#xC804;&#xC790; &#xBC30;&#xC5F4; &#xBD84;&#xC11D; &#xD45C;&#xC2DC; TSC iPSCs &#xC7A0;&#xC7AC;&#xC801;&#xC778; pluripotent &#xCC44;&#xD0DD; &#xD588;&#xB2E4;. &#xBCC0;&#xD658; &#xC18D;&#xB3C4; &#xC544;&#xB9C8; extraembryonic &#xD608;&#xD1B5; &#xACE0;&#xC815; &#xC81C;&#xC5B4; &#xD558;&#xB294; &#xACE0;&#xC720; &#xC720;&#xC804;&#xC790; &#xB124;&#xD2B8;&#xC6CC;&#xD06C; &#xB54C;&#xBB38;&#xC5D0; &#xCCB4;&#xC138;&#xD3EC; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC744; &#xB2E4;&#xC2DC; &#xB9CC;&#xB4E4; &#xC2E4;&#xD5D8; &#xBCF4;&#xB2E4; &#xB0AE;&#xC740;. &#xCCB4; &#xC678;&#xC640; &#xBE44;&#xBCF4;&#xC5D0;, TSC iPSCs &#xADF8; &#xCCB4;&#xC138;&#xD3EC; &#xC6B4;&#xBA85;&#xC73C;&#xB85C; &#xC720;&#xB3C4; &#xB420; &#xC218;&#xB97C; &#xB098;&#xD0C0;&#xB0B4;&#xB294; 3 &#xAC1C;&#xC758; &#xBAA8;&#xB4E0; &#xBC30;&#xC544; &#xC138;&#xADE0; &#xCE35;&#xC744; &#xB300;&#xD45C; &#xD558;&#xB294; &#xC870;&#xC9C1;&#xC73C;&#xB85C; &#xBD84;&#xD654;. &#xB9C8;&#xC9C0;&#xB9C9;&#xC73C;&#xB85C;, TSC iPSCs chimerized &#xBC30;&#xC544; &#xC801;&#xC808; &#xD558; &#xACE0; &#xC950;,&#xC774; &#xC138;&#xD3EC; &#xC804;&#xCCB4; &#xCCB4;&#xC138;&#xD3EC; &#xBD84;&#xD654; &#xC7A0;&#xC7AC;&#xC801;&#xC778; &#xC778;&#xC218; &#xD588;&#xB2E4; &#xB098;&#xD0C0;&#xB0B4;&#xB294; &#xC138;&#xADE0; &#xB77C;&#xC778;&#xC5D0; &#xAE30;&#xC5EC;. &#xC774;&#xB7EC;&#xD55C; &#xACB0;&#xACFC; &#xD3EC;&#xC720;&#xB958;&#xC5D0;&#xC11C; &#xCCAB; &#xBC88;&#xC9F8; &#xB9AC;&#xB2C8;&#xC9C0; &#xACB0;&#xC815;&#xC744; &#xC81C;&#xC5B4; &#xD558;&#xB294; &#xBD84;&#xC790; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC758; &#xB354; &#xB098;&#xC740; &#xC774;&#xD574; &#xC774;&#xC5B4;&#xC9C8;.

&#xB9AC;&#xB2C8;&#xC9C0;&#xB294; murine extraembryonic trophoblast &#xC904;&#xAE30; &#xC138;&#xD3EC; pluripotent &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xBCC0;&#xD658;.

En los mam&iacute;feros, la primera decisi&oacute;n de destino celular es inicializada por polarizaci&oacute;n celular en la fase de 8 a 16 c&eacute;lulas del embri&oacute;n preimplantacional. En esta etapa, c&eacute;lulas exteriores adoptan una suerte de trofectodermo (TE), mientras que el interior de la c&eacute;lula poblaci&oacute;n da lugar a la masa celular interna (ICM). Antes de la implantaci&oacute;n, redes de interacci&oacute;n transcripcional y modificaciones epigen&eacute;ticas dividen el destino extraembrionarias y embrionario irrevocablemente. Aqu&iacute;, Divulgamos que l&iacute;neas de c&eacute;lulas madre (TSC) extraembrionarias trofoblasto se convierten en c&eacute;lulas pluripotentes inducidas (TSC-iPSCs) por overexpressing Oct4, Sox2, Klf4 y cMyc. Metilaci&oacute;n estudios y an&aacute;lisis de la matriz del gene indican que TSC-iPSCs adopt&oacute; un pluripotentes potenciales. La tasa de conversi&oacute;n fue inferior a los de experimentos reprogramaci&oacute;n som&aacute;ticos, probablemente debido a la &uacute;nica red gen&eacute;tica controla la fijaci&oacute;n de linaje extraembrionarias. Tanto in vitro e in vivo, TSC-iPSCs diferenciado en tejidos que representan los tres embrionarias capas germinales, indicando que se pod&iacute;a inducir esa suerte de c&eacute;lulas som&aacute;ticas. Finalmente, TSC-iPSCs chimerized el embri&oacute;n adecuado y contribuy&oacute; a la l&iacute;nea germinal de los ratones, lo que indica que estas c&eacute;lulas hab&iacute;an adquirido potencial completo som&aacute;tica diferenciaci&oacute;n. Estos resultados llevan a una mejor comprensi&oacute;n de los procesos moleculares que rigen la primera decisi&oacute;n de linaje en mam&iacute;feros.

Conversi&oacute;n de linaje de c&eacute;lulas del trofoblasto extraembrionarias murino en c&eacute;lulas madre pluripotentes.

In recent years, knowledge regarding the genetic and epigenetic programmes governing specification, maintenance and differentiation of the extraembryonic lineage has advanced substantially. Establishment and analysis of mice deficient in genes implicated in trophoblast lineage and the option to generate and manipulate murine stem cell lines from the inner cell mass and the trophectoderm in vitro represent major advances. The activating enhancer binding protein 2 (AP2) family of transcription factors is expressed during mammalian development and in certain malignant diseases. This article summarizes the data regarding expression and function of murine Tcfap2 and human TFAP2 in extraembryonic development and differentiation. It also presents a model integrating Tcfap2c into the framework of trophoblast development and highlights the requirement of Tcfap2c to maintain trophoblast stem cells. With regard to human trophoblast cell-lineage restriction, the role of TFAP2C in lineage specification and maintenance is speculated upon. Furthermore, an overview of target genes of AP2 in mouse and human affecting placenta development and function is provided and the evidence suggesting that defects in regulating TFAP2 members might contribute to placental defects is discussed.

The role of transcription factor Tcfap2c/TFAP2C in trophectoderm development.

Trophoblast stem cells (TSCs) are in vitro equivalents to the precursor cells of the placenta. TSCs are cultured in serum-rich medium with fibroblast growth factor 4, heparin, and embryonic-fibroblast-conditioned medium. Here, we developed a simple medium consisting of ten chemically defined ingredients for culture of TSCs on Matrigel or synthetic substrates, named TX medium. Gene expression and DNA methylation profiling demonstrated the faithful propagation of expression profiles and epigenomic characteristics of TSCs cultured in TX. Further, TX medium supported the de novo derivation of TSC lines. Finally, TSCs cultured in TX differentiate into all derivatives of the trophectodermal lineage in vitro, give rise to hemorrhagic lesions in nude mice, and chimerize the placenta, indicating that they retained all hallmarks of TSCs. TX media formulation no longer requires fetal bovine serum and conditioned medium, which facilitates and standardizes the culture of this extraembryonic lineage.

Derivation and maintenance of murine trophoblast stem cells under defined conditions.

Trophoblast stem cells (TSCs) arise from the first cell fate decision in the developing embryo and generate extra-embryonic lineages, giving rise to the fetal portion of the placenta. Mouse embryonic and extra-embryonic lineages are strictly separated by a distinct epigenetic barrier, which is not fully overcome following expression of TSC-determining factors in embryonic stem cells. Here, we show that transient expression of Tfap2c, Gata3, Eomes, and Ets2 is sufficient to reprogram mouse embryonic fibroblasts and post-natal tail-tip-derived fibroblasts into induced TSCs (iTSCs) and surmount the epigenetic barrier separating somatic from extra-embryonic lineages. iTSCs share nearly identical morphological characteristics, gene expression profiles, and DNA methylation patterns with blastocyst-derived TSCs. Furthermore, iTSCs display transgene-independent self-renewal, differentiate along extra-embryonic lineages, and chimerize host placentas following blastocyst injection. These findings provide insights into the transcription factor networks governing TSC identity and opportunities for studying the epigenetic barriers underlying embryonic and extra-embryonic lineage segregation.

Direct Induction of Trophoblast Stem Cells from Murine Fibroblasts.

Loss of TFAP2C in mouse leads to developmental defects in the extra-embryonic compartment with lethality at embryonic day (E)7.5. To investigate the requirement of TFAP2C in later placental development, deletion of TFAP2C was induced throughout extra-embryonic ectoderm at E6.5, leading to severe placental abnormalities caused by reduced trophoblast population and resulting in embryonic retardation by E8.5. Deletion of TFAP2C in TPBPA(+) progenitors at E8.5 results in growth arrest of the junctional zone. TFAP2C regulates its target genes Cdkn1a (previously p21) and Dusp6, which are involved in repression of MAPK signaling. Loss of TFAP2C reduces activation of ERK1/2 in the placenta. Downregulation of Akt1 and reduced activation of phosphorylated AKT in the mutant placenta are accompanied by impaired glycogen synthesis. Loss of TFAP2C led to upregulation of imprinted gene H19 and downregulation of Slc38a4 and Ascl2. The placental insufficiency post E16.5 causes fetal growth restriction, with 19% lighter mutant pups. Knockdown of TFAP2C in human trophoblast choriocarcinoma JAr cells inhibited MAPK and AKT signaling. Thus, we present a model where TFAP2C in trophoblasts controls proliferation by repressing Cdkn1a and activating the MAPK pathway, further supporting differentiation of glycogen cells by activating the AKT pathway.

Caroline Kubaczka

Department of Developmental Pathology

Institute of Pathology, University of Bonn Medical School

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Caroline Kubaczka

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Developmental Pathology

Institute of Pathology, University of Bonn Medical School

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Department of Developmental Pathology