efficient-generation-editing-feeder-free-ipscs-from-human-pancreatic

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

National Institutes of Health

The culture of human embryonic stem cells (hESCs) is limited, both technically and with respect to clinical potential, by the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. The concern over xenogeneic contaminants from the mouse feeder cells may restrict transplantation to humans and the variability in MEFs from batch-to-batch and laboratory-to-laboratory may contribute to some of the variability in experimental results. Finally, use of any feeder layer increases the work load and subsequently limits the large-scale culture of human ES cells. Thus, the development of feeder-free cultures will allow more reproducible culture conditions, facilitate scale-up and potentiate the clinical use of cells differentiated from hESC cultures. In this review, we describe various methods tested to culture cells in the absence of MEF feeder layers and other advances in eliminating xenogeneic products from the culture system.

Toward xeno-free culture of human embryonic stem cells.

The expression and function of several multidrug transporters (including ABCB1 and ABCG2) have been studied in human cancer cells and in mouse and human adult stem cells. However, the expression of ABCG2 in human embryonic stem cells (hESCs) remains unclear. Limited and contradictory results in the literature from two research groups have raised questions regarding its expression and function. In this study, we used quantitative real-time PCR, Northern blots, whole genome RNA sequencing, Western blots, and immunofluorescence microscopy to study ABCG2 expression in hESCs. We found that full-length ABCG2 mRNA transcripts are expressed in undifferentiated hESC lines. However, ABCG2 protein was undetectable even under embryoid body differentiation or cytotoxic drug induction. Moreover, surface ABCG2 protein was coexpressed with the differentiation marker stage-specific embryonic antigen-1 of hESCs, following constant BMP-4 signaling at days 4 and 6. This expression was tightly correlated with the downregulation of two microRNAs (miRNAs) (i.e., hsa-miR-519c and hsa-miR-520h). Transfection of miRNA mimics and inhibitors of these two miRNAs confirmed their direct involvement in the regulation ABCG2 translation. Our findings clarify the controversy regarding the expression of the ABCG2 gene and also provide new insights into translational control of the expression of membrane transporter mRNAs by miRNAs in hESCs.

Regulation and expression of the ATP-binding cassette transporter ABCG2 in human embryonic stem cells.

Regenerative medicine, relying on human embryonic stem cell (hESC) technology, opens promising new avenues for therapy of many severe diseases. However, this approach is restricted by limited production of the desired cells due to the refractory properties of hESC growth in vitro. It is further hindered by insufficient control of cellular stress, growth rates, and heterogeneous cellular states under current culture conditions. In this study, we report a novel cell culture method based on a non-colony type monolayer (NCM) growth. Human ESCs under NCM remain pluripotent as determined by teratoma assays and sustain the potential to differentiate into three germ layers. This NCM culture has been shown to homogenize cellular states, precisely control growth rates, significantly increase cell production, and enhance hESC recovery from cryopreservation without compromising chromosomal integrity. This culture system is simple, robust, scalable, and suitable for high-throughput screening and drug discovery.

Non-colony type monolayer culture of human embryonic stem cells.

&#x5F88;&#x591A;&#x7531;&#x8BF1;&#x5BFC;&#x7684;&#x591A;&#x529F;&#x80FD;&#x5E72;&#x7EC6;&#x80DE;&#x6280;&#x672F;&#x751F;&#x6210;&#x7684;&#x5174;&#x594B;&#x4E0E;&#x5EFA;&#x6A21;&#x4EE5;&#x53CA;&#x6F5C;&#x5728;&#x7684;&#x4E2A;&#x6027;&#x5316;&#x7684;&#x7EC6;&#x80DE;&#x6CBB;&#x7597;&#x7684;&#x75BE;&#x75C5;&#x7684;&#x53EF;&#x80FD;&#x6027;&#x6709;&#x5173;&#x3002;&#x7136;&#x800C;&#xFF0C;&#x8981;&#x8FFD;&#x6C42;&#x8FD9;&#x662F;&#x91CD;&#x8981;&#x7684;&#x662F;&#x8981;&#x4E86;&#x89E3;&#x5305;&#x62EC;&#x5176;&#x56FA;&#x6709;&#x53D8;&#x5F02;&#x6027;&#x591A;&#x80FD;&#x5E72;&#x6B63;&#x5E38;&#x72B6;&#x6001;&#x3002;&#x6211;&#x4EEC;&#x6267;&#x884C;&#x5404;&#x79CD;&#x5206;&#x5B50;&#x5206;&#x6790;&#x5316;&#x9A8C; 21 hESC &#x7EBF;&#x6761;&#x548C; 8 hiPSC &#x884C;&#x6765;&#x751F;&#x6210;&#x5168;&#x9762;&#x7684;&#x591A;&#x80FD;&#x5E72;&#x7EC6;&#x80DE;&#x7684;&#x672A;&#x5206;&#x5316;&#x72B6;&#x6001;&#x7684;&#x5FEB;&#x7167;&#x3002;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x6570;&#x636E;&#x7684;&#x5206;&#x6790;&#x663E;&#x793A;&#xFF0C;&#x6839;&#x636E;&#x524D;&#x51E0;&#x6B21;&#x62A5;&#x544A;&#x6CA1;&#x6709;&#x673A;&#x4F53;&#x529F;&#x80FD;&#x7279;&#x5F02;&#x6027;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x6A21;&#x5F0F;&#x3002;&#x6211;&#x4EEC;&#x8FDB;&#x4E00;&#x6B65;&#x6BD4;&#x8F83;&#x5206;&#x5316;&#x4F5C;&#x4E3A;&#x80DA;&#x4F53; 2 &#x5A92;&#x4F53;&#x62DF;&#x542F;&#x52A8;&#x5BF9;&#x5355;&#x72EC;&#x7684;&#x5355;&#x5143;&#x683C;&#x7684;&#x547D;&#x8FD0;&#xFF0C;&#x4EE5;&#x53CA; 20 &#x6210;&#x4EBA;&#x7EC4;&#x7EC7;&#x5206;&#x5316;&#x7684;&#x7EC6;&#x80DE;&#x3002;&#x4ECE;&#x8FD9;&#x4E00;&#x5206;&#x6790;&#x6211;&#x4EEC;&#x5DF2;&#x7ECF;&#x4EA7;&#x751F;&#x4E86;&#x57FA;&#x56E0;&#x5217;&#x8868;&#x5B9A;&#x4E49;&#x591A;&#x6F5C;&#x80FD;&#xFF0C;&#x4E3A;&#x591A;&#x80FD;&#x5E72;&#x56FD;&#x5BB6;&#x5EFA;&#x7ACB;&#x4E00;&#x4E2A;&#x57FA;&#x7EBF;&#x3002;&#x6700;&#x540E;&#xFF0C;&#x6211;&#x4EEC;&#x63D0;&#x4F9B;&#x5217;&#x8868;&#x663E;&#x793A;&#x72EC;&#x7ACB;&#x5355;&#x5143;&#x683C;&#x547D;&#x8FD0;&#x7684;&#x62DF;&#x8BAE;&#x7684;&#x504F;&#x89C1;&#x8FD9;&#x4E24;&#x79CD;&#x5206;&#x5316;&#x6761;&#x4EF6;&#x4E30;&#x5BCC;&#x7684;&#x57FA;&#x56E0;&#x3002;

StemCellDB&#xFF1A; &#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x6570;&#x636E;&#x5E93;&#x5728;&#x56FD;&#x5BB6;&#x5065;&#x5EB7;&#x7814;&#x7A76;&#x6240;&#x3002;

Much of the excitement generated by induced pluripotent stem cell technology is concerned with the possibility of disease modeling as well as the potential for personalized cell therapy. However, to pursue this it is important to understand the 'normal' pluripotent state including its inherent variability. We have performed various molecular profiling assays for 21 hESC lines and 8 hiPSC lines to generate a comprehensive snapshot of the undifferentiated state of pluripotent stem cells. Analysis of the gene expression data revealed no iPSC-specific gene expression pattern in accordance with previous reports. We further compared cells, differentiated as embryoid bodies in 2 media proposed to initiate differentiation towards separate cell fates, as well as 20 adult tissues. From this analysis we have generated a gene list which defines pluripotency and establishes a baseline for the pluripotent state. Finally, we provide lists of genes enriched under both differentiation conditions which show the proposed bias toward independent cell fates.

StemCellDB: The Human Pluripotent Stem Cell Database at the National Institutes of Health.

Une grande partie de l&#39;enthousiasme g&eacute;n&eacute;r&eacute; par la technologie des cellules souches pluripotentes induites est pr&eacute;occup&eacute;e par la possibilit&eacute; de maladie de mod&eacute;lisation ainsi que le potentiel de la th&eacute;rapie cellulaire personnalis&eacute;. Toutefois, afin de poursuivre ce, il est important de comprendre l&#39;&eacute;tat de pluripotentes &laquo; normal &raquo;, y compris sa variabilit&eacute; inh&eacute;rente. Nous avons effectu&eacute; plusieurs essais mol&eacute;culaires de profilage pour 21 lign&eacute;es de CSEh et 8 lignes de hiPSC pour g&eacute;n&eacute;rer un aper&ccedil;u complet de l&#39;&eacute;tat indiff&eacute;renci&eacute; de cellules souches pluripotentes. Analyse les donn&eacute;es d&#39;expression g&eacute;nique a r&eacute;v&eacute;l&eacute; aucun mod&egrave;le d&#39;expression sp&eacute;cifiques iPSC g&egrave;ne conform&eacute;ment aux pr&eacute;c&eacute;dents rapports. Plus loin, nous avons compar&eacute; les cellules diff&eacute;renci&eacute;es comme corps embryo&iuml;des dans 2 m&eacute;dias propos&eacute;s de lancer la diff&eacute;renciation vers des destins cellulaires distincts, ainsi que 20 tissus adultes. De cette analyse, nous avons g&eacute;n&eacute;r&eacute; une liste de g&egrave;ne qui d&eacute;finit la pluripotence et &eacute;tablit une ligne de base pour l&#39;&eacute;tat pluripotent. Enfin, nous fournissons des listes de g&egrave;nes enrichis dans les deux conditions de diff&eacute;renciation qui montrent le biais propos&eacute; vers les destins de cellules ind&eacute;pendantes.

StemCellDB : La base de donn&eacute;es de cellules souches pluripotentes humaines au niveau National Institutes of Health.

Viel von der Aufregung von induzierten pluripotenten Stammzellen-Technologie befasst sich mit der M&ouml;glichkeit der Modellierung sowie das Potenzial f&uuml;r personalisierte Zelltherapie-Krankheit. Um dies zu verfolgen ist es jedoch wichtig zu verstehen, den &#39;normalen&#39; pluripotenten Zustand einschlie&szlig;lich ihrer inh&auml;renten Variabilit&auml;t. Wir haben verschiedene Molekulare Profilerstellungs-Assays f&uuml;r 21 hESC-Linien und 8 HiPSC Zeilen generieren einen umfassenden Snapshot des undifferenzierten pluripotenten Stammzellen durchgef&uuml;hrt. Analyse der Gen-Ausdruck-Daten ergab kein iPSC-spezifische Gen-Ausdruck-Muster nach fr&uuml;heren Berichten. Wir verglichen weitere Zellen, differenzierte als Embryoid K&ouml;rper in 2 Medien vorgeschlagene Differenzierung gegen&uuml;ber separate Zelle Schicksale sowie 20 Erwachsene Gewebe zu initiieren. Aus dieser Analyse haben wir eine gen-Liste erzeugt, die Pluripotenz definiert und etabliert eine Baseline f&uuml;r den pluripotenten Zustand. Schlie&szlig;lich bieten wir Listen von Genen, die unter beiden Differenzierung-Bedingungen, die die vorgeschlagenen in Richtung unabh&auml;ngiger Zelle Schicksale Einseitigkeit bereichert.

StemCellDB: Die menschliche Pluripotent Stem Cell Datenbank an den National Institutes of Health.

Molto l&#39;entusiasmo suscitato dalla tecnologia delle cellule staminali pluripotenti indotte &egrave; interessato la possibilit&agrave; di malattia modellazione cos&igrave; come il potenziale per la terapia cellulare personalizzato. Tuttavia, per perseguire questo &egrave; importante comprendere lo stato &#39;normale&#39; pluripotenti compresa sua variabilit&agrave; intrinseca. Abbiamo eseguito vari saggi di analisi molecolare per 21 linee di hESC e 8 hiPSC per generare uno snapshot completo dello stato indifferenziato di cellule staminali pluripotenti. Analisi dei dati di espressione genica ha rivelato nessun modello di espressione iPSC specifico gene conformemente a precedenti relazioni. Abbiamo confrontato ulteriormente le cellule, differenziate come corpi embryoid in 2 media proposto di avviare la differenziazione verso destini cella separata, come pure i tessuti adulti 20. Da questa analisi abbiamo abbiamo generato un elenco di gene che definisce la pluripotenza e stabilisce una linea di base per lo stato pluripotenti. Infine, forniamo liste di geni arricchiti sotto entrambe le condizioni di differenziazione che mostrano il bias proposto verso destini cellula indipendente.

StemCellDB: L&#39;umano cellule staminali pluripotenti Database presso i National Institutes of Health.

&#x8A98;&#x5C0E;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x6280;&#x8853;&#x306B;&#x3088;&#x3063;&#x3066;&#x751F;&#x6210;&#x3055;&#x308C;&#x305F;&#x8208;&#x596E;&#x306E;&#x591A;&#x304F;&#x306F;&#x30D1;&#x30FC;&#x30BD;&#x30CA;&#x30E9;&#x30A4;&#x30BA;&#x3055;&#x308C;&#x305F;&#x7D30;&#x80DE;&#x7642;&#x6CD5;&#x306E;&#x53EF;&#x80FD;&#x6027;&#x3060;&#x3051;&#x3067;&#x306A;&#x304F;&#x3001;&#x30E2;&#x30C7;&#x30EA;&#x30F3;&#x30B0;&#x75BE;&#x60A3;&#x306E;&#x53EF;&#x80FD;&#x6027;&#x3092;&#x61F8;&#x5FF5;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;&#x3053;&#x308C;&#x3092;&#x8FFD;&#x6C42;&#x3059;&#x308B;&#x305D;&#x306E;&#x56FA;&#x6709;&#x306E;&#x5909;&#x52D5;&#x3092;&#x542B;&#x3080; &#39;normal&#39; &#x591A;&#x80FD;&#x6027;&#x306E;&#x72B6;&#x614B;&#x3092;&#x7406;&#x89E3;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x91CD;&#x8981;&#x3067;&#x3059;&#x3002;&#x69D8;&#x3005; &#x306A;&#x5206;&#x5B50;&#x30A2;&#x30C3;&#x30BB;&#x30A4; 21 hESC &#x7DDA;&#x3068;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x672A;&#x5206;&#x5316;&#x72B6;&#x614B;&#x306E;&#x5305;&#x62EC;&#x7684;&#x306A;&#x30B9;&#x30CA;&#x30C3;&#x30D7;&#x30B7;&#x30E7;&#x30C3;&#x30C8;&#x3092;&#x751F;&#x6210;&#x3059;&#x308B; 8 &#x304B;&#x3089;&#x4F5C;&#x88FD;&#x3057;&#x305F;&#x30E9;&#x30A4;&#x30F3;&#x3092;&#x884C;&#x3063;&#x305F;&#x3002;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x30C7;&#x30FC;&#x30BF;&#x306E;&#x89E3;&#x6790;&#x306F;&#x3001;&#x4EE5;&#x524D;&#x306E;&#x30EC;&#x30DD;&#x30FC;&#x30C8;&#x306B;&#x5F93;&#x3063;&#x3066; iPSC &#x7279;&#x7570;&#x7684;&#x907A;&#x4F1D;&#x5B50;&#x8868;&#x73FE;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3002;&#x6211;&#x3005; &#x306F;&#x3055;&#x3089;&#x306B;&#x500B;&#x5225;&#x7D30;&#x80DE;&#x904B;&#x547D;&#x3068;&#x3057;&#x3066; 20 &#x6210;&#x4F53;&#x7D44;&#x7E54;&#x3078;&#x306E;&#x5206;&#x5316;&#x3092;&#x958B;&#x59CB;&#x3059;&#x308B;&#x63D0;&#x6848; 2 &#x306E;&#x30E1;&#x30C7;&#x30A3;&#x30A2;&#x306E;&#x80DA;&#x69D8;&#x4F53;&#x3068;&#x3057;&#x3066;&#x5206;&#x5316;&#x3057;&#x305F;&#x7D30;&#x80DE;&#x3092;&#x6BD4;&#x8F03;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x306E;&#x5206;&#x6790;&#x304B;&#x3089;&#x306F;&#x591A;&#x80FD;&#x6027;&#x3092;&#x5B9A;&#x7FA9;&#x3057;&#x3001;&#x591A;&#x80FD;&#x6027;&#x306E;&#x72B6;&#x614B;&#x306E;&#x305F;&#x3081;&#x306E;&#x30D9;&#x30FC;&#x30B9;&#x30E9;&#x30A4;&#x30F3;&#x3092;&#x78BA;&#x7ACB;&#x3059;&#x308B;&#x907A;&#x4F1D;&#x5B50;&#x30EA;&#x30B9;&#x30C8;&#x3092;&#x751F;&#x6210;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x6700;&#x5F8C;&#x306B;&#x3001;&#x63D0;&#x6848;&#x3055;&#x308C;&#x305F;&#x72EC;&#x7ACB;&#x3057;&#x305F;&#x7D30;&#x80DE;&#x904B;&#x547D;&#x30D0;&#x30A4;&#x30A2;&#x30B9;&#x3092;&#x793A;&#x3059;&#x4E21;&#x65B9;&#x306E;&#x5206;&#x5316;&#x306E;&#x6761;&#x4EF6;&#x306E;&#x4E0B;&#x3067;&#x6FC3;&#x7E2E;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x30EA;&#x30B9;&#x30C8;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;

StemCellDB&#xFF1A; &#x56FD;&#x7ACB;&#x885B;&#x751F;&#x7814;&#x7A76;&#x6240;&#x3067;&#x306E;&#x3072;&#x3068;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x30C7;&#x30FC;&#x30BF;&#x30D9;&#x30FC;&#x30B9;&#x3002;

&#xC720;&#xB3C4; &#xB9CC;&#xB2A5; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xAE30;&#xC220;&#xC5D0; &#xC758;&#xD574; &#xC0DD;&#xC131; &#xB41C; &#xD765;&#xBD84;&#xC758; &#xC5F4;&#xAE30;&#xAC00; &#xB9CE;&#xC740; &#xC9C8;&#xBCD1; &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C;, &#xB9DE;&#xCDA4;&#xD615;&#xB41C; &#xC138;&#xD3EC; &#xCE58;&#xB8CC;&#xC5D0; &#xB300; &#xD55C; &#xAC00;&#xB2A5;&#xC131; &#xBAA8;&#xB378;&#xB9C1;&#xC758; &#xAC00;&#xB2A5;&#xC131;&#xACFC; &#xD568;&#xAED8; &#xC6B0;&#xB824; &#xD558;&#xACE0;&#xC788;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xC774;&#xAC83;&#xC744; &#xCD94;&#xAD6C; &#xD558;&#xB294; &#xADF8;&#xAC83;&#xC740; &#xADF8;&#xAC83;&#xC758; &#xB0B4;&#xC7AC; &#xBCC0;&#xB3D9;&#xC131;&#xC744; &#xD3EC;&#xD568; &#xD55C; &#39;&#xC815;&#xC0C1;&#39; pluripotent &#xC0C1;&#xD0DC;&#xB97C; &#xC774;&#xD574; &#xD558;&#xB294; &#xAC83;&#xC774; &#xC911;&#xC694;&#xC785;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC; 21 hESC &#xB77C;&#xC778;&#xACFC; pluripotent &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC758; undifferentiated &#xC0C1;&#xD0DC;&#xC758; &#xC885;&#xD569;&#xC801;&#xC778; &#xC2A4;&#xB0C5;&#xC20F;&#xC744; &#xC0DD;&#xC131; 8 hiPSC &#xB77C;&#xC778;&#xC5D0; &#xB300; &#xD55C; &#xB2E4;&#xC591; &#xD55C; &#xBD84;&#xC790; &#xD504;&#xB85C; &#xD30C;&#xC77C;&#xB9C1; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC744; &#xC218;&#xD589; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC720;&#xC804;&#xC790; &#xD45C;&#xD604; &#xB370;&#xC774;&#xD130; &#xBD84;&#xC11D; iPSC &#xD2B9;&#xC815; &#xC720;&#xC804;&#xC790; &#xC2DD; &#xD328;&#xD134;&#xC744; &#xB530;&#xB77C; &#xC774;&#xC804; &#xBCF4;&#xACE0;&#xC11C;&#xB97C; &#xACF5;&#xAC1C; &#xD588;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xB354; &#xC140;, 2 &#xBBF8;&#xB514;&#xC5B4; 20 &#xC131;&#xC778; &#xC870;&#xC9C1;&#xC73C;&#xB85C; &#xBCC4;&#xB3C4; &#xC140; &#xC6B4;&#xBA85;&#xC744; &#xD5A5;&#xD574; &#xCC28;&#xBCC4;&#xD654; &#xC2DC;&#xC791; &#xC81C;&#xC548; embryoid &#xC2DC;&#xCCB4;&#xB85C; &#xCC28;&#xBCC4;&#xD654; &#xB41C; &#xBE44;&#xAD50;. &#xC774; &#xBD84;&#xC11D;&#xC5D0;&#xC11C; &#xC6B0;&#xB9AC; &#xC815;&#xC758; pluripotency pluripotent &#xC0C1;&#xD0DC;&#xC5D0; &#xB300; &#xD55C; &#xCD08;&#xAE30; &#xACC4;&#xD68D;&#xC744; &#xC124;&#xC815; &#xD558;&#xB294; &#xC720;&#xC804;&#xC790; &#xBAA9;&#xB85D;&#xC744; &#xC0DD;&#xC131;. &#xB9C8;&#xC9C0;&#xB9C9;&#xC73C;&#xB85C;, &#xC6B0;&#xB9AC;&#xB294; &#xB3C5;&#xB9BD;&#xC801;&#xC778; &#xC138;&#xD3EC; &#xC6B4;&#xBA85; &#xD5A5;&#xD574; &#xC81C;&#xC548; &#xB41C; &#xBC14;&#xC774;&#xC5B4;&#xC2A4;&#xB97C; &#xBCF4;&#xC5EC; &#xB450; &#xBD84;&#xD654; &#xC870;&#xAC74; &#xD558;&#xC5D0;&#xC11C; &#xB18D;&#xCD95; &#xD558;&#xB294; &#xC720;&#xC804;&#xC790;&#xC758; &#xBAA9;&#xB85D;&#xC744; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;.

StemCellDB: &#xC778;&#xAC04;&#xC758; Pluripotent &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xB370;&#xC774;&#xD130;&#xBCA0;&#xC774;&#xC2A4; &#xAC74;&#xAC15;&#xC758; &#xAD6D;&#xB9BD; &#xC5F0;&#xAD6C;&#xC18C;&#xC5D0;&#xC11C;.

Gran parte de la emoci&oacute;n generada por la tecnolog&iacute;a de c&eacute;lulas madre pluripotentes inducidas se refiere a la posibilidad de enfermedad modelado as&iacute; como el potencial de la terapia celular personalizado. Sin embargo, para dedicarse a esto es importante comprender el estado pluripotente &#39;normal&#39;, incluyendo su variabilidad inherente. Hemos realizado varios ensayos perfiles moleculares para 21 hESCs y 8 hiPSC l&iacute;neas al generar una instant&aacute;nea completa del estado indiferenciado de c&eacute;lulas madre pluripotentes. El an&aacute;lisis de los datos de expresi&oacute;n gen&eacute;tica no revel&oacute; ning&uacute;n patr&oacute;n de expresi&oacute;n de genes espec&iacute;ficos de iPSC seg&uacute;n informes anteriores. Comparamos m&aacute;s c&eacute;lulas diferenciadas como cuerpos embryoid en 2 medios propuestos iniciar la diferenciaci&oacute;n hacia destinos separados de la c&eacute;lula, as&iacute; como 20 tejidos adultos. De este an&aacute;lisis hemos generado una lista del gen que define la pluripotencia y establece un punto de partida para el estado pluripotente. Por &uacute;ltimo, proporcionamos listas de genes enriquecidos en ambas condiciones de diferenciaci&oacute;n que demuestran el sesgo propuesto hacia destinos de c&eacute;lulas independientes.

StemCellDB: El humano Pluripotent Stem Cell base en los institutos nacionales de salud.

Many studies have compared the genetic and epigenetic profiles of human induced pluripotent stem cells (hiPSCs) to human embryonic stem cells (hESCs) and yet the picture remains unclear. To address this, we derived a population of neural precursor cells (NPCs) from the H1 (WA01) hESC line and generated isogenic iPSC lines by reprogramming. The gene expression and methylation profile of three lines were compared to the parental line and intermediate NPC population. We found no gene probe with expression that differed significantly between hESC and iPSC samples under undifferentiated or differentiated conditions. Analysis of the global methylation pattern also showed no significant difference between the two PSC populations. Both undifferentiated populations were distinctly different from the intermediate NPC population in both gene expression and methylation profiles. One point to note is that H1 is a male line and so extrapolation to female lines should be cautioned. However, these data confirm our previous findings that there are no significant differences between hESCs and hiPSCs at the gene expression or methylation level.

Comparison of the molecular profiles of human embryonic and induced pluripotent stem cells of isogenic origin.

Human pluripotent stem cells (hPSCs) provide powerful resources for application in regenerative medicine and pharmaceutical development. In the past decade, various methods have been developed for large-scale hPSC culture that rely on combined use of multiple growth components, including media containing various growth factors, extracellular matrices, 3D environmental cues, and modes of multicellular association. In this Protocol Review, we dissect these growth components by comparing cell culture methods and identifying the benefits and pitfalls associated with each one. We further provide criteria, considerations, and suggestions to achieve optimal cell growth for hPSC expansion, differentiation, and use in future therapeutic applications.

Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics.

Human pluripotent stem cells (hPSCs) have two potentially attractive applications: cell replacement-based therapies and drug discovery. Both require the efficient generation of large quantities of clinical-grade stem cells that are free from harmful genomic alterations. The currently employed colony-type culture methods often result in low cell yields, unavoidably heterogeneous cell populations, and substantial chromosomal abnormalities. Here, we shed light on the structural relationship between hPSC colonies/embryoid bodies and early-stage embryos in order to optimize current culture methods based on the insights from developmental biology. We further highlight core signaling pathways that underlie multiple epithelial-to-mesenchymal transitions (EMTs), cellular heterogeneity, and chromosomal instability in hPSCs. We also analyze emerging methods such as non-colony type monolayer (NCM) and suspension culture, which provide alternative growth models for hPSC expansion and differentiation. Furthermore, based on the influence of cell-cell interactions and signaling pathways, we propose concepts, strategies, and solutions for production of clinical-grade hPSCs, stem cell precursors, and miniorganoids, which are pivotal steps needed for future clinical applications.

Developmental insights from early mammalian embryos and core signaling pathways that influence human pluripotent cell growth and differentiation.

Human pluripotent stem cell (hPSC) lines exhibit repeated patterns of genetic variation, which can alter in vitro properties as well as suitability for clinical use. We examined associations between copy-number variations (CNVs) on chromosome 17 and hPSC mesodiencephalic dopaminergic (mDA) differentiation. Among 24 hPSC lines, two karyotypically normal lines, BG03 and CT3, and BG01V2, with trisomy 17, exhibited amplification of the WNT3/WNT9B region and rapid mDA differentiation. In hPSC lines with amplified WNT3/WNT9B, basic fibroblast growth factor (bFGF) signaling through mitogen-activated protein kinase (MAPK)/ERK amplifies canonical WNT signaling by phosphorylating LRP6, resulting in enhanced undifferentiated proliferation. When bFGF is absent, noncanonical WNT signaling becomes dominant due to upregulation of SIAH2, enhancing JNK signaling and promoting loss of pluripotency. When bFGF is present during mDA differentiation, stabilization of canonical WNT signaling causes upregulation of LMX1A and mDA induction. Therefore, CNVs in 17q21.31, a "hot spot" for genetic variation, have multiple and complex effects on hPSC cellular phenotype.

Functional consequences of 17q21.31/WNT3-WNT9B amplification in hPSCs with respect to neural differentiation.

Adoptive transfer of T cells genetically modified to express chimeric antigen receptors (CARs) targeting the CD19 B cell-associated protein have demonstrated potent activity against relapsed/refractory B-lineage acute lymphoblastic leukemia (B-ALL). Not all patients respond, and CD19-negative relapses have been observed. Overexpression of the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) occurs in a subset of adults and children with B-ALL and confers a high risk of relapse. Recent data suggest the TSLPR signaling axis is functionally important, suggesting that TSLPR would be an ideal immunotherapeutic target. We constructed short and long CARs targeting TSLPR and tested efficacy against CRLF2-overexpressing B-ALL. Both CARs demonstrated activity in vitro, but only short TSLPR CAR T cells mediated leukemia regression. In vivo activity of the short CAR was also associated with long-term persistence of CAR-expressing T cells. Short TSLPR CAR treatment of mice engrafted with a TSLPR-expressing ALL cell line induced leukemia cytotoxicity with efficacy comparable with that of CD19 CAR T cells. Short TSLPR CAR T cells also eradicated leukemia in 4 xenograft models of human CRLF2-overexpressing ALL. Finally, TSLPR has limited surface expression on normal tissues. TSLPR-targeted CAR T cells thus represent a potent oncoprotein-targeted immunotherapy for high-risk ALL.

Eradication of B-ALL using chimeric antigen receptor-expressing T cells targeting the TSLPR oncoprotein.

Because of unavoidable confounding variables in the direct study of human subjects, it has been difficult to unravel the effects of prenatal cocaine exposure on the human fetal brain, as well as the cellular and biochemical mechanisms involved. Here, we propose a novel approach using a human pluripotent stem cell (hPSC)-based 3D neocortical organoid model. This model retains essential features of human neocortical development by encompassing a single self-organized neocortical structure, without including an animal-derived gelatinous matrix. We reported previously that prenatal cocaine exposure to rats during the most active period of neural progenitor proliferation induces cytoarchitectural changes in the embryonic neocortex. We also identified a role of CYP450 and consequent oxidative ER stress signaling in these effects. However, because of differences between humans and rodents in neocorticogenesis and brain CYP metabolism, translation of the research findings from the rodent model to human brain development is uncertain. Using hPSC 3D neocortical organoids, we demonstrate that the effects of cocaine are mediated through CYP3A5-induced generation of reactive oxygen species, inhibition of neocortical progenitor cell proliferation, induction of premature neuronal differentiation, and interruption of neural tissue development. Furthermore, knockdown of CYP3A5 reversed these cocaine-induced pathological phenotypes, suggesting CYP3A5 as a therapeutic target to mitigate the deleterious neurodevelopmental effects of prenatal cocaine exposure in humans. Moreover, 3D organoid methodology provides an innovative platform for identifying adverse effects of abused psychostimulants and pharmaceutical agents, and can be adapted for use in neurodevelopmental disorders with genetic etiologies.

Barbara Mallon

NIH Stem Cell Unit,
Bethesda,
NIH Stem Cell Unit, Bethesda

National Institutes of Health

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Barbara Mallon

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National Institutes of Health

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

NIH Stem Cell Unit,
Bethesda,
NIH Stem Cell Unit, Bethesda