Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 ...

Boston University and Boston Medical Center

Boston University School of Medicine

&#x7EE7;&#x627F;&#x4EBA;&#x7C7B; &alpha; 1-antitrypsin (AAT) &#x57FA;&#x56E0;&#x7A81;&#x53D8;&#x5BFC;&#x81F4; AAT &#x86CB;&#x767D;&#x4E0D;&#x8DB3;&#x5FAA;&#x73AF;&#x6C34;&#x5E73;&#x548C;&#x53D1;&#x5C55;&#x4E2D;&#x56FD;&#x5BB6;&#x80BA;&#x6C14;&#x80BF;&#x7684;&#x503E;&#x5411;&#x3002;&#x4E2A;&#x4EBA;&#x5B66;&#x80FD;&#x6D4B;&#x9A8C;&#x4E2D;&#x6709;&#x7F3A;&#x9677;&#x7684;&#x57FA;&#x56E0;&#x6CBB;&#x7597;&#x662F;&#x4E00;&#x4E2A;&#x6709;&#x5438;&#x5F15;&#x529B;&#x7684;&#x76EE;&#x6807;&#xFF0C;&#x56E0;&#x4E3A;&#x4EFB;&#x4F55;&#x80FD;&#x591F;&#x5206;&#x6CCC; AAT &#x7684;&#x5355;&#x5143;&#x683C;&#x7C7B;&#x578B;&#x6B63;&#x5E38; AAT &#x57FA;&#x56E0;&#x8F6C;&#x5165;&#x5E94;&#x626D;&#x8F6C;&#x4E0D;&#x8DB3;&#x5B66;&#x80FD;&#x6D4B;&#x9A8C;&#x6C34;&#x5E73;&#x548C;&#x8870;&#x51CF;&#x7684;&#x80BA;&#x90E8;&#x75BE;&#x75C5;&#x7684;&#x7814;&#x7A76;&#x8FDB;&#x5C55;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x63D0;&#x51FA;&#x57FA;&#x4E8E; lentivirally &#x8F6C;&#x5BFC;&#x9020;&#x8840;&#x5E72;&#x7EC6;&#x80DE; (&#x661F;) &#x79FB;&#x690D; AAT &#x57FA;&#x56E0;&#x8F6C;&#x79FB;&#x7684;&#x4E00;&#x79CD;&#x65B9;&#x6CD5;&#x3002;&#x6211;&#x4EEC;&#x5F00;&#x53D1;&#x4E86;&#x4E00;&#x79CD;&#x65B0;&#x578B;&#x7684;&#x53CC;&#x542F;&#x52A8;&#x5B50;&#x6162;&#x75C5;&#x6BD2;&#x7CFB;&#x7EDF;&#xFF0C;&#x4F20;&#x8F93;&#x6B63;&#x5E38;&#x4EBA;&#x7C7B; AAT &#x57FA;&#x56E0;&#x4EE5;&#x53CA;&#x8367;&#x5149;&#x589E;&#x5165;&#x5C0F;&#x9F20;&#x661F;&#x8DDF;&#x8E2A;&quot;&#x8BB0;&#x8005;&#x57FA;&#x56E0;&quot;&#x3002;&#x79FB;&#x690D; 3000 &#x7684;&#x8F6C;&#x5BFC;&#x661F;&#x4E3A;&#x8F90;&#x7167;&#x7684;&#x9F20;&#x6807;&#x6536;&#x4EF6;&#x4EBA;&#x540E;&#xFF0C;&#x6211;&#x4EEC;&#x8868;&#x73B0;&#x51FA;&#x8FD9;&#x4E24;&#x4E2A;&#x57FA;&#x56E0;&#x5728;&#x5C0F;&#x9F20;&#x4F53;&#x5185;&#x81F3;&#x5C11; 31 &#x5468;&#x540C;&#x65F6;&#x548C;&#x6301;&#x7EED;&#x7CFB;&#x7EDF;&#x6027;&#x7684;&#x8868;&#x8FBE;&#x3002;&#x672C;&#x8BAE;&#x5B9A;&#x4E66;&#x8F6C;&#x67D3;&#x5E72;&#x7EC6;&#x80DE;&#x4FDD;&#x6301;&#x8111;&#x795E;&#x7ECF;&#x3001; &#x81EA;&#x6211;&#x66F4;&#x65B0;&#x7684;&#x6F5C;&#x529B;&#x548C;&#x80FD;&#x529B;&#x6765;&#x91CD;&#x5EFA;&#x5C0F;&#x5B66;&#x548C;&#x4E2D;&#x5B66;&#x7684;&#x53D7;&#x52A9;&#x4EBA;&#x7684;&#x9020;&#x8840;&#x7CFB;&#x7EDF;&#x3002;&#x8FD9;&#x4E2A;&#x57FA;&#x4E8E;&#x6162;&#x75C5;&#x6BD2;&#x7684;&#x7CFB;&#x7EDF;&#x53EF;&#x7528;&#x4E8E;&#x9700;&#x8981;&#x7CFB;&#x7EDF;&#x6027;&#x7684; anti-proteases &#x6216;&#x5404;&#x79CD;&#x80BA;&#x90E8;&#x75BE;&#x75C5;&#x7684;&#x53D1;&#x75C5;&#x673A;&#x5236;&#x4E0E;&#x76F8;&#x5173;&#x7684;&#x7EC6;&#x80DE;&#x56E0;&#x5B50;&#x5206;&#x6CCC;&#x7684;&#x8C03;&#x67E5;&#x3002;

1 &#x53D7;&#x4F53;&#x53CA;&#x5176; antitrypsin &#x7684;&#x64CD;&#x7EB5;&#x9020;&#x8840;&#x5E72;&#x7EC6;&#x80DE;&#x79FB;&#x690D;&#x540E;&#x7684;&#x6301;&#x7EED;&#x7684;&#x8868;&#x8FBE;&#x3002;

Inherited mutations in the human alpha(1)-antitrypsin (AAT) gene lead to deficient circulating levels of AAT protein and a predisposition to developing emphysema. Gene therapy for individuals deficient in AAT is an attractive goal, because transfer of a normal AAT gene into any cell type able to secrete AAT should reverse deficient AAT levels and attenuate progression of lung disease. Here we present an approach for AAT gene transfer based on the transplantation of lentivirally transduced hematopoietic stem cells (HSCs). We develop a novel dual-promoter lentiviral system to transfer normal human AAT cDNA as well as a fluorescent tracking "reporter gene" into murine HSCs. After transplantation of 3,000 transduced HSCs into irradiated mouse recipients, we demonstrate simultaneous and sustained systemic expression of both genes in vivo for at least 31 weeks. The stem cells transduced with this protocol maintain multipotency, self-renewal potential, and the ability to reconstitute the hematopoietic systems of both primary and secondary recipients. This lentiviral-based system may be useful for investigations requiring the systemic secretion of anti-proteases or cytokines relevant to the pathogenesis of a variety of lung diseases.

Sustained expression of alpha1-antitrypsin after transplantation of manipulated hematopoietic stem cells.

H&eacute;r&eacute;ditaires mutations du g&egrave;ne humain alpha 1-antitrypsine (AAT) entra&icirc;ner d&eacute;ficients taux circulants de prot&eacute;ine AAT et une pr&eacute;disposition au d&eacute;veloppement de l&#39;emphys&egrave;me. La th&eacute;rapie g&eacute;nique pour les personnes d&eacute;ficientes en AAT est un objectif attrayant, parce que le transfert d&#39;un g&egrave;ne normal de AAT dans n&#39;importe quel type de cellule capable de s&eacute;cr&eacute;ter l&#39;AAT doit s&#39;inverser d&eacute;ficient AAT ainsi att&eacute;nuer l&#39;&eacute;volution des maladies pulmonaires. Nous pr&eacute;sentons une approche pour le transfert de g&egrave;nes d&#39;AAT bas&eacute; sur la greffe de lentivirally transduits des cellules souches h&eacute;matopo&iuml;&eacute;tiques (CSH). Nous d&eacute;veloppons un nouveau syst&egrave;me LENTIVIRAUX dual-promoteur pour transf&eacute;rer le cDNA de AAT humaine normale, mais aussi un fluorescent &laquo; g&egrave;ne rapporteur &raquo; de suivi dans les CSH murin. Apr&egrave;s que transplantation de 3,000 transduites CSH en destinataires des souris irradi&eacute;es, nous d&eacute;montrons l&#39;expression syst&eacute;mique simultan&eacute;e et soutenue des deux g&egrave;nes in vivo pour au moins 31 semaines. Les cellules souches transduites avec ce protocole maintenir multipotentialit&eacute;, autorenouvellement potentiel et la capacit&eacute; de reconstituer les syst&egrave;mes h&eacute;matopo&iuml;&eacute;tiques des b&eacute;n&eacute;ficiaires primaires et secondaires. Ce syst&egrave;me bas&eacute; sur des g&egrave;nes peut &ecirc;tre utile pour les enqu&ecirc;tes n&eacute;cessitant la s&eacute;cr&eacute;tion syst&eacute;mique des anti-prot&eacute;ases ou cytokines pertinentes &agrave; la pathogen&egrave;se d&#39;une vari&eacute;t&eacute; de maladies pulmonaires.

Expression durable de l&#39;alpha1-antitrypsine apr&egrave;s transplantation de manipuler les cellules souches h&eacute;matopo&iuml;&eacute;tiques.

Vererbte Mutationen im menschlichen alpha 1-Antitrypsin (AAT)-Gen f&uuml;hren zu mangelhaft zirkulierenden Niveaus des AAT-Proteins und eine Pr&auml;disposition zur Entwicklung von Emphysem. Gentherapie f&uuml;r Einzelpersonen, die einen Mangel an AAT ist ein attraktives Ziel, da Transfer eines normalen AAT-Gens in jeder Zelle Art in der Lage, AAT sezernieren mangelhaft AAT-Ebenen und D&auml;mpfen Fortschreiten der Lungenerkrankung umkehren sollte. Hier pr&auml;sentieren wir ein Konzept f&uuml;r AAT Gentransfer basierend auf die Transplantation von lentivirally transduzierten h&auml;matopoetischen Stammzellen (HSCs). Wir entwickeln ein neuartiges Dual-Projekttr&auml;ger Lentivirale System zum normalen menschlichen AAT cDNA sowie eine Leuchtstofflampe tracking &quot;Reporter-gen&quot; in murinen HSCs &uuml;bertragen. Nach der Transplantation von 3.000 HSCs in bestrahlten Maus Empf&auml;nger transduzierten, demonstrieren wir gleichzeitige und nachhaltige systemische Expression beide Gene in-vivo mindestens 31 Wochen. Die Stammzellen, die mit diesem Protokoll transduzierten pflegen Multipotenz, Selbsterneuerung Potenzial und die F&auml;higkeit, die h&auml;matopoetischen Systeme der prim&auml;ren und sekund&auml;ren Empf&auml;nger wieder zusammenzusetzen. Diese Lentiviralen basierendes System n&uuml;tzlich f&uuml;r Untersuchungen erfordern die systemische Sekretion von Anti-proteases oder Zytokine, die relevant f&uuml;r die Pathogenese einer Vielzahl von Lungenkrankheiten.

Nachhaltig Ausdruck der alpha1-Antitrypsin nach der Transplantation von manipulierten hematopoietic Stammzellen.

Ereditato mutazioni l&#39;alfa umana (1)-portare antitripsina (AAT) gene carenti livelli circolanti di proteina AAT e una predisposizione allo sviluppo di enfisema. Terapia genica per individui carenti di AAT &egrave; un obiettivo attraente, perch&eacute; il trasferimento di un gene normale AAT in qualsiasi tipo di cellula in grado di secernere AAT dovrebbe invertire carenti livelli AAT e attenuare progressione della malattia polmonare. Qui di seguito presentiamo un approccio basato sul trapianto di lentivirally trasdotte cellule staminali ematopoietiche (HSCs) del trasferimento del gene AAT. Sviluppiamo un romanzo sistema lentivirale dual-promotore per trasferimento normale umano cDNA AAT, nonch&eacute; un fluorescente &quot;gene reporter&quot; di rilevamento in murino HSCs. Dopo il trapianto di 3.000 transduced HSCs in destinatari del mouse irradiati, dimostriamo simultanea e sostenuta espressione sistemica di entrambi i geni in vivo per almeno 31 settimane. Le cellule staminali transduced con questo protocollo mantenere multipotency, potenziale di auto-rinnovamento e la capacit&agrave; di ricostituire gli ematopoietici sistemi dei destinatari primari e secondari. Questo sistema basato su lentivirali possa essere utile per le indagini che richiedono la sistemica secrezione di citochine rilevanti per la patogenesi di una variet&agrave; di malattie polmonari o di anti-proteases.

Espressione sostenuta di alpha1-antitripsina dopo il trapianto di cellule staminali ematopoietiche manipolati.

&#x4EBA;&#x9593;&#x306E;&#x30A2;&#x30EB;&#x30D5;&#x30A1; (1) &#x306E;&#x7A81;&#x7136;&#x5909;&#x7570;&#x3092;&#x7D99;&#x627F;-antitrypsin (AAT) &#x907A;&#x4F1D;&#x5B50;&#x925B;&#x6B20;&#x4E4F; AAT &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3068;&#x80BA;&#x6C17;&#x816B;&#x3092;&#x958B;&#x767A;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x7D20;&#x56E0;&#x306E;&#x30EC;&#x30D9;&#x30EB;&#x3092;&#x5FAA;&#x74B0;&#x3057;&#x307E;&#x3059;&#x3002;AAT &#x3092;&#x5206;&#x6CCC;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x4EFB;&#x610F;&#x306E;&#x30BB;&#x30EB;&#x578B;&#x306B; AAT &#x306E;&#x6B63;&#x5E38;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x8EE2;&#x9001;&#x306F; AAT &#x306E;&#x6B20;&#x4E4F;&#x5024;&#x3068;&#x6E1B;&#x8870;&#x9032;&#x884C;&#x80BA;&#x75BE;&#x60A3;&#x306E;&#x9006;&#x3079;&#x304D;&#x306E;&#x3067;&#x500B;&#x4EBA; AAT &#x306E;&#x6B20;&#x4E4F;&#x306E;&#x305F;&#x3081;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x6CBB;&#x7642;&#x306F;&#x9B45;&#x529B;&#x7684;&#x306A;&#x76EE;&#x6A19;&#x3067;&#x3059;&#x3002;Lentivirally &#x5F62;&#x8CEA;&#x5C0E;&#x5165;&#x3055;&#x308C;&#x305F;&#x9020;&#x8840;&#x5E79;&#x7D30;&#x80DE; (HSCs) &#x306E;&#x79FB;&#x690D;&#x306B;&#x57FA;&#x3065;&#x3044;&#x3066; AAT &#x907A;&#x4F1D;&#x5B50;&#x4F1D;&#x9054;&#x306E;&#x624B;&#x6CD5;&#x3092;&#x63D0;&#x6848;&#x3057;&#x307E;&#x3059;&#x3002;&#x6B63;&#x5E38;&#x30D2;&#x30C8; AAT cDNA &#x3068;&#x3057;&#x3066;&#x300C;&#x30EC;&#x30DD;&#x30FC;&#x30BF;&#x30FC;&#x907A;&#x4F1D;&#x5B50;&#x300D;&#x30DE;&#x30A6;&#x30B9;&#x306E; HSCs &#x306B;&#x8FFD;&#x8DE1;&#x86CD;&#x5149;&#x3092;&#x8EE2;&#x9001;&#x3059;&#x308B;&#x65B0;&#x898F;&#x30C7;&#x30E5;&#x30A2;&#x30EB; &#x30D7;&#x30ED;&#x30E2;&#x30FC;&#x30BF;&#x30FC; &#x30EC;&#x30F3;&#x30C1; &#x30A6;&#x30A4;&#x30EB;&#x30B9; &#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x3092;&#x958B;&#x767A;&#x3057;&#x307E;&#x3059;&#x3002;3,000 &#x306E;&#x79FB;&#x690D; HSCs &#x7167;&#x5C04;&#x30DE;&#x30A6;&#x30B9;&#x53D7;&#x4FE1;&#x8005;&#x306B;&#x5C0E;&#x5165;&#x3057;&#x305F;&#x5F8C;&#x3001;&#x6211;&#x3005; &#x4E21;&#x65B9;&#x5C11;&#x306A;&#x304F;&#x3068;&#x3082; 31 &#x9031;&#x9593;&#x306E; in vivo &#x3067;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x540C;&#x6642;&#x304B;&#x3064;&#x6301;&#x7D9A;&#x7684;&#x306A;&#x5168;&#x8EAB;&#x306E;&#x5F0F;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x30D7;&#x30ED;&#x30C8;&#x30B3;&#x30EB;&#x306E;&#x5C0E;&#x5165;&#x5E79;&#x7D30;&#x80DE;&#x5206;&#x5316;&#x7387;&#x3001;&#x81EA;&#x5DF1;&#x8907;&#x88FD;&#x306E;&#x6F5C;&#x5728;&#x7684;&#x306A;&#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30EA;&#x3068;&#x30BB;&#x30AB;&#x30F3;&#x30C0;&#x30EA;&#x306E;&#x4E21;&#x65B9;&#x306E;&#x53D7;&#x4FE1;&#x8005;&#x306E;&#x9020;&#x8840;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x3092;&#x518D;&#x69CB;&#x6210;&#x3059;&#x308B;&#x80FD;&#x529B;&#x3092;&#x7DAD;&#x6301;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x30EC;&#x30F3;&#x30C1; &#x30A6;&#x30A4;&#x30EB;&#x30B9; &#x30D9;&#x30FC;&#x30B9;&#x306E;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x306F;&#x3001;anti-proteases &#x307E;&#x305F;&#x306F;&#x69D8;&#x3005; &#x306A;&#x80BA;&#x75BE;&#x60A3;&#x306E;&#x75C5;&#x56E0;&#x306B;&#x95A2;&#x9023;&#x3059;&#x308B;&#x30B5;&#x30A4;&#x30C8;&#x30AB;&#x30A4;&#x30F3;&#x306E;&#x5168;&#x8EAB;&#x306E;&#x5206;&#x6CCC;&#x3092;&#x5FC5;&#x8981;&#x3068;&#x3059;&#x308B;&#x8ABF;&#x67FB;&#x306E;&#x305F;&#x3081;&#x5F79;&#x306B;&#x7ACB;&#x3064;&#x304B;&#x3082;&#x3057;&#x308C;&#x307E;&#x305B;&#x3093;&#x3002;

&#x64CD;&#x4F5C;&#x3055;&#x308C;&#x305F;&#x9020;&#x8840;&#x5E79;&#x7D30;&#x80DE;&#x79FB;&#x690D;&#x5F8C;&#x306E;&#x6301;&#x7D9A;&#x7684;&#x306A;&#x5F0F;&#x306E;&#x30A2;&#x30EB;&#x30D5;&#x30A1; 1-&#x30A2;&#x30F3;&#x30C1;&#x30C8;&#x30EA;&#x30D7;&#x30B7;&#x30F3;&#x3002;

&#xC778;&#xAC04;&#xC758; &#xC54C;&#xD30C; (1)-antitrypsin (AAT) &#xC720;&#xC804;&#xC790;&#xC5D0; &#xC0C1;&#xC18D; &#xB41C; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xBC1C;&#xC0DD;&#xD560; AAT &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xACB0;&#xD54D; &#xC21C;&#xD658; &#xB808;&#xBCA8;&#xACFC; &#xAE30;&#xC885; &#xAC1C;&#xBC1C;&#xC744; &#xD0C0;&#xACE0; &#xB0AC;&#xB2E4;. &#xAC1C;&#xC778; AAT &#xACB0;&#xD54D;&#xC5D0; &#xB300; &#xD55C; &#xC720;&#xC804;&#xC790; &#xCE58;&#xB8CC; &#xB54C;&#xBB38;&#xC5D0; &#xBAA8;&#xB4E0; &#xC140; &#xD615;&#xC2DD; AAT &#xBD84; &#xBE44; &#xC218;&#xB85C; &#xC815;&#xC0C1;&#xC801;&#xC778; AAT &#xC720;&#xC804;&#xC790;&#xC758; &#xC591;&#xB3C4; &#xD574;&#xC57C; &#xC5ED; &#xACB0;&#xD54D; AAT &#xC218;&#xC900; &#xBC0F; &#xD3D0; &#xC9C8;&#xD658;&#xC758; attenuate &#xC9C4;&#xD589; &#xB9E4;&#xB825;&#xC801;&#xC778; &#xBAA9;&#xD45C; &#xC774;&#xB2E4;. &#xC5EC;&#xAE30;&#xC5D0; &#xC6B0;&#xB9AC;&#xAC00; &#xD604;&#xC7AC; lentivirally transduced &#xC870; &#xD608; &#xC904;&#xAE30; &#xC138;&#xD3EC; (HSCs)&#xC758; &#xC774;&#xC2DD;&#xC5D0; &#xB530;&#xB77C; AAT &#xC720;&#xC804;&#xC790; &#xC774;&#xB3D9;&#xC5D0; &#xB300; &#xD55C; &#xC811;&#xADFC;. &#xC6B0;&#xB9AC;&#xB294; &#xC815;&#xC0C1;&#xC801;&#xC778; &#xC778;&#xAC04;&#xC758; AAT cDNA murine Hscs&#xB85C; &quot;&#xB9AC;&#xD3EC;&#xD130; &#xC720;&#xC804;&#xC790;&quot;&#xB97C; &#xCD94;&#xC801; &#xD558;&#xB294; &#xD615;&#xAD11;&#xC744; &#xC804;&#xC1A1; &#xD558;&#xB3C4;&#xB85D; &#xC18C;&#xC124; &#xB4C0;&#xC5BC; &#xBAA8;&#xD130; lentiviral &#xC2DC;&#xC2A4;&#xD15C;&#xC744; &#xAC1C;&#xBC1C;. &#xD6C4; &#xC774;&#xC2DD; 3000&#xC758; &#xBC29;&#xC0AC;&#xB2A5;&#xB41C; &#xB9C8;&#xC6B0;&#xC2A4; &#xBC1B;&#xB294; &#xC0AC;&#xB78C;&#xC73C;&#xB85C; HSCs &#xBD88;&#xB9AC;&#xACE0;, &#xC801;&#xC5B4;&#xB3C4; 31 &#xC8FC;&#xC5D0; &#xB300; &#xD55C; vivo&#xC5D0;&#xC11C; &#xB450; &#xC720;&#xC804;&#xC790;&#xC758; &#xB3D9;&#xC2DC;&#xC640; &#xC9C0;&#xC18D;&#xC801;&#xC778; &#xC870;&#xC9C1;&#xC758; &#xC2DD;&#xC744; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. &#xC774; &#xD504;&#xB85C;&#xD1A0;&#xCF5C;&#xC744; &#xBD88;&#xB9AC;&#xACE0; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xC720;&#xC9C0; multipotency, &#xC790;&#xAE30; &#xAC31;&#xC2E0; &#xAC00;&#xB2A5;&#xC131; &#xBC0F; &#xBAA8;&#xB450; &#xAE30;&#xBCF8; &#xBC0F; &#xBCF4;&#xC870; &#xBC1B;&#xB294; &#xC0AC;&#xB78C; &#xC870; &#xD608; &#xC2DC;&#xC2A4;&#xD15C;&#xC744; &#xC7AC;&#xAD6C;&#xC131;&#xD560; &#xC218; &#xC788;&#xAC8C;. &#xC774; lentiviral &#xAE30;&#xBC18; &#xC2DC;&#xC2A4;&#xD15C; anti-proteases &#xB610;&#xB294; &#xB2E4;&#xC591; &#xD55C; &#xD3D0; &#xC9C8;&#xD658;&#xC758; &#xBCD1; &#xC778;&#xC5D0; &#xAD00;&#xB828; &#xB41C; &#xD06C;&#xB9B0; &#xC2DC; &#xD1A0; &#xD0A8;&#xC758; &#xC870;&#xC9C1;&#xC758; &#xBD84; &#xBE44;&#xB97C; &#xC694;&#xAD6C; &#xD558;&#xB294; &#xC218;&#xC0AC;&#xB97C; &#xC704;&#xD574; &#xC720;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xC870;&#xC791;&#xB41C; &#xC870; &#xD608; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xC774;&#xC2DD; &#xD6C4; alpha1-antitrypsin &#xC9C0;&#xC18D;&#xC801;&#xC778;&#xB41C; &#xD45C;&#xD604;.

Heredado mutaciones en el gene humano alfa 1-antitripsina (AAT) conducen a deficientes niveles circulantes de la prote&iacute;na AAT y una predisposici&oacute;n al desarrollo de enfisema. Terapia g&eacute;nica para las personas deficientes en AAT es un objetivo atractivo, porque la transferencia de un gen normal de la AAT en cualquier tipo de c&eacute;lulas capaces de secretar AAT debe invertir deficientes niveles AAT y atenuada de progresi&oacute;n de la enfermedad pulmonar. Aqu&iacute; presentamos un enfoque para la transferencia de genes de la AAT basado en el trasplante de lentivirally transduced c&eacute;lulas madre hematopoy&eacute;ticas (HSCs). Desarrollamos un novedoso sistema lentivirales dual-promotor para transferir el cDNA AAT humana normal as&iacute; como un fluorescente &quot;gen reportero&quot; de seguimiento en HSCs murinas. Despu&eacute;s de trasplante de 3.000 transduced HSCs en recipientes de rat&oacute;n irradiados, demostramos la expresi&oacute;n sist&eacute;mica sostenida y simult&aacute;nea de ambos genes in vivo durante por lo menos 31 semanas. Las c&eacute;lulas transduced con este protocolo mantener multipotencia, auto-renovaci&oacute;n potencial y la capacidad de reconstituir los sistemas hematopoy&eacute;ticos de destinatarios primarios y secundarios. Este sistema basado en lentivirales puede ser &uacute;til para investigaciones que requieren la secreci&oacute;n sist&eacute;mica de anti-proteases o citoquinas pertinentes a la patogenesia de una variedad de enfermedades pulmonares.

Expresi&oacute;n sostenida de alfa-1-antitripsina despu&eacute;s del trasplante de c&eacute;lulas hematopoy&eacute;ticas manipuladas.

Although RNA interference (RNAi) has become a ubiquitous laboratory tool since its discovery 12 years ago, in vivo delivery to selected cell types remains a major technical challenge. Here, we report the use of lentiviral vectors for long-term in vivo delivery of RNAi selectively to resident alveolar macrophages (AMs), key immune effector cells in the lung. We demonstrate the therapeutic potential of this approach by RNAi-based downregulation of p65 (RelA), a component of the pro-inflammatory transcriptional regulator, nuclear factor κB (NF-κB) and a key participant in lung disease pathogenesis. In vivo RNAi delivery results in decreased induction of NF-κB and downstream neutrophilic chemokines in transduced AMs as well as attenuated lung neutrophilia following stimulation with lipopolysaccharide (LPS). Through concurrent delivery of a novel lentiviral reporter vector (lenti-NF-κB-luc-GFP) we track in vivo expression of NF-κB target genes in real time, a critical step towards extending RNAi-based therapy to longstanding lung diseases. Application of this system reveals that resident AMs persist in the airspaces of mice following the resolution of LPS-induced inflammation, thus allowing these localized cells to be used as effective vehicles for prolonged RNAi delivery in disease settings.

Lentiviral delivery of RNAi for in vivo lineage-specific modulation of gene expression in mouse lung macrophages.

Directed differentiation is a powerful cell culture technique where developmental pathways are applied to a pluripotent progenitor in order to generate specific terminally differentiated cell populations. Here, we describe a serum-free protocol using growth factors in defined concentrations to derive iPSC-hepatic cells starting from both feeder and feeder-free conditions. The generated iPSC-hepatic cells are developmentally similar to fetal stage hepatocytes, and when generated from patients with genetic mutations such as alpha-1 antitrypsin deficiency recapitulate pathologic changes associated with clinical disease, such as protein misfolding, intracellular retention of misfolded proteins, and elevated levels of ER stress.

Protocol for Directed Differentiation of Human Induced Pluripotent Stem Cells (iPSCs) to a Hepatic Lineage.

Alpha-1 antitrypsin (AAT) is an acute phase protein that possesses immune-regulatory and anti-inflammatory functions independent of antiprotease activity. AAT deficiency (AATD) is associated with early-onset emphysema and chronic obstructive pulmonary disease. Of interest are the AATD nonsense mutations (termed null or Q0), the majority of which arise from premature termination codons in the mRNA coding region. We have recently demonstrated that plasma from an AATD patient homozygous for the Null Bolton allele ( ) contains AAT protein of truncated size. Although the potential to alleviate the phenotypic consequences of AATD by increasing levels of truncated protein holds therapeutic promise, protein functionality is key. The goal of this study was to evaluate the structural features and anti-inflammatory capacity of Q0-AAT. A low-abundance, truncated AAT protein was confirmed in plasma of a Q0-AATD patient and was secreted by patient-derived induced pluripotent stem cell-hepatic cells. Functional assays confirmed the ability of purified Q0-AAT protein to bind neutrophil elastase and to inhibit protease activity. Q0-AAT bound IL-8 and leukotriene B, comparable to healthy control M-AAT, and significantly decreased leukotriene B-induced neutrophil adhesion ( = 0.04). Through a mechanism involving increased mRNA stability ( = 0.007), ataluren treatment of HEK-293 significantly increased  mRNA expression ( = 0.03) and Q0-AAT truncated protein secretion ( = 0.04). Results support the rationale for treatment with pharmacological agents that augment levels of functional Q0-AAT protein, thus offering a potential therapeutic option for AATD patients with rare mutations of similar theratype.

Circulating Truncated Alpha-1 Antitrypsin Glycoprotein in Patient Plasma Retains Anti-Inflammatory Capacity.

Individuals with the genetic disorder alpha-1 antitrypsin deficiency (AATD) are at risk of developing lung and liver disease. Patient induced pluripotent stem cells (iPSCs) have been found to model features of AATD pathogenesis but only a handful of AATD patient iPSC lines have been published. To capture the significant phenotypic diversity of the patient population, we describe here the establishment and characterization of a curated repository of AATD iPSCs with associated disease-relevant clinical data. To highlight the utility of the repository, we selected a subset of iPSC lines for functional characterization. Selected lines were differentiated to generate both hepatic and lung cell lineages and analyzed by RNA sequencing. In addition, two iPSC lines were targeted using CRISPR/Cas9 editing to accomplish scarless repair. Repository iPSCs are available to investigators for studies of disease pathogenesis and therapeutic discovery.

A Highly Phenotyped Open Access Repository of Alpha-1 Antitrypsin Deficiency Pluripotent Stem Cells.

A hallmark of severe COVID-19 pneumonia is SARS-CoV-2 infection of the facultative progenitors of lung alveoli, the alveolar epithelial type 2 cells (AT2s). However, inability to access these cells from patients, particularly at early stages of disease, limits an understanding of disease inception. Here, we present an in vitro human model that simulates the initial apical infection of alveolar epithelium with SARS-CoV-2 by using induced pluripotent stem cell-derived AT2s that have been adapted to air-liquid interface culture. We find a rapid transcriptomic change in infected cells, characterized by a shift to an inflammatory phenotype with upregulation of NF-κB signaling and loss of the mature alveolar program. Drug testing confirms the efficacy of remdesivir as well as TMPRSS2 protease inhibition, validating a putative mechanism used for viral entry in alveolar cells. Our model system reveals cell-intrinsic responses of a key lung target cell to SARS-CoV-2 infection and should facilitate drug development.

SARS-CoV-2 Infection of Pluripotent Stem Cell-Derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response.

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.

Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2.

Alpha-1 antitrypsin deficiency (AATD) is most commonly caused by the Z mutation, a single-base substitution that leads to AAT protein misfolding and associated liver and lung disease. In this study, we apply adenine base editors to correct the Z mutation in patient induced pluripotent stem cells (iPSCs) and iPSC-derived hepatocytes (iHeps). We demonstrate that correction of the Z mutation in patient iPSCs reduces aberrant AAT accumulation and increases its secretion. Adenine base editing (ABE) of differentiated iHeps decreases ER stress in edited cells, as demonstrated by single-cell RNA sequencing. We find ABE to be highly efficient in iPSCs and do not identify off-target genomic mutations by whole-genome sequencing. These results reveal the feasibility and utility of base editing to correct the Z mutation in AATD patient cells.

Andrew A. Wilson

Center for Regenerative Medicine

Boston University and Boston Medical Center

Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

Andrew A. Wilson

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Boston University and Boston Medical Center

Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

Center for Regenerative Medicine