generation-human-induced-pluripotent-stem-cells-from-peripheral-blood

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

efficient-ips-cell-generation-from-blood-using-episomes-hdac

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Children's Hospital of Philadelphia

The Children's Hospital of Philadelphia

Lo sviluppo di metodi per realizzare efficienti riprogrammazione delle cellule umane, evitando la presenza permanente di riprogrammazione transgeni rappresenta un passo fondamentale verso l&#39;uso di cellule staminali pluripotenti indotte (iPSC) per scopi clinici, quali la malattia di modellazione o ricostituire le terapie. Anche se esistono metodi diversi per la generazione di iPSC libero di riprogrammazione transgeni da mouse cellule o tessuti umani normali neonatali, un sistema di riprogrammazione sufficientemente efficiente &egrave; ancora necessario per raggiungere la derivazione diffusa delle malattie specifiche iPSC dagli esseri umani con malattie ereditarie o degenerative. Qui, segnaliamo l&#39;uso di una versione umanizzata di un singolo vettore lentivirale &quot;staminali cassetta cella&quot; per realizzare efficienti riprogrammazione dei fibroblasti della pelle normale o malati ottenuti dagli esseri umani di qualsiasi et&agrave;. Trasferimento simultaneo di tre o quattro fattori di riprogrammazione nelle cellule bersaglio umano utilizzando questo singolo vettore permette di derivazione umana iPSC contenente una singola integrazione virale soggetti ad accisa che su rimozione genera umano iPSC libero dei transgeni integrato. Come prova di principio, qui applichiamo questa strategia per generare &amp;gt; 100 linee di iPSC specifici per la malattia polmonare da individui con una variet&agrave; di malattie che colpiscono i compartimenti epiteliali, endoteliali o interstiziali del polmone, fibrosi cistica, &alpha;-1 antitripsina deficit correlati enfisema, sclerodermia e falciforme. Inoltre, abbiamo dimostrato che iPSC umani generati con questo approccio hanno la capacit&agrave; di differenziarsi robustamente in endoderma definitivo in vitro, tessuto precursore dello sviluppo degli epiteli polmonari.

Generazione del polmone privo di transgene specifici per la malattia umana indotta staminali pluripotenti usando una cassetta singola ad accisa lentivirali stem cell.

&#x53D1;&#x5C55;&#x7684;&#x5B9E;&#x73B0;&#x540C;&#x65F6;&#x907F;&#x514D;&#x6C38;&#x4E45;&#x5B58;&#x5728;&#x7684;&#x91CD;&#x7F16;&#x7A0B;&#x8F6C;&#x57FA;&#x56E0;&#x7EC6;&#x80DE;&#x7684;&#x9AD8;&#x6548;&#x91CD;&#x7F16;&#x7A0B;&#x7684;&#x65B9;&#x6CD5;&#x8868;&#x793A;&#x8D70;&#x5411;&#x4E34;&#x5E8A;&#x7528;&#x9014;&#xFF0C;&#x4F8B;&#x5982;&#x75BE;&#x75C5;&#x5EFA;&#x6A21;&#x6216;&#x91CD;&#x7EC4;&#x7597;&#x6CD5;&#x8BF1;&#x5BFC;&#x7684;&#x591A;&#x529F;&#x80FD;&#x5E72;&#x7EC6;&#x80DE; (&#x5BA1;&#x9605;) &#x4F7F;&#x7528;&#x7684;&#x4E00;&#x4E2A;&#x5173;&#x952E;&#x6B65;&#x9AA4;&#x3002;&#x5C3D;&#x7BA1;&#x751F;&#x6210;&#x673A;&#x4F53;&#x529F;&#x80FD;&#x514D;&#x8D39;&#x7684;&#x91CD;&#x7F16;&#x7A0B;&#x8F6C;&#x57FA;&#x56E0;&#x5C0F;&#x9F20;&#x7EC6;&#x80DE;&#x6216;&#x65B0;&#x751F;&#x513F;&#x6B63;&#x5E38;&#x7684;&#x4EBA;&#x4F53;&#x7EC4;&#x7EC7;&#x5B58;&#x5728;&#x7684;&#x51E0;&#x79CD;&#x65B9;&#x6CD5;&#xFF0C;&#x5145;&#x5206;&#x6709;&#x6548;&#x7684;&#x7F16;&#x7A0B;&#x7CFB;&#x7EDF;&#x4ECD;&#x7136;&#x9700;&#x8981;&#x5B9E;&#x73B0;&#x7684;&#x5177;&#x4F53;&#x75BE;&#x75C5;&#x673A;&#x4F53;&#x529F;&#x80FD;&#x4ECE;&#x7EE7;&#x627F;&#x6216;&#x9000;&#x884C;&#x6027;&#x75BE;&#x75C5;&#x4E0E;&#x4EBA;&#x7C7B;&#x7684;&#x666E;&#x904D;&#x63A8;&#x5BFC;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x62A5;&#x544A;&#x4F7F;&#x7528;&#x7684;&#x5355;&#x4E00;&quot;&#x5E72;&#x7EC6;&#x80DE;&#x5361;&#x5E26;&quot;&#x8F7D;&#x4F53;&#x6765;&#x5B8C;&#x6210;&#x6B63;&#x5E38;&#x6216;&#x75C5;&#x53D8;&#x7684;&#x76AE;&#x80A4;&#x6210;&#x7EA4;&#x7EF4;&#x7EC6;&#x80DE;&#x4ECE;&#x51E0;&#x4E4E;&#x4EFB;&#x4F55;&#x5E74;&#x9F84;&#x7684;&#x4EBA;&#x83B7;&#x5F97;&#x6709;&#x6548;&#x91CD;&#x7F16;&#x7A0B;&#x7684;&#x4EBA;&#x6027;&#x5316;&#x7248;&#x672C;&#x3002;&#x4E09;&#x4E2A;&#x6216;&#x56DB;&#x4E2A;&#x7F16;&#x7A0B;&#x56E0;&#x7D20;&#x7EB3;&#x5165;&#x4EBA;&#x7C7B;&#x76EE;&#x6807;&#x5355;&#x5143;&#x683C;&#x4F7F;&#x7528;&#x6B64;&#x5355;&#x77E2;&#x91CF;&#x7684;&#x540C;&#x65F6;&#x4F20;&#x8F93;&#x5141;&#x8BB8;&#x5305;&#x542B;&#x5355;&#x4E2A;&#x7684;&#x53EF;&#x62BD;&#x7A0E;&#x75C5;&#x6BD2;&#x6027;&#x4E00;&#x4F53;&#x5316;&#xFF0C;&#x53BB;&#x9664;&#x751F;&#x6210;&#x4EBA;&#x7C7B;&#x673A;&#x4F53;&#x529F;&#x80FD;&#x514D;&#x8D39;&#x7684;&#x7EFC;&#x5408;&#x8F6C;&#x57FA;&#x56E0;&#x4EBA;&#x7C7B;&#x673A;&#x4F53;&#x529F;&#x80FD;&#x7684;&#x63A8;&#x5BFC;&#x3002;&#x4F5C;&#x4E3A;&#x8BC1;&#x660E;&#x7684;&#x539F;&#x5219;&#xFF0C;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x8FD0;&#x7528;&#x8FD9;&#x4E2A;&#x7B56;&#x7565;&#x6765;&#x751F;&#x6210; &gt; 100 &#x80BA;&#x75BE;&#x75C5;&#x7279;&#x5B9A;&#x673A;&#x4F53;&#x529F;&#x80FD;&#x884C;&#x4ECE;&#x5404;&#x79CD;&#x5404;&#x6837;&#x7684;&#x5F71;&#x54CD;&#x80BA;&#xFF0C;&#x5305;&#x62EC;&#x56CA;&#x6027;&#x7EA4;&#x7EF4;&#x5316;&#x3001; &alpha;-1 antitrypsin &#x7F3A;&#x4E4F;&#x6709;&#x5173;&#x80BA;&#x6C14;&#x80BF;&#x3001; &#x786C;&#x76AE;&#x75C5;&#x3001; &#x9570;&#x72B6;&#x7EC6;&#x80DE;&#x75C5;&#x7684;&#x4E0A;&#x76AE;&#x3001; &#x5185;&#x76AE;&#x7EC6;&#x80DE;&#xFF0C;&#x6216;&#x95F4;&#x8D28;&#x8F66;&#x53A2;&#x7684;&#x75BE;&#x75C5;&#x7684;&#x4E2A;&#x4EBA;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x6211;&#x4EEC;&#x8868;&#x660E;&#xFF0C;&#x4EBA;&#x7C7B;&#x7684;&#x673A;&#x4F53;&#x529F;&#x80FD;&#x4E0E;&#x6B64;&#x751F;&#x6210;&#x7684;&#x65B9;&#x6CD5;&#x6709;&#x80FD;&#x529B;&#x5F3A;&#x52B2;&#x5206;&#x5316;&#x6210;&#x5185;&#x80DA;&#x6700;&#x7EC8;&#x5C42;&#x4F53;&#x5916;&#xFF0C;&#x80BA;&#x4E0A;&#x76AE;&#x7EC6;&#x80DE;&#x7684;&#x53D1;&#x80B2;&#x524D;&#x4F53;&#x7EC4;&#x7EC7;&#x3002;

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The development of methods to achieve efficient reprogramming of human cells while avoiding the permanent presence of reprogramming transgenes represents a critical step toward the use of induced pluripotent stem cells (iPSC) for clinical purposes, such as disease modeling or reconstituting therapies. Although several methods exist for generating iPSC free of reprogramming transgenes from mouse cells or neonatal normal human tissues, a sufficiently efficient reprogramming system is still needed to achieve the widespread derivation of disease-specific iPSC from humans with inherited or degenerative diseases. Here, we report the use of a humanized version of a single lentiviral "stem cell cassette" vector to accomplish efficient reprogramming of normal or diseased skin fibroblasts obtained from humans of virtually any age. Simultaneous transfer of either three or four reprogramming factors into human target cells using this single vector allows derivation of human iPSC containing a single excisable viral integration that on removal generates human iPSC free of integrated transgenes. As a proof of principle, here we apply this strategy to generate >100 lung disease-specific iPSC lines from individuals with a variety of diseases affecting the epithelial, endothelial, or interstitial compartments of the lung, including cystic fibrosis, Î±-1 antitrypsin deficiency-related emphysema, scleroderma, and sickle-cell disease. Moreover, we demonstrate that human iPSC generated with this approach have the ability to robustly differentiate into definitive endoderm in vitro, the developmental precursor tissue of lung epithelia.

Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette.

Le d&eacute;veloppement de m&eacute;thodes pour atteindre efficace de reprogrammation des cellules humaines tout en &eacute;vitant la pr&eacute;sence permanente de reprogrammation des transg&egrave;nes repr&eacute;sente une &eacute;tape cruciale vers l&#39;utilisation des cellules souches pluripotentes induites (iPSC) &agrave; des fins cliniques, telles que la maladie de mod&eacute;lisation ou de reconstituer des th&eacute;rapies. Bien que plusieurs m&eacute;thodes existent pour g&eacute;n&eacute;rer l&#39;iPSC libre de reprogrammation des transg&egrave;nes de cellules de souris ou de tissus humains normaux n&eacute;onatales, un syst&egrave;me suffisamment efficace de reprogrammation est encore n&eacute;cessaire pour atteindre la d&eacute;rivation g&eacute;n&eacute;ralis&eacute;e de l&#39;iPSC sp&eacute;cifiques &agrave; certaines maladies chez les humains atteints de maladies h&eacute;r&eacute;ditaires ou d&eacute;g&eacute;n&eacute;ratives. Nous rapportons ici, l&#39;utilisation d&#39;une version humanis&eacute;e d&#39;un unique vecteur lentiviral &laquo; cassette de cellules de la tige &raquo; pour accomplir une reprogrammation efficace des fibroblastes de peau normale ou malades provenant des humains de pratiquement n&#39;importe quel &acirc;ge. Transfert simultan&eacute; de trois ou quatre facteurs de reprogrammation en cellules de cible humaine &agrave; l&#39;aide de ce vecteur unique permet de d&eacute;rivation de l&#39;iPSC humain contenant une seule int&eacute;gration virale soumis &agrave; accises g&eacute;n&eacute;rant iPSC humain libre de transg&egrave;nes int&eacute;gr&eacute;s sur l&#39;enl&egrave;vement. Comme une preuve de principe, ici, nous appliquons cette strat&eacute;gie pour g&eacute;n&eacute;rer &gt; 100 lignes d&#39;iPSC sp&eacute;cifiques &agrave; une maladie pulmonaire d&#39;individus atteints de diverses maladies affectant les compartiments &eacute;pith&eacute;liales, endoth&eacute;liales ou interstitielles du poumon, y compris de la fibrose kystique, &alpha; 1 antitrypsine li&eacute;es &agrave; une carence en l&#39;emphys&egrave;me, la scl&eacute;rodermie et dr&eacute;panocytose. De plus, nous d&eacute;montrons qu&#39;iPSC humaine g&eacute;n&eacute;r&eacute;e avec cette approche ont la capacit&eacute; de diff&eacute;rencier robuste en d&eacute;finitive endoderme in vitro, le tissu pr&eacute;curseur du d&eacute;veloppement de l&#39;&eacute;pith&eacute;lium pulmonaire.

G&eacute;n&eacute;ration de poumon transg&egrave;ne libres des cellules souches sp&eacute;cifiques &agrave; certaines maladies humaines pluripotentes induites &agrave; l&#39;aide d&#39;une seule cellule de tige des g&egrave;nes soumis &agrave; accises cassette.

Die Entwicklung von Methoden zu erreichen, effiziente Reprogrammierung menschlicher Zellen ohne die st&auml;ndige Pr&auml;senz der Reprogrammierung transgene stellt einen entscheidenden Schritt in Richtung der Nutzung von induzierten pluripotenten Stammzellen (iPSC) f&uuml;r klinische Zwecke, wie Krankheit, modellieren oder Rekonstituierung der Therapien. Zwar es mehrere Methoden zum Generieren von iPSC frei gibt von Neuprogrammierung transgenen Maus Zellen oder neonatale normalen menschlichen Geweben, ist ein ausreichend effizientes Neuprogrammierung System erforderlich, um die weit verbreitete Ableitung des krankheitsspezifischen iPSC von Menschen mit ererbten oder degenerativen Krankheiten zu erreichen. Hier teilen wir Ihnen die Verwendung einer humanisierten Version eines einzigen Lentivirale &quot;Vorbau Zelle Kassette&quot; Vector, effiziente Neuprogrammierung der normalen oder kranken Hautfibroblasten gewonnenen Menschen nahezu jeden Alters zu erreichen. Gleichzeitige &Uuml;bertragung entweder drei oder vier Neuprogrammierung Faktoren in human Target-Zellen, die unter Verwendung dieser einzigen Vektor erm&ouml;glicht die Ableitung der menschlichen iPSC, enth&auml;lt eine einzelne verbrauchsteuerpflichtige virale-Integration, die &uuml;ber die Entfernung frei von integrierten transgenen menschlichen iPSC generiert. Als Beweis des Prinzips, hier wenden wir diese Strategie generiert &gt; 100 Lunge krankheitsspezifische iPSC-Linien von Personen, die mit einer Vielzahl von Krankheiten, die das Epithel, Endothel oder interstitielle F&auml;cher der Lunge, einschlie&szlig;lich Mukoviszidose, &alpha;-1-antitrypsin-Mangel-bezogene-Emphysem, Sklerodermie und Sichelzellenan&auml;mie. Dar&uuml;ber hinaus zeigen wir, dass menschliche iPSC generiert mit diesem Ansatz haben die F&auml;higkeit, robust in der endg&uuml;ltigen Endodermis in vitro der Entwicklungsbiologie Vorl&auml;ufer-Gewebe der Lunge Epithelien zu unterscheiden.

Generation von Transgen-freie Lunge krankheitsspezifische menschlichen induzierten pluripotenten Stammzellen, die mit eine einzigen verbrauchsteuerpflichtiger Lentivirale Stammzell-Kassette.

Transgenes &#x30EA;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30DF;&#x30F3;&#x30B0;&#x306E;&#x6052;&#x4E45;&#x7684;&#x306A;&#x5B58;&#x5728;&#x3092;&#x56DE;&#x907F;&#x3057;&#x306A;&#x304C;&#x3089;&#x52B9;&#x7387;&#x7684;&#x306A;&#x3072;&#x3068;&#x7D30;&#x80DE;&#x306E;&#x30EA;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30DF;&#x30F3;&#x30B0;&#x3092;&#x5B9F;&#x73FE;&#x3059;&#x308B;&#x65B9;&#x6CD5;&#x306E;&#x958B;&#x767A;&#x306F;&#x3001;&#x30E2;&#x30C7;&#x30EA;&#x30F3;&#x30B0;&#x3084;&#x30BB;&#x30E9;&#x30D4;&#x30FC;&#x306E;&#x518D;&#x69CB;&#x6210;&#x75BE;&#x60A3;&#x306A;&#x3069;&#x306E;&#x81E8;&#x5E8A;&#x76EE;&#x7684;&#x306E;&#x8A98;&#x5C0E;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE; (iPSC) &#x306E;&#x4F7F;&#x7528;&#x306B;&#x5411;&#x3051;&#x305F;&#x91CD;&#x8981;&#x306A;&#x30B9;&#x30C6;&#x30C3;&#x30D7;&#x3092;&#x8868;&#x3057;&#x307E;&#x3059;&#x3002;IPSC transgenes &#x30DE;&#x30A6;&#x30B9;&#x7D30;&#x80DE;&#x307E;&#x305F;&#x306F;&#x65B0;&#x751F;&#x5150;&#x306E;&#x6B63;&#x5E38;&#x30D2;&#x30C8;&#x7D44;&#x7E54;&#x304B;&#x3089;&#x30EA;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30DF;&#x30F3;&#x30B0;&#x306E;&#x81EA;&#x7531;&#x3092;&#x751F;&#x6210;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x65B9;&#x6CD5;&#x304C;&#x5B58;&#x5728;&#x3059;&#x308B;&#x304C;&#x3001;&#x5341;&#x5206;&#x306B;&#x52B9;&#x7387;&#x7684;&#x306A;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30E0;&#x3057;&#x76F4;&#x3059;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x306F;&#x307E;&#x3060;&#x75BE;&#x60A3;&#x7279;&#x7570; iPSC &#x4EBA;&#x9593;&#x7D99;&#x627F;&#x307E;&#x305F;&#x306F;&#x5909;&#x6027;&#x75BE;&#x60A3;&#x304B;&#x3089;&#x306E;&#x5E83;&#x7BC4;&#x306A;&#x5C0E;&#x51FA;&#x3092;&#x9054;&#x6210;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x5FC5;&#x8981;&#x3067;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x5B9F;&#x8CEA;&#x7684;&#x306B;&#x3042;&#x3089;&#x3086;&#x308B;&#x5E74;&#x9F62;&#x306E;&#x4EBA;&#x9593;&#x304B;&#x3089;&#x5F97;&#x3089;&#x308C;&#x305F;&#x6B63;&#x5E38;&#x307E;&#x305F;&#x306F;&#x60A3;&#x90E8;&#x306E;&#x76AE;&#x819A;&#x7DDA;&#x7DAD;&#x82BD;&#x7D30;&#x80DE;&#x306E;&#x52B9;&#x7387;&#x7684;&#x306A;&#x66F8;&#x304D;&#x63DB;&#x3048;&#x3092;&#x9054;&#x6210;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3001;&#x30B7;&#x30F3;&#x30B0;&#x30EB;&#x300C;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x30AB;&#x30BB;&#x30C3;&#x30C8;&#x300D;&#x30EC;&#x30F3;&#x30C1;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x30D9;&#x30AF;&#x30BF;&#x30FC;&#x306E;&#x30D2;&#x30C8;&#x5316;&#x30D0;&#x30FC;&#x30B8;&#x30E7;&#x30F3;&#x4F7F;&#x7528;&#x3092;&#x5831;&#x544A;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E; 1 &#x3064;&#x306E;&#x30D9;&#x30AF;&#x30C8;&#x30EB;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x4EBA;&#x9593;&#x306E;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8; &#x30BB;&#x30EB;&#x306B; 3 &#x3064;&#x307E;&#x305F;&#x306F; 4 &#x3064;&#x306E;&#x30EA;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30DF;&#x30F3;&#x30B0;&#x8981;&#x56E0;&#x306E;&#x540C;&#x6642;&#x8EE2;&#x9001;&#x306F;&#x9664;&#x53BB;&#x306B;&#x95A2;&#x3059;&#x308B;&#x4EBA;&#x9593; iPSC &#x7D71;&#x5408; transgenes &#x306E;&#x7121;&#x6599;&#x751F;&#x6210;&#x30B7;&#x30F3;&#x30B0;&#x30EB;&#x304C;&#x7269;&#x54C1;&#x7A0E;&#x8AB2;&#x7A0E;&#x5BFE;&#x8C61;&#x306E;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x7D71;&#x5408;&#x3092;&#x542B;&#x3080;&#x4EBA;&#x9593;&#x306E; iPSC &#x306E;&#x5C0E;&#x51FA;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;&#x539F;&#x5247;&#x306E;&#x8A3C;&#x62E0;&#x3068;&#x3057;&#x3066;&#x306F;&#x3001;&#x3053;&#x3053;&#x3067;&#x6211;&#x3005; &#x3092;&#x751F;&#x6210;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x3053;&#x306E;&#x6226;&#x7565;&#x3092;&#x9069;&#x7528;&#x76EE;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x3001;&#x4E0A;&#x76AE;&#x3084;&#x9593;&#x8CEA;&#x30B3;&#x30F3;&#x30D1;&#x30FC;&#x30C8;&#x30E1;&#x30F3;&#x30C8; &alpha; 1 antitrypsin &#x6B20;&#x4E4F;&#x95A2;&#x9023;&#x80BA;&#x6C17;&#x816B;&#x3001;&#x5F37;&#x76AE;&#x75C7;&#x3001;&#x56A2;&#x80DE;&#x6027;&#x7DDA;&#x7DAD;&#x75C7;&#x3001;&#x938C;&#x72B6;&#x8D64;&#x8840;&#x7403;&#x75C5;&#x3092;&#x542B;&#x3080;&#x80BA;&#x306B;&#x5F71;&#x97FF;&#x3092;&#x4E0E;&#x3048;&#x308B;&#x75C5;&#x6C17;&#x306E;&#x69D8;&#x3005; &#x306A;&#x500B;&#x4EBA;&#x304B;&#x3089;&#x306E; 100 &#x306E;&#x80BA;&#x75BE;&#x60A3;&#x7279;&#x7570; iPSC &#x884C;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x6211;&#x3005; &#x3053;&#x308C;&#x3092;&#x751F;&#x6210;&#x305D;&#x306E;&#x4EBA;&#x9593; iPSC &#x3092;&#x793A;&#x3059;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x306F;&#x9811;&#x5065;&#x6C7A;&#x5B9A;&#x7684;&#x306A;&#x80DA;&#x8449;&#x306B; in vitro &#x80BA;&#x4E0A;&#x76AE;&#x306E;&#x767A;&#x9054;&#x524D;&#x99C6;&#x4F53;&#x7D44;&#x7E54;&#x3092;&#x533A;&#x5225;&#x3059;&#x308B;&#x80FD;&#x529B;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

&#x5358;&#x4E00;&#x306E;&#x30EC;&#x30F3;&#x30C1; &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x5E79;&#x7D30;&#x80DE;&#x304C;&#x7269;&#x54C1;&#x7A0E;&#x8AB2;&#x7A0E;&#x5BFE;&#x8C61;&#x306E;&#x30AB;&#x30BB;&#x30C3;&#x30C8;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x7121;&#x6599;&#x306E; transgene &#x80BA;&#x75BE;&#x60A3;&#x7279;&#x7570;&#x3072;&#x3068;&#x8A98;&#x5C0E;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x4E16;&#x4EE3;&#x3002;

Transgenes &#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D;&#xC758; &#xC601;&#xC6D0;&#xD55C; &#xC874;&#xC7AC;&#xB97C; &#xD53C;&#xD558; &#xBA74; &#xC11C; &#xC778;&#xAC04; &#xC138;&#xD3EC;&#xC758; &#xD6A8;&#xC728;&#xC801;&#xC778; &#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D;&#xC744; &#xB2EC;&#xC131; &#xD558;&#xB294; &#xBC29;&#xBC95;&#xC758; &#xAC1C;&#xBC1C; &#xC9C8;&#xBCD1; &#xBAA8;&#xB378;&#xB9C1; &#xB610;&#xB294; &#xC7AC;&#xAD6C;&#xC131; &#xCE58;&#xB8CC; &#xB4F1;&#xC758; &#xC784;&#xC0C1; &#xC6A9;&#xB3C4;&#xB85C; &#xC720;&#xB3C4; &#xB9CC;&#xB2A5; &#xC904;&#xAE30; &#xC138;&#xD3EC; (iPSC)&#xC758; &#xC0AC;&#xC6A9;&#xC744; &#xD5A5;&#xD574; &#xC911;&#xC694; &#xD55C; &#xB2E8;&#xACC4;&#xB97C; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;. IPSC &#xB9C8;&#xC6B0;&#xC2A4; &#xC140; &#xB610;&#xB294; &#xC2E0;&#xC0DD;&#xC544; &#xC815;&#xC0C1;&#xC801;&#xC778; &#xC778;&#xAC04;&#xC758; &#xC870;&#xC9C1;&#xC5D0;&#xC11C; transgenes &#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D;&#xC758; &#xC790;&#xC720;&#xB97C; &#xC0DD;&#xC131; &#xD558;&#xAE30; &#xC704;&#xD55C; &#xC5EC;&#xB7EC; &#xAC00;&#xC9C0; &#xBC29;&#xBC95;&#xC774; &#xC788;&#xC5B4;&#xB3C4; &#xC0C1;&#xC18D; &#xB418;&#xAC70;&#xB098; &#xD1F4;&#xD589; &#xC131; &#xC9C8;&#xBCD1;&#xC744; &#xAC00;&#xC9C4; &#xC778;&#xAC04;&#xC5D0;&#xC11C; &#xC9C8;&#xBCD1; &#xAD00;&#xB828; Ipsc&#xC758; &#xAD11;&#xBC94;&#xC704; &#xD558; &#xAC8C; &#xC720;&#xB3C4; &#xB2EC;&#xC131; &#xD558;&#xAE30; &#xC704;&#xD574; &#xCDA9;&#xBD84;&#xD788; &#xD6A8;&#xC728;&#xC801;&#xC778; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC744; &#xB2E4;&#xC2DC; &#xB9CC;&#xB4E4; &#xC2DC;&#xC2A4;&#xD15C; &#xC5EC;&#xC804;&#xD788; &#xD544;&#xC694; &#xD569;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xAC00; &#xAC70;&#xC758; &#xBAA8;&#xB4E0; &#xB098;&#xC774;&#xC758; &#xC778;&#xAC04;&#xC5D0;&#xC11C; &#xC5BB;&#xC740; &#xC815;&#xC0C1; &#xB610;&#xB294; &#xBCD1;&#xC5D0; &#xAC78;&#xB9B0; &#xD53C;&#xBD80; &#xC12C;&#xC720; &#xC544; &#xC138;&#xD3EC;&#xC758; &#xD6A8;&#xC728;&#xC801;&#xC778; &#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D;&#xC744; &#xB2EC;&#xC131; &#xD558;&#xAE30; &#xC704;&#xD574; &#xB2E8;&#xC77C; lentiviral &quot;&#xC904;&#xAE30; &#xC138;&#xD3EC; &#xCE74;&#xC138;&#xD2B8;&quot; &#xBCA1;&#xD130;&#xC758; humanized &#xBC84;&#xC804;&#xC758; &#xC0AC;&#xC6A9;&#xC744; &#xBCF4;&#xACE0;. &#xC774; &#xB2E8;&#xC77C; &#xBCA1;&#xD130;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC778;&#xAC04;&#xC758; &#xB300;&#xC0C1; &#xC140;&#xC5D0; 3 &#xAC1C; &#xB610;&#xB294; 4 &#xAC1C;&#xC758; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC744; &#xB2E4;&#xC2DC; &#xB9CC;&#xB4E4; &#xC694;&#xC778;&#xC758; &#xB3D9;&#xC2DC; &#xC804;&#xC1A1; &#xC81C;&#xAC70;&#xC5D0; &#xC778;&#xAC04;&#xC758; iPSC &#xBB34;&#xB8CC; &#xD1B5;&#xD569;&#xB41C; transgenes&#xB97C; &#xC0DD;&#xC131; &#xD558;&#xB294; &#xB2E8;&#xC77C; excisable &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC131; &#xD1B5;&#xD569;&#xC744; &#xD3EC;&#xD568; &#xD558;&#xB294; &#xC778;&#xAC04;&#xC758; Ipsc&#xC758; &#xD30C;&#xC0DD; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC6D0;&#xB9AC;&#xC758; &#xC99D;&#xAC70;&#xB85C; &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC;&#xAC00; &#xC0DD;&#xC131; &#xD558;&#xB294;&#xC774; &#xC804;&#xB7B5; &#xC801;&#xC6A9; &amp;gt; &#xB0AD; &#xC131; &#xC12C;&#xC720; &#xC99D;, &alpha;-1 antitrypsin &#xACB0;&#xD54D; &#xAD00;&#xB828; &#xAE30;&#xC885;, scleroderma, &#xB0AB; &#xC138;&#xD3EC; &#xC9C8;&#xBCD1; &#xB4F1; &#xD3D0;&#xC758; &#xC0C1;&#xD53C;, &#xB0B4; &#xD53C;, &#xB610;&#xB294; &#xAC04; &#xC9C8; &#xC131; &#xAD6C;&#xD68D;&#xC5D0; &#xC601;&#xD5A5;&#xC744; &#xBBF8;&#xCE58;&#xB294; &#xC9C8;&#xBCD1;&#xC758; &#xB2E4;&#xC591; &#xD55C; &#xAC1C;&#xC778; &#xC73C;&#xB85C;&#xBD80;&#xD130; 100 &#xD3D0; &#xC9C8;&#xD658; &#xAD00;&#xB828; iPSC &#xB77C;&#xC778;. &#xB610;&#xD55C;, &#xC6B0;&#xB9AC;&#xB294; &#xC778;&#xAC04;&#xC758; Ipsc&#xC774; &#xC0DD;&#xC131; &#xBCF4;&#xC5EC; &#xC811;&#xADFC; &#xC5C4;&#xACA9; &#xD558; &#xAC8C; &#xCD5C;&#xC885; endoderm &#xC2DC;&#xD5D8;&#xAD00;&#xC5D0;&#xC11C; &#xD3D0; &#xC0C1;&#xD53C;&#xC758; &#xBC1C;&#xB2EC; &#xC804;&#xC870; &#xC870;&#xC9C1;&#xC73C;&#xB85C; &#xBD84;&#xD654; &#xD558;&#xB294; &#xAE30;&#xB2A5;&#xC774; &#xC788;&#xB2E4;.

Transgene &#xBB34;&#xB8CC; &#xD3D0; &#xC9C8;&#xD658; &#xAD00;&#xB828; &#xC778;&#xAC04; &#xC720;&#xB3C4; &#xB9CC;&#xB2A5; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xB2E8;&#xC77C; excisable lentiviral &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xCE74;&#xC138;&#xD2B8;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC0DD;&#xC131; &#xD569;&#xB2C8;&#xB2E4;.

El desarrollo de m&eacute;todos para lograr eficiente reprogramaci&oacute;n de c&eacute;lulas humanas, evitando la presencia permanente de reprogramaci&oacute;n transgenes representa un paso crucial hacia el uso de c&eacute;lulas pluripotentes inducidas (iPSC) fines cl&iacute;nicos, tales como enfermedad de modelado o terapias de reconstituci&oacute;n. Aunque existen varios m&eacute;todos para la generaci&oacute;n de iPSC de reprogramaci&oacute;n de los transgenes de las c&eacute;lulas de rat&oacute;n o tejidos humanos normales neonatales, todav&iacute;a es necesario un sistema de reprogramaci&oacute;n suficientemente eficiente para lograr la derivaci&oacute;n generalizada de iPSC enfermedad-espec&iacute;fica de los seres humanos con enfermedades hereditarias o degenerativas. Aqu&iacute;, Divulgamos el uso de una versi&oacute;n humanizada de un vector lentivirales &quot;cassette de la c&eacute;lula de v&aacute;stago&quot; solo para lograr la reprogramaci&oacute;n eficiente de fibroblastos de la piel normal o enfermo de los seres humanos de cualquier edad. Transferencia simult&aacute;nea de tres o cuatro factores reprogramaci&oacute;n en c&eacute;lulas de human target utilizando este vector solo permite derivaci&oacute;n de iPSC humano que contiene una &uacute;nica integraci&oacute;n viral los que en el retiro genera iPSC humana libre de transgenes integrados. Como una prueba del principio, aqu&iacute; aplicamos esta estrategia para generar &gt; 100 l&iacute;neas de iPSC espec&iacute;ficos de la enfermedad pulmonar de individuos con una variedad de enfermedades que afectan a los compartimientos epiteliales, endoteliales o intersticiales del pulm&oacute;n, como la fibrosis qu&iacute;stica, enfisema de relacionados con la deficiencia de &alpha;-1-antitripsina, esclerodermia y enfermedad de c&eacute;lulas falciformes. Por otra parte, demostramos que iPSC humano generado con este enfoque tienen la capacidad de distinguir s&oacute;lidamente en definitiva endodermo in vitro, el tejido del precursor del desarrollo del epitelio pulmonar.

Generaci&oacute;n de c&eacute;lulas pluripotentes inducidas humanas de la enfermedad-espec&iacute;fica pulm&oacute;n libre de transgenes usando un cassette de c&eacute;lula madre lentivirales los.

El uso de c&eacute;lulas madre pluripotentes humanas para los estudios de laboratorio y terapia celular est&aacute; obstaculizado por su capacidad potencial y limitada tumor-formaci&oacute;n para generar poblaciones puras de tipos de c&eacute;lulas diferenciadas in vitro. Para enfrentar estos problemas, hemos establecido l&iacute;neas de c&eacute;lulas progenitoras endod&eacute;rmico (EP) de c&eacute;lulas madre pluripotentes inducida y embrionarias humanas. Se establecieron condiciones de crecimiento optimizado que permite cerca ilimitada (&gt; 10(16)) EP celular auto-renovaci&oacute;n en que muestran un patr&oacute;n de expresi&oacute;n morfolog&iacute;a y gen caracter&iacute;stico del endodermo definitivo. Sobre la manipulaci&oacute;n de sus condiciones de cultivo in vitro o trasplante en ratones, clonalmente derivadas EP de c&eacute;lulas se diferencian en numerosos linajes endod&eacute;rmicos, incluyendo monohormonal &beta;-c&eacute;lulas pancre&aacute;ticas glucosa-sensible, hepatocitos y epitelio intestinal. Lo importante, las c&eacute;lulas de la EP son nontumorigenic en vivo. As&iacute;, las c&eacute;lulas de la EP representan una poderosa herramienta para estudiar la especificaci&oacute;n del endodermo y ofrecen una fuente potencialmente segura de tejidos de origen endod&eacute;rmico para terapias de trasplante.

L&iacute;neas de progenitor endodermal renueva a s&iacute; misma generadas a partir de c&eacute;lulas madre pluripotentes humanas.

&#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x7528;&#x4E8E;&#x5B9E;&#x9A8C;&#x5BA4;&#x7814;&#x7A76;&#x548C;&#x57FA;&#x4E8E;&#x7EC6;&#x80DE;&#x7597;&#x6CD5;&#x88AB;&#x53D7;&#x5230;&#x5176;&#x80BF;&#x7624;&#x5F62;&#x6210;&#x6F5C;&#x5728;&#x548C;&#x6709;&#x9650;&#x80FD;&#x529B;&#x6765;&#x751F;&#x6210;&#x4E0D;&#x540C;&#x7684;&#x7C7B;&#x578B;&#x7684;&#x7EC6;&#x80DE;&#x4F53;&#x5916;&#x7EAF;&#x4EBA;&#x53E3;&#x3002;&#x4E3A;&#x4E86;&#x89E3;&#x51B3;&#x8FD9;&#x4E9B;&#x95EE;&#x9898;&#xFF0C;&#x6211;&#x4EEC;&#x5EFA;&#x7ACB;&#x4E86;&#x4ECE;&#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x548C;&#x8BF1;&#x5BFC;&#x591A;&#x80FD;&#x5E72;&#x7EC6;&#x80DE;&#x80DA;&#x7956; (EP) &#x7EC6;&#x80DE;&#x682A;&#x3002;&#x4F18;&#x5316;&#x7684;&#x751F;&#x957F;&#x6761;&#x4EF6;&#x5EFA;&#x7ACB;&#x4E86;&#x4F7F;&#x9644;&#x8FD1;&#x65E0;&#x9650; (&gt; 10(16)) EP &#x7EC6;&#x80DE;&#x81EA;&#x6211;&#x66F4;&#x65B0;&#xFF0C;&#x5B83;&#x4EEC;&#x5C06;&#x663E;&#x793A;&#x7279;&#x5F81;&#x660E;&#x786E;&#x7684;&#x5185;&#x80DA;&#x5C42;&#x7684;&#x5F62;&#x6001;&#x5B66;&#x548C;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x6A21;&#x5F0F;&#x3002;&#x540E;&#x5176;&#x4F53;&#x5916;&#x57F9;&#x517B;&#x6761;&#x4EF6;&#x6216;&#x79FB;&#x690D;&#x5230;&#x5C0F;&#x9F20;&#x7684;&#x64CD;&#x7EB5;&#xFF0C;&#x65E0;&#x6027;&#x7E41;&#x6B96;&#x6D3E;&#x751F;&#x7684; EP &#x7EC6;&#x80DE;&#x5206;&#x5316;&#x4E3A;&#x8BB8;&#x591A;&#x80DA;&#x8C31;&#x7CFB;&#xFF0C;&#x5305;&#x62EC; monohormonal &#x8461;&#x8404;&#x7CD6;&#x53CD;&#x5E94;&#x80F0;&#x817A; &beta; &#x7EC6;&#x80DE;&#x3001; &#x809D;&#x7EC6;&#x80DE;&#x548C;&#x80A0;&#x4E0A;&#x76AE;&#x7EC6;&#x80DE;&#x3002;&#x91CD;&#x8981;&#x7684;&#x662F;&#xFF0C;EP &#x7EC6;&#x80DE;&#x662F;&#x6B63;&#x5E38;&#x7684;&#x4F53;&#x5185;&#x3002;&#x56E0;&#x6B64;&#xFF0C;EP &#x5355;&#x5143;&#x683C;&#x4EE3;&#x8868;&#x7814;&#x7A76;&#x5185;&#x80DA;&#x5C42;&#x89C4;&#x8303;&#x5E76;&#x63D0;&#x4F9B;&#x6D3E;&#x751F;&#x7684;&#x80DA;&#x7EC4;&#x7EC7;&#x79FB;&#x690D;&#x7597;&#x6CD5;&#x53EF;&#x80FD;&#x5B89;&#x5168;&#x6E90;&#x7684;&#x5F3A;&#x5927;&#x5DE5;&#x5177;&#x3002;

&#x4ECE;&#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x751F;&#x6210;&#x7684;&#x80DA;&#x7956;&#x884C;&#x81EA;&#x6211;&#x66F4;&#x65B0;&#x3002;

The use of human pluripotent stem cells for laboratory studies and cell-based therapies is hampered by their tumor-forming potential and limited ability to generate pure populations of differentiated cell types in vitro. To address these issues, we established endodermal progenitor (EP) cell lines from human embryonic and induced pluripotent stem cells. Optimized growth conditions were established that allow near unlimited (>10(16)) EP cell self-renewal in which they display a morphology and gene expression pattern characteristic of definitive endoderm. Upon manipulation of their culture conditions in vitro or transplantation into mice, clonally derived EP cells differentiate into numerous endodermal lineages, including monohormonal glucose-responsive pancreatic Î²-cells, hepatocytes, and intestinal epithelia. Importantly, EP cells are nontumorigenic in vivo. Thus, EP cells represent a powerful tool to study endoderm specification and offer a potentially safe source of endodermal-derived tissues for transplantation therapies.

Self-renewing endodermal progenitor lines generated from human pluripotent stem cells.

L&#39;utilisation de cellules souches pluripotentes humaines pour des &eacute;tudes en laboratoire et les th&eacute;rapies cellulaires est entrav&eacute;e par leur capacit&eacute; potentielle et limit&eacute;e formant des tumeurs pour g&eacute;n&eacute;rer des populations pures de types de cellules diff&eacute;renci&eacute;es in vitro. Pour r&eacute;soudre ces probl&egrave;mes, nous avons cr&eacute;&eacute; des lign&eacute;es de cellules prog&eacute;nitrices endodermiques (EP) de cellules souches humaines pluripotentes embryonnaires et induits. Avons &eacute;tabli les conditions de croissance optimis&eacute;e qui permet pr&egrave;s illimit&eacute; (&gt; 10(16)) EP cell autorenouvellement dans lequel ils affichent un mod&egrave;le d&#39;expression morphologie et g&egrave;ne caract&eacute;ristique de l&#39;endoderme d&eacute;finitif. Lors de la manipulation de leurs conditions de culture in vitro ou de la transplantation chez la souris, par clonage d&eacute;riv&eacute;es EP cellules se diff&eacute;rencient en nombreuses lign&eacute;es endodermiques, notamment monohormonal &beta;-cellules pancr&eacute;atiques glucose sensibles et des h&eacute;patocytes &eacute;pith&eacute;lium intestinal. Ce qui est important, les cellules de l&#39;EP sont tumorig&eacute;niques in vivo. Ainsi, les cellules de EP repr&eacute;sentent un outil puissant pour &eacute;tudier la sp&eacute;cification de l&#39;endoderme et offrir une source potentiellement s&ucirc;re des tissus endodermiques d&eacute;riv&eacute;es pour les th&eacute;rapies de transplantation.

Lignes de prog&eacute;niteurs endodermique autorenouvellement g&eacute;n&eacute;r&eacute;s &agrave; partir de cellules souches pluripotentes humaines.

Die Verwendung von menschlichen pluripotenten Stammzellen f&uuml;r Laborstudien und zellbasierte Therapien wird durch ihre Tumor-bildenden Potenzial und begrenzte F&auml;higkeit zum Generieren von reiner Populations von differenzierten Zelltypen in-vitro-behindert. Um diese Probleme anzugehen, haben wir endodermalen Vorl&auml;uferzellen (EP)-Zelllinien aus menschlichen embryonalen und induzierte pluripotente Stammzellen gegr&uuml;ndet. Optimale Wachstumsbedingungen entstanden, erlauben, in der N&auml;he von unbegrenzten (&gt; 10(16)) EP cell Selbsterneuerung in dem sie ein Morphologie und gen Ausdrucksmuster charakteristisch f&uuml;r definitive Endodermis anzeigen. Auf Manipulation ihrer in-vitro-Kultur-Zust&auml;nde oder Transplantation in M&auml;use differenzieren sich klonal abgeleitete EP Zellen in zahlreichen endodermalen Linien, einschlie&szlig;lich Monohormonal Glukose-responsive Pankreas-&beta;-Zellen, Hepatozyten und intestinale Epithelien. Wichtig ist die EP-Zellen in Vivo nontumorigenic. Daher stellen EP Zellen ein leistungsf&auml;higes Tool zum studieren Endodermis-Spezifikation und bieten eine potentiell sichere Quelle von endodermalen abgeleiteten Gewebe f&uuml;r Transplantation Therapien dar.

Erneuernde endodermalen Vorl&auml;uferzellen Linien aus menschlichen pluripotenten Stammzellen erzeugt.

L&#39;uso di cellule staminali pluripotenti umane per gli studi di laboratorio e le terapie cell-based &egrave; ostacolata dalla loro formazione tumorale potenziali e limitato la capacit&agrave; di generare pure popolazioni di tipi di cellule differenziate in vitro. Per risolvere questi problemi, abbiamo stabilito le linee di cellule progenitrici endodermico (EP) da cellule staminali pluripotenti indotte ed embrionale umano. Condizioni di crescita ottimizzata sono state stabilite vicino illimitato, che consentono (&amp;gt; 10(16)) EP cell self-renewal in cui mostrano un modello di espressione genica e morfologia caratteristico dell&#39;endoderma definitivo. Sulla manipolazione delle loro condizioni di coltura in vitro o trapianto in topi, clonandosi derivate EP le cellule si differenziano in numerosi lignaggi endodermico, compreso monohormonal reattivo-glucosio &beta;-cellule pancreatiche, epatociti ed epitelio intestinale. Importante, le cellule EP sono nontumorigenic in vivo. Cos&igrave;, le cellule EP rappresentano uno strumento potente per studiare specifiche endoderma e offrono una fonte potenzialmente sicura dei tessuti derivati da endodermico per terapie di trapianto.

Linee progenitrici endodermico autorinnovabile generate da cellule staminali pluripotenti umane.

&#x3072;&#x3068;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x4F7F;&#x7528;&#x7814;&#x7A76;&#x306E;&#x5B9F;&#x9A13;&#x5BA4;&#x304A;&#x3088;&#x3073;&#x30BB;&#x30EB; &#x30D9;&#x30FC;&#x30B9;&#x306E;&#x6CBB;&#x7642;&#x306E;&#x305F;&#x3081;&#x7D14;&#x7C8B;&#x306A;&#x4EBA;&#x53E3;&#x306E; in vitro &#x306E;&#x5206;&#x5316;&#x3057;&#x305F;&#x7D30;&#x80DE;&#x306E;&#x7A2E;&#x985E;&#x3092;&#x751F;&#x6210;&#x3059;&#x308B;&#x816B;&#x760D;&#x5F62;&#x6210;&#x9650;&#x5B9A;&#x7684;&#x6F5C;&#x5728;&#x80FD;&#x529B;&#x306B;&#x3088;&#x3063;&#x3066;&#x59A8;&#x3052;&#x3089;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x554F;&#x984C;&#x306B;&#x5BFE;&#x51E6;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x4EBA;&#x9593;&#x306E;&#x840C;&#x82BD;&#x671F;&#x304A;&#x3088;&#x3073;&#x8A98;&#x5C0E;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x304B;&#x3089;&#x80DA;&#x8449;&#x524D;&#x99C6; (EP) &#x30BB;&#x30EB;&#x30E9;&#x30A4;&#x30F3;&#x3092;&#x8A2D;&#x7ACB;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x6700;&#x9069;&#x5316;&#x3055;&#x308C;&#x305F;&#x6210;&#x9577;&#x6761;&#x4EF6;&#x304C;&#x78BA;&#x7ACB;&#x3055;&#x308C;&#x305F;&#x8A31;&#x53EF;&#x7121;&#x5236;&#x9650;&#x8FD1;&#x304F; &#xFF08;&#x76EE; 10(16)) EP &#x7D30;&#x80DE;&#x306E;&#x81EA;&#x5DF1;&#x8907;&#x88FD;&#x5F7C;&#x3089;&#x8868;&#x793A;&#x5F62;&#x614B;&#x3068;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x6C7A;&#x5B9A;&#x7684;&#x306A;&#x5185;&#x80DA;&#x8449;&#x306E;&#x7279;&#x6027;&#x3002;&#x5F7C;&#x3089;&#x306E; in vitro &#x57F9;&#x990A;&#x6761;&#x4EF6;&#x307E;&#x305F;&#x306F;&#x30DE;&#x30A6;&#x30B9;&#x3078;&#x79FB;&#x690D;&#x306E;&#x64CD;&#x4F5C;&#x6642;&#x306B;&#x3001;clonally &#x6D3E;&#x751F; EP &#x7D30;&#x80DE; monohormonal &#x306E;&#x30B0;&#x30EB;&#x30B3;&#x30FC;&#x30B9;&#x5FDC;&#x7B54;&#x6027;&#x81B5; &beta; &#x7D30;&#x80DE;&#x3001;&#x809D;&#x7D30;&#x80DE;&#x3001;&#x8178;&#x7BA1;&#x4E0A;&#x76AE;&#x7D30;&#x80DE;&#x306A;&#x3069;&#x6570;&#x591A;&#x304F;&#x306E;&#x5185;&#x80DA;&#x8449;&#x7CFB;&#x3078;&#x306E;&#x5206;&#x5316;&#x3057;&#x307E;&#x3059;&#x3002;&#x91CD;&#x8981;&#x306A;&#x306F;&#x3001;EP &#x7D30;&#x80DE;&#x306F;&#x751F;&#x4F53;&#x5185;&#x306E; nontumorigenic &#x3067;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;EP &#x30BB;&#x30EB;&#x5185;&#x80DA;&#x8449;&#x306E;&#x4ED5;&#x69D8;&#x306B;&#x95A2;&#x3059;&#x308B;&#x7814;&#x7A76;&#x3057;&#x3001;&#x5185;&#x80DA;&#x8449;&#x7531;&#x6765;&#x7D44;&#x7E54;&#x79FB;&#x690D;&#x6CBB;&#x7642;&#x306E;&#x305F;&#x3081;&#x306E;&#x6F5C;&#x5728;&#x7684;&#x306B;&#x5B89;&#x5168;&#x306A;&#x30BD;&#x30FC;&#x30B9;&#x3092;&#x63D0;&#x4F9B;&#x3059;&#x308B;&#x5F37;&#x529B;&#x306A;&#x30C4;&#x30FC;&#x30EB;&#x3092;&#x8868;&#x3057;&#x307E;&#x3059;&#x3002;

&#x3072;&#x3068;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x304B;&#x3089;&#x751F;&#x6210;&#x3055;&#x308C;&#x305F;&#x53D6;&#x3063;&#x3066;&#x4EE3;&#x308F;&#x308C;&#x308B;&#x5185;&#x76AE;&#x524D;&#x99C6;&#x884C;&#x3002;

&#xC2E4;&#xD5D8;&#xC2E4; &#xC5F0;&#xAD6C; &#xBC0F; &#xC138;&#xD3EC; &#xAE30;&#xBC18; &#xC694;&#xBC95;&#xC5D0; &#xB300; &#xD55C; &#xC778;&#xAC04;&#xC758; pluripotent &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC758; &#xC0AC;&#xC6A9;&#xC740; &#xADF8;&#xB4E4;&#xC758; &#xC885;&#xC591; &#xD615;&#xC131; &#xAC00;&#xB2A5;&#xC131; &#xBC0F; &#xC81C;&#xD55C; &#xB41C; &#xC0DD;&#xC131; &#xD558;&#xB294; &#xAE30;&#xB2A5; &#xC0DD;&#xCCB4; &#xC678;&#xC5D0;&#xC11C; &#xCC28;&#xBCC4;&#xD654; &#xB41C; &#xC138;&#xD3EC; &#xC720;&#xD615;&#xC758; &#xC21C;&#xC218;&#xD55C; &#xC778;&#xAD6C;&#xC5D0; &#xC758;&#xD574; &#xBC29;&#xD574;. &#xC774;&#xB7EC;&#xD55C; &#xBB38;&#xC81C;&#xB97C; &#xD574;&#xACB0; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC6B0;&#xB9AC;&#xB294; &#xC778;&#xAC04; &#xBC30;&#xC544; &#xBC0F; &#xC720;&#xB3C4; &#xB9CC;&#xB2A5; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; endodermal &#xC870;&#xC0C1; (EP) &#xC140; &#xB77C;&#xC778; &#xC124;&#xB9BD;. &#xCD5C;&#xC801;&#xD654; &#xB41C; &#xC131;&#xC7A5; &#xC870;&#xAC74; &#xADFC;&#xCC98; &#xBB34;&#xC81C;&#xD55C; &#xD5C8;&#xC6A9; &#xD558;&#xB294; &#xC124;&#xCE58; &#xB418;&#xC5C8;&#xB2E4; (&amp;gt; 10(16)) EP &#xC790;&#xAE30; &#xAC31;&#xC2E0; &#xD615;&#xD0DC;&#xD559; &#xBC0F; &#xC9C4; &#xC2DD; &#xD328;&#xD134; &#xD655;&#xC2E4; &#xD55C; endoderm&#xC758; &#xD2B9;&#xC131;&#xC744; &#xD45C;&#xC2DC; &#xD558;&#xB294; &#xC140;. &#xADF8;&#xB4E4;&#xC758; &#xBB38;&#xD654; &#xC870;&#xAC74; &#xCCB4; &#xC678; &#xB610;&#xB294; &#xC950;&#xC5D0; &#xC774;&#xC2DD;&#xC758; &#xC870;&#xC791;&#xC5D0; &#xB530;&#xB77C; clonally &#xD30C;&#xC0DD;&#xB41C; EP &#xC140; monohormonal &#xD3EC;&#xB3C4; &#xB2F9; &#xBC18;&#xC751; &#xCDCC; &#xC7A5; &beta;-&#xC138;&#xD3EC;, &#xC7A5;&#xB0B4; &#xC0C1;&#xD53C;, hepatocytes, &#xB4F1; &#xC218;&#xB9CE;&#xC740; endodermal &#xD608;&#xD1B5;&#xC73C;&#xB85C; &#xCC28;&#xBCC4;&#xD654;. &#xC911;&#xC694; &#xD55C; &#xAC83;&#xC740;, EP &#xC138;&#xD3EC;&#xB294; &#xBE44;&#xBCF4;&#xC5D0; nontumorigenic. &#xB530;&#xB77C;&#xC11C;, EP &#xC140; endoderm &#xBA85;&#xC138;&#xB97C; &#xACF5;&#xBD80; &#xD558; &#xACE0; endodermal&#xC5D0;&#xC11C; &#xD30C;&#xC0DD; &#xB41C; &#xC870;&#xC9C1;&#xC758; &#xC774;&#xC2DD; &#xC694;&#xBC95;&#xC5D0; &#xB300; &#xD55C; &#xC7A0;&#xC7AC;&#xC801;&#xC73C;&#xB85C; &#xC548;&#xC804;&#xD55C; &#xC18C;&#xC2A4;&#xB97C; &#xC81C;&#xACF5; &#xD558;&#xB294; &#xAC15;&#xB825;&#xD55C; &#xB3C4;&#xAD6C;&#xB97C; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;.

&#xD558;&#xB2F5;&#xB2C8;&#xB2E4; endodermal &#xC870;&#xC0C1; &#xC120; &#xC778;&#xAC04;&#xC758; pluripotent &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xC0DD;&#xC131; &#xB41C;.

&#x5510;&#x6C0F;&#x7EFC;&#x5408;&#x5F81; &#xFF08;21 &#x4E09;&#x4F53;&#x7EFC;&#x5408;&#x5F81;&#xFF0C;T21&#xFF09; &#x60A3;&#x8005;&#x6709;&#x4E00;&#x751F;&#x7684;&#x8840;&#x6DB2;&#x5B66;&#x5F02;&#x5E38;&#x3002;&#x65B0;&#x751F;&#x513F;&#x7ECF;&#x5E38;&#x8868;&#x73B0;&#x51FA;&#x5F02;&#x5E38;&#x8840;&#x7EC6;&#x80DE;&#x8BA1;&#x6570;&#x548C;&#x65E0;&#x6027;&#x7CFB;&#x7684;&#x75C5;&#x53F2;&#x3002;&#x80CE;&#x513F;&#x809D;&#x810F;&#x542B;&#x6709;&#x4EBA;&#x7C7B; T21 &#x6269;&#x5927; erythro &#x5DE8;&#x6838;&#x524D;&#x4F53;&#x589E;&#x6B96;&#x80FD;&#x529B;&#x5F97;&#x5230;&#x63D0;&#x9AD8;&#x3002;T21 &#x5BF9;&#x80DA;&#x80CE;&#x9020;&#x8840;&#x529F;&#x80FD;&#x7684;&#x6700;&#x65E9;&#x9636;&#x6BB5;&#x7684;&#x5F71;&#x54CD;&#x662F;&#x672A;&#x77E5;&#x7684;&#x51E0;&#x4E4E;&#x4E0D;&#x53EF;&#x80FD;&#x5728;&#x4EBA;&#x4F53;&#x7684;&#x68C0;&#x67E5;&#x3002;&#x6211;&#x4EEC;&#x4EFF;&#x7167; T21 &#x5375;&#x9EC4;&#x56CA;&#x9020;&#x8840;&#x529F;&#x80FD;&#x4F7F;&#x7528;&#x4EBA;&#x7C7B;&#x8BF1;&#x5BFC;&#x591A;&#x80FD;&#x5E72;&#x7EC6;&#x80DE; (&#x9B54;&#x9B3C;)&#x3002;&#x8840;&#x7956;&#x4EBA;&#x53E3;&#x4ECE; T21 &#x9B54;&#x9B3C;&#x751F;&#x6210;&#x51FA;&#x5E2D;&#x6B63;&#x5E38;&#x9891;&#x7387;&#xFF0C;&#x800C;&#x901A;&#x5E38;&#x6FC0;&#x589E;&#x3002;&#x7136;&#x800C;&#xFF0C;&#x5177;&#x6709;&#x589E;&#x5F3A;&#x7EA2;&#x7EC6;&#x80DE;&#x751F;&#x6210;&#x548C;&#x8F7B;&#x5EA6;&#x964D;&#x4F4E;&#xFF0C;&#x4F46;&#x6B63;&#x5E38;&#x7684;&#x5DE8;&#x6838;&#x7EC6;&#x80DE;&#x751F;&#x4EA7;&#x6539;&#x53D8;&#x4E86;&#x4ED6;&#x4EEC;&#x7684;&#x53D1;&#x5C55;&#x6F5C;&#x529B;&#x3002;&#x8FD9;&#x4E9B;&#x5F02;&#x5E38;&#x4E0E; T21 &#x80CE;&#x513F;&#x809D;&#x810F;&#xFF0C;&#x91CD;&#x53E0;&#xFF0C;&#x4F46;&#x4E5F;&#x53CD;&#x6620;&#x51FA;&#x91CD;&#x8981;&#x7684;&#x5DEE;&#x5F02;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x663E;&#x793A; T21 &#x8D4B;&#x4E88;&#x4E0D;&#x540C;&#x53D1;&#x80B2;&#x9636;&#x6BB5;-&#x548C; species-specific &#x9020;&#x8840;&#x7F3A;&#x9677;&#x3002;&#x66F4;&#x4E00;&#x822C;&#x5730;&#x8BF4;&#xFF0C;&#x6211;&#x4EEC;&#x8BF4;&#x660E;&#x4E86;&#x9B54;&#x9B3C;&#x5982;&#x4F55;&#x53EF;&#x4EE5;&#x6D1E;&#x5BDF;&#x6B63;&#x5E38;&#x548C;&#x75C5;&#x7406;&#x7684;&#x4EBA;&#x7C7B;&#x53D1;&#x5C55;&#x7684;&#x65E9;&#x671F;&#x9636;&#x6BB5;&#x3002;

21 &#x4E09;&#x4F53;&#x7EFC;&#x5408;&#x5F81;&#x76F8;&#x5173;&#x7684;&#x7F3A;&#x9677;&#xFF0C;&#x5728;&#x901A;&#x8FC7;&#x63ED;&#x793A;&#x4EBA;&#x7C7B;&#x539F;&#x59CB;&#x9020;&#x8840;&#x8BF1;&#x5BFC;&#x591A;&#x80FD;&#x5E72;&#x7EC6;&#x80DE;&#x3002;

Patients with Down syndrome (trisomy 21, T21) have hematologic abnormalities throughout life. Newborns frequently exhibit abnormal blood counts and a clonal preleukemia. Human T21 fetal livers contain expanded erythro-megakaryocytic precursors with enhanced proliferative capacity. The impact of T21 on the earliest stages of embryonic hematopoiesis is unknown and nearly impossible to examine in human subjects. We modeled T21 yolk sac hematopoiesis using human induced pluripotent stem cells (iPSCs). Blood progenitor populations generated from T21 iPSCs were present at normal frequency and proliferated normally. However, their developmental potential was altered with enhanced erythropoiesis and reduced myelopoiesis, but normal megakaryocyte production. These abnormalities overlap with those of T21 fetal livers, but also reflect important differences. Our studies show that T21 confers distinct developmental stage- and species-specific hematopoietic defects. More generally, we illustrate how iPSCs can provide insight into early stages of normal and pathological human development.

Trisomy 21-associated defects in human primitive hematopoiesis revealed through induced pluripotent stem cells.

Les patients avec le syndrome de Down (trisomie 21, T21) ont des anomalies h&eacute;matologiques tout au long de la vie. Nouveaux-n&eacute;s pr&eacute;sentent fr&eacute;quemment anormales num&eacute;ration globulaire et une pr&eacute;leuc&eacute;mie clonale. T21 humaine contiennent du foie foetal &eacute;largi &eacute;rythro-megakaryocytic pr&eacute;curseurs avec renforcement de la capacit&eacute; prolif&eacute;rative. L&#39;impact de la T21 sur les premiers stades de l&#39;h&eacute;matopo&iuml;&egrave;se embryonnaire est inconnu et presque impossible d&#39;examiner des sujets humains. Nous avons mod&eacute;lis&eacute; T21 sac vitellin h&eacute;matopo&iuml;&egrave;se utilisant des cellules souches humaines pluripotentes induites (CISP). Les populations de prog&eacute;niteurs de sang produites de T21 CISP &eacute;taient pr&eacute;sentes &agrave; la fr&eacute;quence normale et prolif&egrave;rent normalement. Toutefois, leur potentiel de d&eacute;veloppement a &eacute;t&eacute; modifi&eacute;e avec l&#39;&eacute;rythropo&iuml;&egrave;se accrue et myelopoiesis r&eacute;duit, mais la production normale m&eacute;gacaryocyte. Ces anomalies se chevauchent avec celles du foie foetal T21, mais &eacute;galement tenir compte des diff&eacute;rences importantes. Nos &eacute;tudes montrent que la T21 conf&egrave;re distincts du d&eacute;veloppement stade - et esp&egrave;ces des d&eacute;fauts h&eacute;matopo&iuml;&eacute;tique. Plus g&eacute;n&eacute;ralement, nous illustrons comment CISP peut fournir la perspicacit&eacute; dans les premiers stades du d&eacute;veloppement humain normal et pathologique.

Associ&eacute;es &agrave; la trisomie 21 vices humain h&eacute;matopo&iuml;&egrave;se primitif, r&eacute;v&eacute;l&eacute; par le biais de cellules souches pluripotentes induites.

Patienten mit Down-Syndrom (Trisomie 21, T21) haben h&auml;matologische Anomalien im ganzen Leben. Neugeborene zeigen h&auml;ufig abnorme Blut z&auml;hlt und einer klonalen Preleukemia. Menschliche T21 enthaltenen fetalen Leber erweitert Diastereomerie megakaryocytic Vorstufen mit verbesserten proliferative Kapazit&auml;t. Der Einfluss ist von T21 auf den fr&uuml;hesten Stadien der embryonalen Blutbildung unbekannt und fast unm&ouml;glich der Versuchspersonen untersucht. Wir modelliert T21 Dottersack Blutbildung mit menschlichen induzierten pluripotenten Stammzellen (iPSCs). Blut Stammvater Populationen aus T21 iPSCs generiert bei normaler Frequenz anwesend waren und normalerweise vermehren. Jedoch &auml;nderte sich ihre Entwicklungs-Potenzial mit verst&auml;rkte Erythropoese und reduzierte Myelopoiesis, sondern normale Megakaryozyt Produktion. Diese Anomalien &uuml;berschneiden sich mit denen des T21 fetalen Leber, aber auch wichtige Unterschiede widerspiegeln. Unsere Studien zeigen, dass T21 unterschiedliche Entwicklungs-Stufe - und Arten - spezifische h&auml;matopoetischen M&auml;ngel verleiht. Generell zeigen wir wie iPSCs Einblick in fr&uuml;hen Phasen der normalen und pathologischen menschlichen Entwicklung bieten kann.

Trisomie 21-assoziierte Defekte in menschlichen primitiven Blutbildung durch induzierte pluripotente Stammzellen offenbart.

I pazienti con sindrome di Down (trisomia 21, T21) presentano anomalie ematologiche per tutta la vita. I neonati presentano frequentemente anormale conta ematica e un preleukemia clonale. T21 umano fegato fetale contenga ampliato eritro-megakaryocytic precursori con maggiore capacit&agrave; proliferativa. L&#39;impatto di T21 sulle prime fasi dell&#39;emopoiesi embrionale &egrave; sconosciuto e quasi impossibile da esaminare nei soggetti umani. Abbiamo modellato T21 vitellino ematopoiesi utilizzando cellule staminali umane pluripotenti indotte (iPSCs). Sangue progenitore popolazioni generate da T21 iPSCs erano presenti alla frequenza normale e proliferavano normalmente. Tuttavia, il loro potenziale dello sviluppo &egrave; stata alterata con maggiore eritropoiesi e myelopoiesis ridotta, ma produzione dei megacariociti normali. Queste anomalie si sovrappongono con quelli di fegato fetale T21, ma anche riflettano differenze importanti. I nostri studi mostrano che il T21 conferisce difetti ematopoietiche a distinti dello sviluppo fase species - specifico e. Pi&ugrave; in generale, ci illustrano come iPSCs pu&ograve; fornire insight nelle prime fasi dello sviluppo umano normale e patologico.

Trisomia 21-associati difetti umani primitivi ematopoiesi rivelato attraverso le cellule staminali pluripotenti indotte.

&#x30C0;&#x30A6;&#x30F3;&#x75C7;&#x5019;&#x7FA4; &#xFF08;21 &#x30C8;&#x30EA;&#x30BD;&#x30DF;&#x30FC;&#x3001;T21) &#x60A3;&#x8005;&#x306E;&#x751F;&#x6DAF;&#x3092;&#x901A;&#x3058;&#x3066;&#x8840;&#x6DB2;&#x5B66;&#x7684;&#x7570;&#x5E38;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x65B0;&#x751F;&#x5150;&#x306F;&#x3001;&#x983B;&#x7E41;&#x306B;&#x7570;&#x5E38;&#x306A;&#x8840;&#x6DB2;&#x306E;&#x30AB;&#x30A6;&#x30F3;&#x30C8;&#x3068;&#x30AF;&#x30ED;&#x30FC;&#x30F3; preleukemia &#x3092;&#x5C55;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x80CE;&#x5150;&#x809D;&#x81D3;&#x3092;&#x542B;&#x3080;&#x4EBA;&#x9593;&#x306E; T21 &#x30A8;&#x30EA;&#x30B9;&#x30ED;&#x5DE8;&#x6838;&#x7403;&#x524D;&#x99C6;&#x4F53;&#x5F37;&#x5316;&#x306E;&#x5897;&#x6B96;&#x80FD;&#x529B;&#x3092;&#x62E1;&#x5927;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;T21 &#x306E;&#x80DA;&#x9020;&#x8840;&#x306E;&#x521D;&#x671F;&#x6BB5;&#x968E;&#x3078;&#x306E;&#x5F71;&#x97FF;&#x306F;&#x672A;&#x77E5;&#x306E;&#x4EBA;&#x9593;&#x306E;&#x984C;&#x6750;&#x3092;&#x691C;&#x8A0E;&#x3059;&#x308B;&#x307B;&#x307C;&#x4E0D;&#x53EF;&#x80FD;&#x3067;&#x3059;&#x3002;T21 &#x5375;&#x9EC4;&#x56A2;&#x9020;&#x8840;&#x306E;&#x3072;&#x3068;&#x8A98;&#x5C0E;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE; (ips &#x7D30;&#x80DE;) &#x306E;&#x4F7F;&#x7528;&#x3092;&#x30E2;&#x30C7;&#x30EB;&#x5316;&#x3002;T21 ips &#x7D30;&#x80DE;&#x304B;&#x3089;&#x751F;&#x6210;&#x3057;&#x305F;&#x8840;&#x6DB2;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x96C6;&#x56E3;&#x306F;&#x901A;&#x5E38;&#x306E;&#x5468;&#x6CE2;&#x6570;&#x3067;&#x5B58;&#x5728;&#x3057;&#x3066;&#x3044;&#x305F;&#x3057;&#x3001;&#x901A;&#x5E38;&#x5897;&#x6B96;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;&#x305D;&#x306E;&#x767A;&#x9054;&#x306E;&#x53EF;&#x80FD;&#x6027;&#x306F;&#x3001;&#x5F37;&#x5316;&#x3055;&#x308C;&#x305F;&#x8D64;&#x8840;&#x7403;&#x304A;&#x3088;&#x3073;&#x6E1B;&#x5C11;&#x3057;&#x305F; down &#x304C;&#x901A;&#x5E38;&#x5DE8;&#x6838;&#x7403;&#x751F;&#x7523;&#x5909;&#x66F4;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x7570;&#x5E38;&#x306F;&#x3001;T21 &#x80CE;&#x5150;&#x809D;&#x81D3;&#x3068;&#x91CD;&#x8907;&#x304C;&#x307E;&#x305F;&#x91CD;&#x8981;&#x306A;&#x9055;&#x3044;&#x3092;&#x53CD;&#x6620;&#x3057;&#x307E;&#x3059;&#x3002;&#x79C1;&#x305F;&#x3061;&#x306E;&#x7814;&#x7A76; T21 &#x7570;&#x306A;&#x308B;&#x767A;&#x9054;&#x6BB5;&#x968E;- &#x3068;&#x7A2E;&#x30FB;&#x7A2E;&#x9020;&#x8840;&#x6B20;&#x9665;&#x3092;&#x4E0E;&#x3048;&#x308B;. &#x3057;&#x307E;&#x3059;&#x3002;&#x4E00;&#x822C;&#x7684;&#x306B;&#x3001;ips &#x7D30;&#x80DE;&#x304C;&#x6B63;&#x5E38;&#x304A;&#x3088;&#x3073;&#x75C5;&#x7684;&#x3072;&#x3068;&#x958B;&#x767A;&#x306E;&#x521D;&#x671F;&#x6BB5;&#x968E;&#x3078;&#x306E;&#x6D1E;&#x5BDF;&#x529B;&#x3092;&#x63D0;&#x4F9B;&#x3059;&#x308B;&#x65B9;&#x6CD5;&#x3092;&#x8AAC;&#x660E;&#x3057;&#x307E;&#x3059;&#x3002;

21 &#x30C8;&#x30EA;&#x30BD;&#x30DF;&#x30FC;&#x95A2;&#x9023;&#x6B20;&#x9665;&#x4EBA;&#x9593;&#x306E;&#x539F;&#x59CB;&#x9020;&#x8840;&#x306E;&#x8A98;&#x5C0E;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;

Choroideremia (CHM) is an X- linked retinal degeneration that is symptomatic in the 1(st) or 2(nd) decade of life causing nyctalopia and loss of peripheral vision. The disease progresses through mid-life, when most patients become blind. CHM is a favorable target for gene augmentation therapy, as the disease is due to loss of function of a protein necessary for retinal cell health, Rab Escort Protein 1 (REP1).The CHM cDNA can be packaged in recombinant adeno-associated virus (rAAV), which has an established track record in human gene therapy studies, and, in addition, there are sensitive and quantitative assays to document REP1 activity. An animal model that accurately reflects the human condition is not available. In this study, we tested the ability to restore REP1 function in personalized in vitro models of CHM: lymphoblasts and induced pluripotent stems cells (iPSCs) from human patients. The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified. Delivery of the CHM cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking. The gene transfer is efficient and the preliminary safety data are encouraging. These studies pave the way for a human clinical trial of gene therapy for CHM.

AAV-mediated gene therapy for choroideremia: preclinical studies in personalized models.

Diamond Blackfan anemia (DBA) is a congenital disorder with erythroid (Ery) hypoplasia and tissue morphogenic abnormalities. Most DBA cases are caused by heterozygous null mutations in genes encoding ribosomal proteins. Understanding how haploinsufficiency of these ubiquitous proteins causes DBA is hampered by limited availability of tissues from affected patients. We generated induced pluripotent stem cells (iPSCs) from fibroblasts of DBA patients carrying mutations in RPS19 and RPL5. Compared with controls, DBA fibroblasts formed iPSCs inefficiently, although we obtained 1 stable clone from each fibroblast line. RPS19-mutated iPSCs exhibited defects in 40S (small) ribosomal subunit assembly and production of 18S ribosomal RNA (rRNA). Upon induced differentiation, the mutant clone exhibited globally impaired hematopoiesis, with the Ery lineage affected most profoundly. RPL5-mutated iPSCs exhibited defective 60S (large) ribosomal subunit assembly, accumulation of 12S pre-rRNA, and impaired erythropoiesis. In both mutant iPSC lines, genetic correction of ribosomal protein deficiency via complementary DNA transfer into the "safe harbor" AAVS1 locus alleviated abnormalities in ribosome biogenesis and hematopoiesis. Our studies show that pathological features of DBA are recapitulated by iPSCs, provide a renewable source of cells to model various tissue defects, and demonstrate proof of principle for genetic correction strategies in patient stem cells.

Ribosomal and hematopoietic defects in induced pluripotent stem cells derived from Diamond Blackfan anemia patients.

Induced pluripotent stem cells (iPSCs) hold great promise for modeling human hematopoietic diseases. However, intrinsic variability in the capacities of different iPSC lines for hematopoietic development complicates comparative studies and is currently unexplained. We created and analyzed 3 separate iPSC clones from fibroblasts of 3 different normal individuals using a standardized approach that included excision of integrated reprogramming genes by Cre-Lox mediated recombination. Gene expression profiling and hematopoietic differentiation assays showed that independent lines from the same individual were generally more similar to one another than those from different individuals. However, one iPSC line (WT2.1) exhibited a distinctly different gene expression, proliferation rate, and hematopoietic developmental potential relative to all other iPSC lines. This "outlier" clone also acquired extensive copy number variations (CNVs) during reprogramming, which may be responsible for its divergent properties. Our data indicate how inherent and acquired genetic differences can influence iPSC properties, including hematopoietic potential.

Clonal genetic and hematopoietic heterogeneity among human-induced pluripotent stem cell lines.

Megakaryocyte-specific transgene expression in patient-derived induced pluripotent stem cells (iPSCs) offers a new approach to study and potentially treat disorders affecting megakaryocytes and platelets. By using a Gp1ba promoter, we developed a strategy for achieving a high level of protein expression in human megakaryocytes. The feasibility of this approach was demonstrated in iPSCs derived from two patients with Glanzmann thrombasthenia (GT), an inherited platelet disorder caused by mutations in integrin αIIbβ3. Hemizygous insertion of Gp1ba promoter-driven human αIIb complementary DNA into the AAVS1 locus of iPSCs led to high αIIb messenger RNA and protein expression and correction of surface αIIbβ3 in megakaryocytes. Agonist stimulation of these cells displayed recovery of integrin αIIbβ3 activation. Our findings demonstrate a novel approach to studying human megakaryocyte biology as well as functional correction of the GT defect, offering a potential therapeutic strategy for patients with diseases that affect platelet function.

High-level transgene expression in induced pluripotent stem cell-derived megakaryocytes: correction of Glanzmann thrombasthenia.

The Wnt gene family consists of structurally related genes encoding secreted signaling molecules that have been implicated in many developmental processes, including regulation of cell fate and patterning during embryogenesis. Previously, we found that Wnt signaling is required for primitive or yolk sac-derived-erythropoiesis using the murine embryonic stem cell (ESC) system. Here, we examine the effect of Wnt signaling on the formation of early hematopoietic progenitors derived from human ESCs. The first hematopoietic progenitor cells in the human ESC system express the pan-hematopoietic marker CD41 and the erythrocyte marker, glycophorin A or CD235. We have developed a novel serum-free, feeder-free, adherent differentiation system that can efficiently generate large numbers of CD41+CD235+ cells. We demonstrate that this cell population contains progenitors not just for primitive erythroid and megakaryocyte cells but for the myeloid lineage as well and term this population the primitive common myeloid progenitor (CMP). Treatment of mesoderm-specified cells with Wnt3a led to a loss of hematopoietic colony-forming ability while the inhibition of canonical Wnt signaling with DKK1 led to an increase in the number of primitive CMPs. Canonical Wnt signaling also inhibits the expansion and/or survival of primitive erythrocytes and megakaryocytes, but not myeloid cells, derived from this progenitor population. These findings are in contrast to the role of Wnt signaling during mouse ESC differentiation and demonstrate the importance of the human ESC system in studying species-specific differences in development.

The negative impact of Wnt signaling on megakaryocyte and primitive erythroid progenitors derived from human embryonic stem cells.

Human pluripotent stem cells offer a powerful system to study human biology and disease. Here, we report a system to both express transgenes specifically in ES cell derived hematopoietic cells and knockdown gene expression stably throughout the differentiation of ES cells. We characterize a CD43 promoter construct that when inserted into the AAVS1 "safe harbor" locus utilizing a zinc finger nuclease specifically drives GFP expression in hematopoietic cells derived from a transgenic ES cell line and faithfully recapitulates endogenous CD43 expression. In addition, using the same gene targeting strategy we demonstrate that constitutive expression of short hairpin RNAs within a microRNA backbone can suppress expression of PU.1, an important regulator of myeloid cell development. We show that PU.1 knockdown cell lines display an inhibition in myeloid cell formation and skewing towards erythroid development. Overall, we have generated a powerful system to track hematopoietic development from pluripotent stem cells and study gene function through hematopoietic specific gene expression and constitutive gene knockdown.

Utilization of the AAVS1 safe harbor locus for hematopoietic specific transgene expression and gene knockdown in human ES cells.

This chapter describes a two-dimensional "monolayer" system for differentiating human pluripotent stem cells (PSCs) into "primitive" hematopoietic progenitor cells (HPCs) resembling those produced in vivo by the early embryonic yolk sac. This experimental system utilizes defined conditions without serum or feeder cells. Cytokines are added sequentially to stimulate the formation of mesoderm and its subsequent patterning to hematopoietic progenitors. The HPCs produced by this protocol have multi-lineage potential (erythroid, megakaryocyte, and myeloid) and can be isolated as a homogeneous population for use in standard hematopoietic studies including liquid expansion to mature lineages and colony assays. In addition, the HPCs can be cryopreserved for distribution or analysis at later times. The HPCs generated by this protocol have been used successfully to better define intrinsic variation in hematopoietic potential between different PSC lines and to model human hematopoietic diseases using patient-derived induced pluripotent stem cells.

ArticleTotal : 2	Year
Publication title Cited by 80	2012
Publication title Cited by 18	2014

Article	Year
Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette. Stem cells (Dayton, Ohio)\| PubMed ID: 20715179	2010
Self-renewing endodermal progenitor lines generated from human pluripotent stem cells. Cell stem cell\| PubMed ID: 22482503	2012
Trisomy 21-associated defects in human primitive hematopoiesis revealed through induced pluripotent stem cells. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 23045704	2012
AAV-mediated gene therapy for choroideremia: preclinical studies in personalized models. PloS one\| PubMed ID: 23667438	2013
Ribosomal and hematopoietic defects in induced pluripotent stem cells derived from Diamond Blackfan anemia patients. Blood\| PubMed ID: 23744582	2013
Clonal genetic and hematopoietic heterogeneity among human-induced pluripotent stem cell lines. Blood\| PubMed ID: 23940280	2013
High-level transgene expression in induced pluripotent stem cell-derived megakaryocytes: correction of Glanzmann thrombasthenia. Blood\| PubMed ID: 24335497	2014
The negative impact of Wnt signaling on megakaryocyte and primitive erythroid progenitors derived from human embryonic stem cells. Stem cell research\| PubMed ID: 24412757	2014
Utilization of the AAVS1 safe harbor locus for hematopoietic specific transgene expression and gene knockdown in human ES cells. Stem cell research\| PubMed ID: 24631742	2014
Hematopoietic differentiation of pluripotent stem cells in culture. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 25062629	2014

Jason A. Mills

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