A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long ...

Icahn School of Medicine at Mount Sinai

Schizophrenia (SCZD) is a debilitating neurological disorder with a world-wide prevalence of 1%; there is a strong genetic component, with an estimated heritability of 80-85%. Although post-mortem studies have revealed reduced brain volume, cell size, spine density and abnormal neural distribution in the prefrontal cortex and hippocampus of SCZD brain tissue and neuropharmacological studies have implicated dopaminergic, glutamatergic and GABAergic activity in SCZD, the cell types affected in SCZD and the molecular mechanisms underlying the disease state remain unclear. To elucidate the cellular and molecular defects of SCZD, we directly reprogrammed fibroblasts from SCZD patients into human induced pluripotent stem cells (hiPSCs) and subsequently differentiated these disorder-specific hiPSCs into neurons (Supplementary Fig. 1). SCZD hiPSC neurons showed diminished neuronal connectivity in conjunction with decreased neurite number, PSD95-protein levels and glutamate receptor expression. Gene expression profiles of SCZD hiPSC neurons identified altered expression of many components of the cyclic AMP and WNT signalling pathways. Key cellular and molecular elements of the SCZD phenotype were ameliorated following treatment of SCZD hiPSC neurons with the antipsychotic loxapine. To date, hiPSC neuronal pathology has only been demonstrated in diseases characterized by both the loss of function of a single gene product and rapid disease progression in early childhood. We now report hiPSC neuronal phenotypes and gene expression changes associated with SCZD, a complex genetic psychiatric disorder.

Modelling schizophrenia using human induced pluripotent stem cells.

&#x901A;&#x8FC7;&#x6A21;&#x4EFF;&#x80DA;&#x80CE;&#x53D1;&#x80B2;&#x7684;&#x9020;&#x8840;&#x7CFB;&#x7EDF;&#xFF0C;&#x6211;&#x4EEC;&#x5F00;&#x53D1;&#x4E86;&#x9020;&#x8840;&#x7684;&#x8840;&#x7EDF;&#x548C;&#x539F;&#x59CB;&#x7684;&#x9020;&#x8840;&#x7EC6;&#x80DE;&#xFF0C;&#x4ECE;&#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE; &#xFF08;ESC&#xFF09; &#x548C;&#x8BF1;&#x5BFC;&#x591A;&#x80FD;&#x5E72;&#x7EC6;&#x80DE; &#xFF08;&#x9B54;&#x9B3C;&#xFF09; &#x7684;&#x524D;&#x4F53;&#x7684;&#x4EE3;&#x7684;&#x4F18;&#x5316;&#x4F53;&#x5916;&#x5206;&#x5316;&#x8BAE;&#x5B9A;&#x4E66; &#x300B;&#x3002;&#x7EC6;&#x80DE;&#x56E0;&#x5B50;&#x3001; &#x7EC6;&#x80DE;&#x5916;&#x57FA;&#x8D28;&#x6210;&#x5206;&#xFF0C;&#x548C;&#x5C0F;&#x5206;&#x5B50;&#xFF0C;&#x4EE5;&#x53CA;&#x65F6;&#x6001;&#x5173;&#x8054;&#x548C;&#x8FD9;&#x4E9B;&#x56E0;&#x7D20;&#x7684;&#x6D53;&#x5EA6;&#x7B49;&#x56E0;&#x7D20;&#x5BF9;&#x4E03;&#x4E2A;&#x4E0D;&#x540C;&#x7684;&#x4EBA;&#x7C7B; ESC &#x548C;&#x673A;&#x4F53;&#x529F;&#x80FD;&#x7EBF;&#x8FDB;&#x884C;&#x4E86;&#x6D4B;&#x8BD5;&#x3002;&#x6211;&#x4EEC;&#x62A5;&#x544A;&#x8FBE; 84%&#x4EBA;&#x7C7B; CD45 + &#x7EC6;&#x80DE;&#x5206;&#x5316; &#xFF08;&#x5E73;&#x5747; 41%&plusmn; 16%&#xFF0C;&#x4ECE;&#x4E03;&#x591A;&#x80FD;&#x5E72;&#x884C;&#xFF09; &#x4ECE;&#x5206;&#x5316;&#x6587;&#x5316;&#xFF0C;&#x5176;&#x4E2D;&#x5305;&#x62EC;&#x76F8;&#x5F53;&#x6570;&#x91CF;&#x7684;&#x539F;&#x59CB; CD45 + /cd34 + &#x548C; CD45 + / CD34 + CD38 &#x9020;&#x8840;&#x7956;&#x7EC6;&#x80DE;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x9020;&#x8840;&#x7956;&#x7EC6;&#x80DE;&#x751F;&#x6210;&#xFF0C;&#x83CC;&#x843D;&#x5F62;&#x6210;&#x5355;&#x4F4D;&#x68C0;&#x6D4B;&#x65B9;&#x6CD5;&#xFF0C;&#x4EE5;&#x8861;&#x91CF;&#x6570;&#x5B57;&#x662F;&#x76F8;&#x5F53;&#x4E8E;&#x4ECE;&#x65B0;&#x9C9C;&#x8110;&#x5E26;&#x8840;&#x5355;&#x4E2A;&#x6838;&#x7EC6;&#x80DE;&#x682A;&#x4E0A;&#x83B7;&#x5F97;&#x7684;&#x6570;&#x5B57;&#x6BCF; CD45 + &#x7EC6;&#x80DE;&#x57FA;&#x7840;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x65B9;&#x6CD5;&#x6F14;&#x793A;&#x591A;&#x80FD;&#x5E72;&#x9020;&#x8840;&#x7956;&#x7EC6;&#x80DE;&#x4E0E;&#x62A5;&#x544A;&#x5230;&#x76EE;&#x524D;&#x4E3A;&#x6B62;&#x7684;&#x6700;&#x9AD8;&#x6548;&#x7387;&#x4E4B;&#x95F4;&#x7684;&#x9AD8;&#x6548;&#x53D1;&#x7535; (CD45 + /cd34 +) &#x4EBA;&#x7C7B; ESC &#x548C;&#x673A;&#x4F53;&#x529F;&#x80FD;&#x7684;&#x82E5;&#x5E72;&#x884C;&#x4E0A;&#x4F7F;&#x7528;&#x5355;&#x4E2A;&#x6807;&#x51C6;&#x5316;&#x7684;&#x5206;&#x5316;&#x7684;&#x534F;&#x8BAE;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x6570;&#x636E;&#x6DFB;&#x52A0;&#x5230;&#x84C4;&#x8BC1;&#x636E;&#x5B58;&#x5728;&#x7684;&#x4F53;&#x5916;&#x9020;&#x8840;&#x5E72;&#x7EC6;&#x80DE; &#xFF08;HSC&#xFF09; &#x7684;&#x6D3E;&#x751F;&#x524D;&#x5146;&#x5AC1;&#x63A5;&#x79FB;&#x690D;&#x5C0F;&#x9F20;&#x7684;&#x80FD;&#x529B;&#x6709;&#x9650;&#xFF0C;&#x4F46;&#x6709;&#x591A;&#x80FD;&#x5E72;&#x9020;&#x8840;&#x7684;&#x53EF;&#x80FD;&#x3002;&#x56E0;&#x4E3A;&#x672C;&#x8BAE;&#x5B9A;&#x4E66;&#x6709;&#x6548;&#x5730;&#x6269;&#x5927;&#x4E86; preblood &#x524D;&#x4F53;&#x548C;&#x9020;&#x8840;&#x7956;&#x7EC6;&#x80DE;&#xFF0C;&#x5B83;&#x975E;&#x5E38;&#x9002;&#x5408;&#x4E8E;&#x6D4B;&#x8BD5;&#x65B0;&#x578B;&#x56E0;&#x7D20;&#x7684;&#x4EA7;&#x751F;&#x548C;&#x6269;&#x5C55;&#x7684;&#x6700;&#x7EC8;&#x661F;&#x4E0E;&#x957F;&#x671F; repopulating &#x80FD;&#x529B;&#x3002;

&#x7B80;&#x62A5;&#xFF1A; &#x9AD8;&#x6548;&#x751F;&#x6210;&#x9020;&#x8840;&#x524D;&#x4F53;&#x53CA;&#x4ECE;&#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x7684;&#x7956;&#x7EC6;&#x80DE;&#x3002;

By mimicking embryonic development of the hematopoietic system, we have developed an optimized in vitro differentiation protocol for the generation of precursors of hematopoietic lineages and primitive hematopoietic cells from human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSCs). Factors such as cytokines, extra cellular matrix components, and small molecules as well as the temporal association and concentration of these factors were tested on seven different human ESC and iPSC lines. We report the differentiation of up to 84% human CD45+ cells (average 41% Â± 16%, from seven pluripotent lines) from the differentiation culture, including significant numbers of primitive CD45+/CD34+ and CD45+/CD34+/CD38- hematopoietic progenitors. Moreover, the numbers of hematopoietic progenitor cells generated, as measured by colony forming unit assays, were comparable to numbers obtained from fresh umbilical cord blood mononuclear cell isolates on a per CD45+ cell basis. Our approach demonstrates highly efficient generation of multipotent hematopoietic progenitors with among the highest efficiencies reported to date (CD45+/CD34+) using a single standardized differentiation protocol on several human ESC and iPSC lines. Our data add to the cumulating evidence for the existence of an in vitro derived precursor to the hematopoietic stem cell (HSC) with limited engrafting ability in transplanted mice but with multipotent hematopoietic potential. Because this protocol efficiently expands the preblood precursors and hematopoietic progenitors, it is ideal for testing novel factors for the generation and expansion of definitive HSCs with long-term repopulating ability.

Brief report: efficient generation of hematopoietic precursors and progenitors from human pluripotent stem cell lines.

En imitant le d&#xE9;veloppement embryonnaire du syst&#xE8;me h&#xE9;matopo&#xEF;&#xE9;tique, nous avons d&#xE9;velopp&#xE9; un. Optimis&#xE9; dans le protocole de diff&#xE9;renciation in vitro pour la g&#xE9;n&#xE9;ration des pr&#xE9;curseurs des lign&#xE9;es h&#xE9;matopo&#xEF;&#xE9;tiques et les cellules h&#xE9;matopo&#xEF;&#xE9;tiques primitives &#xE0; partir de cellules souches embryonnaires (ESC) et cellules souches pluripotentes induites (CISP) Des facteurs tels que les cytokines, des composants matriciels suppl&#xE9;mentaires cellulaires, et des petites mol&#xE9;cules ainsi que l&#x26;#39;association temporelle et de la concentration de ces facteurs ont &#xE9;t&#xE9; test&#xE9;s sur sept diff&#xE9;rents CSE humaines et des lignes IPSC. Nous rapportons la diff&#xE9;renciation d&#x26;#39;un maximum de 84% humaine CD45 + cellules (en moyenne 41% &#xB1; 16%, passant de sept lignes pluripotentes) de la culture de diff&#xE9;renciation, y compris un nombre important de CD45 primitive + / CD34 + et CD45 + / CD34 + / CD38-h&#xE9;matopo&#xEF;&#xE9;tiques prog&#xE9;nitrices. En outre, le nombre de cellules prog&#xE9;nitrices h&#xE9;matopo&#xEF;&#xE9;tiques g&#xE9;n&#xE9;r&#xE9;s, tels que mesur&#xE9;s par des tests Unit&#xE9; Formant Colonie, &#xE9;taient comparables &#xE0; des num&#xE9;ros obtenus &#xE0; partir de nouvelles souches du cordon ombilical des cellules sanguines mononucl&#xE9;aires sur un CD45 + par la base de cellules. Notre approche d&#xE9;montre g&#xE9;n&#xE9;ration hautement efficace de prog&#xE9;niteurs h&#xE9;matopo&#xEF;&#xE9;tiques multipotentes avec parmi les meilleurs rendements rapport&#xE9;s &#xE0; ce jour (CD45 + / CD34 +) en utilisant un protocole de diff&#xE9;renciation unique standardis&#xE9; sur plusieurs lignes et les CSE humaines IPSC. Nos donn&#xE9;es ajouter &#xE0; la preuve de cumuler de l&#x26;#39;existence d&#x26;#39;un embryon in vitro d&#xE9;riv&#xE9;e pr&#xE9;curseur de la cellule souche h&#xE9;matopo&#xEF;&#xE9;tique (CSH) avec la capacit&#xE9; limit&#xE9;e en greffant des souris transplant&#xE9;es, mais avec un potentiel h&#xE9;matopo&#xEF;&#xE9;tique pluripotente. Parce que ce protocole &#xE9;tend efficacement les pr&#xE9;curseurs preblood et prog&#xE9;niteurs h&#xE9;matopo&#xEF;&#xE9;tiques, il est id&#xE9;al pour tester de nouveaux facteurs pour la g&#xE9;n&#xE9;ration et l&#x26;#39;expansion des cellules souches h&#xE9;matopo&#xEF;&#xE9;tiques d&#xE9;finitifs &#xE0; long terme la capacit&#xE9; de repeupler.

Bref rapport: la production efficace des pr&#xE9;curseurs h&#xE9;matopo&#xEF;&#xE9;tiques et les prog&#xE9;niteurs de lignes cellules souches pluripotentes humaines

Durch die Nachahmung von embryonale Entwicklung des h&auml;matopoetischen Systems, haben wir eine optimierte in-vitro-Differenzierung-Protokoll f&uuml;r die Generation der Vorl&auml;ufer der h&auml;matopoetischen &Uuml;bertragungslinien und primitiven blutbildender Zellen aus menschlichen embryonalen Stammzellen (ESC) und induzierten pluripotenten Stammzellen (iPSCs) entwickelt. Faktoren wie Zytokine, zus&auml;tzliche zellul&auml;re Matrix-Komponenten, und kleine Molek&uuml;le sowie die zeitliche Zuordnung und Konzentration dieser Faktoren wurden auf sieben verschiedenen menschlichen ESC und iPSC Linien getestet. Wir berichten die Differenzierung von bis zu 84 % CD45 + Humanzellen (durchschnittlich 41 % &plusmn; 16 %, aus sieben Pluripotent Linien) aus der Differenzierung-Kultur, die erhebliche Anzahl dieser primitiven CD45 + / CD34 + und CD45 + / CD34 + / CD38 - h&auml;matopoetischen Vorl&auml;uferzellen. Dar&uuml;ber hinaus waren die Nummern der h&auml;matopoetischen Vorl&auml;uferzellen generiert, gemessen an der Kolonie bilden Einheit Proben, vergleichbar mit Zahlen aus frischen Nabelschnur Blut mononukle&auml;ren Zellen pur auf ein pro Zelle Basis CD45 +. Unsere Vorgehensweise hoch effiziente Erzeugung von multipotenten h&auml;matopoetischen Vorl&auml;uferzellen mit unter die h&ouml;chste Wirkungsgrade bisher gemeldet (CD45 + / CD34 +) mit einem einheitlichen standardisierten Differenzierung-Protokoll auf verschiedene menschliche ESC und iPSC-Linien. Unsere Daten hinzuf&uuml;gen der cumulating Beweis f&uuml;r die Existenz eines in-vitro-abgeleiteten Vorl&auml;ufer der h&auml;matopoetischen Stammzellen (HSC) mit begrenzten engrafting F&auml;higkeit transplantierten M&auml;usen aber mit multipotenten h&auml;matopoetischen Potenzial. Da dieses Protokoll effizient die preblood Grundstoffe und h&auml;matopoetischen Vorl&auml;uferzellen erweitert, ist es ideal zum Testen von Roman Faktoren f&uuml;r die Generierung und Erweiterung des endg&uuml;ltigen HSCs mit langfristigen repopulating F&auml;higkeit.

Kurzer Bericht: effiziente Erzeugung von h&auml;matopoetischen Vorl&auml;ufer und Vorl&auml;uferzellen aus menschlichen pluripotenten Stammzelllinien.

Imitando lo sviluppo embrionale del sistema ematopoietico, abbiamo sviluppato un protocollo ottimizzato differenziazione in vitro per la generazione dei precursori dei lignaggi ematopoietici e primitive cellule ematopoietiche da cellule staminali embrionali umane (ESC) e cellule staminali pluripotenti indotte (iPSCs). Fattori come citochine, componenti della matrice extra cellulare e piccole molecole come pure l&#39;associazione temporale e concentrazione di questi fattori sono stati testati su sette umani ESC e iPSC linee diverse. Riportiamo la differenziazione di fino a 84% CD45 + cellule umane (media 41% &plusmn; 16%, da sette linee pluripotenti) dalla cultura di differenziazione, tra cui un numero significativo di primitivo CD45 + CD34 + e CD45 + CD34 + CD38 - progenitori ematopoietici. Inoltre, il numero di cellule progenitrici ematopoietiche generati, come misurato dalla colonia formando dosaggi di unit&agrave;, era paragonabile ai numeri ottenuti dal cordone ombelicale fresco sangue cellule mononucleate isolati su un CD45 + base al cell. Il nostro approccio dimostra altamente efficiente generazione di progenitrici emopoietiche multipotenti con tra le pi&ugrave; alte efficienze riferite alla data (CD45 + CD34 +) utilizza un protocollo unico differenziazione standardizzato su diverse linee ESC e iPSC umane. I nostri dati aggiungere le cumulating prove dell&#39;esistenza di un precursore della derivata in vitro le cellule staminali ematopoietiche (HSC) con limitate capacit&agrave; di attecchimento in topi trapiantati ma con potenziale emopoietiche multipotenti. Perch&eacute; questo protocollo si espande in modo efficiente i precursori preblood e progenitori ematopoietici, &egrave; ideale per testare nuovi fattori per la generazione e l&#39;espansione di HSCs definitivo con capacit&agrave; ripopolante a lungo termine.

Breve relazione: generazione efficiente di precursori ematopoietici e progenitori da linee di cellule staminali pluripotenti umane.

&#x9020;&#x8840;&#x7CFB;&#x306E;&#x80DA;&#x767A;&#x751F;&#x3092;&#x6A21;&#x5023;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x3001;&#x6211;&#x3005;&#x306F;&#x3001;&#x9020;&#x8840;&#x7CFB;&#x7D71;&#x304A;&#x3088;&#x3073;&#x30D2;&#x30C8;&#x80DA;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#xFF08;ESC&#xFF09;&#x3068;&#x4EBA;&#x5DE5;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#xFF08;&#x6027;IPSC&#xFF09;&#x304B;&#x3089;&#x539F;&#x59CB;&#x9020;&#x8840;&#x7D30;&#x80DE;&#x306E;&#x524D;&#x99C6;&#x4F53;&#x3092;&#x751F;&#x6210;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;in vitro&#x3067;&#x306E;&#x5206;&#x5316;&#x30D7;&#x30ED;&#x30C8;&#x30B3;&#x30EB;&#x306B;&#x6700;&#x9069;&#x5316;&#x3055;&#x308C;&#x958B;&#x767A;&#x3057;&#x3066;&#x304D;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x306E;&#x3088;&#x3046;&#x306A;&#x30B5;&#x30A4;&#x30C8;&#x30AB;&#x30A4;&#x30F3;&#x3001;&#x7D30;&#x80DE;&#x5916;&#x30DE;&#x30C8;&#x30EA;&#x30C3;&#x30AF;&#x30B9;&#x6210;&#x5206;&#x3001;&#x304A;&#x3088;&#x3073;&#x5C0F;&#x5206;&#x5B50;&#x3068;&#x540C;&#x69D8;&#x306B;&#x3053;&#x308C;&#x3089;&#x306E;&#x8981;&#x56E0;&#x306E;&#x6642;&#x9593;&#x7684;&#x95A2;&#x9023;&#x6027;&#x3084;&#x6FC3;&#x5EA6;&#x306A;&#x3069;&#x306E;&#x8981;&#x56E0;&#x306F;&#x3001;7&#x3064;&#x306E;&#x7570;&#x306A;&#x308B;&#x30D2;&#x30C8;ESC&#x3068;IPSC&#x30E9;&#x30A4;&#x30F3;&#x3067;&#x30C6;&#x30B9;&#x30C8;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x6211;&#x3005;&#x306F;&#x539F;&#x59CB;&#x7684;CD45&#x306E;&#x91CD;&#x8981;&#x306A;&#x6570;&#x5B57;+ / CD34 +&#x304A;&#x3088;&#x3073;CD45 + / CD34 + / CD38-&#x9020;&#x8840;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x3092;&#x542B;&#x3080;&#x5206;&#x5316;&#x57F9;&#x990A;&#x6DB2;&#x304B;&#x3089;&#x6700;&#x5927;84&#xFF05;&#x30D2;&#x30C8;CD45&#x306E;&#x967D;&#x6027;&#x7D30;&#x80DE;&#xFF08;&#x5E73;&#x5747;41&#xFF05;&#xB1;16&#xFF05;&#x3001;7&#x591A;&#x80FD;&#x30E9;&#x30A4;&#x30F3;&#x304B;&#x3089;&#xFF09;&#x306E;&#x5206;&#x5316;&#x3092;&#x5831;&#x544A;&#x3057;&#x307E;&#x3059;&#x3002;&#x307E;&#x305F;&#x3001;&#x751F;&#x6210;&#x3055;&#x308C;&#x305F;&#x9020;&#x8840;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x306E;&#x6570;&#x306F;&#x3001;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x306E;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x3092;&#x30B3;&#x30ED;&#x30CB;&#x30FC;&#x5F62;&#x6210;&#x306B;&#x3088;&#x3063;&#x3066;&#x6E2C;&#x5B9A;&#x3055;&#x308C;&#x305F;&#x3001;&#x4E00;&#x4EBA;&#x5F53;&#x305F;&#x308A;CD45 +&#x7D30;&#x80DE;&#x3054;&#x3068;&#x306B;&#x65B0;&#x9BAE;&#x306A;&#x81CD;&#x5E2F;&#x8840;&#x5358;&#x6838;&#x7D30;&#x80DE;&#x306E;&#x5206;&#x96E2;&#x682A;&#x304B;&#x3089;&#x5F97;&#x3089;&#x308C;&#x305F;&#x6570;&#x5024;&#x306B;&#x5339;&#x6575;&#x3057;&#x305F;&#x3002;&#x6211;&#x3005;&#x306E;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x306F;&#x3053;&#x308C;&#x307E;&#x3067;&#x306B;&#x5831;&#x544A;&#x3055;&#x308C;&#x305F;&#x6700;&#x9AD8;&#x306E;&#x52B9;&#x7387;&#x306E;&#x9593;&#x3067;&#x591A;&#x80FD;&#x6027;&#x9020;&#x8840;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x306E;&#x9AD8;&#x52B9;&#x7387;&#x751F;&#x6210;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#xFF08;CD45 + / CD34 +&#xFF09;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x30D2;&#x30C8;ESC&#x3068;IPSC&#x30E9;&#x30A4;&#x30F3;&#x4E0A;&#x306E;&#x5358;&#x4E00;&#x306E;&#x6A19;&#x6E96;&#x5316;&#x3055;&#x308C;&#x305F;&#x5206;&#x5316;&#x30D7;&#x30ED;&#x30C8;&#x30B3;&#x30EB;&#x3092;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x3002;&#x6211;&#x3005;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x3001;&#x79FB;&#x690D;&#x30DE;&#x30A6;&#x30B9;&#x3067;&#x306F;&#x9650;&#x3089;&#x308C;&#x305F;&#x79FB;&#x690D;&#x80FD;&#x529B;&#x3092;&#x6301;&#x3064;&#x304C;&#x591A;&#x80FD;&#x6027;&#x9020;&#x8840;&#x6027;&#x306E;&#x3042;&#x308B;&#x9020;&#x8840;&#x5E79;&#x7D30;&#x80DE;&#xFF08;HSC&#xFF09;&#x306B;&#x524D;&#x99C6;&#x4F53;&#x7531;&#x6765;&#x306E;in vitro&#x3067;&#x306E;&#x5B58;&#x5728;&#x306E;&#x305F;&#x3081;&#x306E;&#x7D2F;&#x7A4D;&#x8A3C;&#x62E0;&#x306B;&#x8FFD;&#x52A0;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x30D7;&#x30ED;&#x30C8;&#x30B3;&#x30EB;&#x306F;&#x52B9;&#x7387;&#x7684;&#x306B;preblood&#x524D;&#x99C6;&#x4F53;&#x304A;&#x3088;&#x3073;&#x9020;&#x8840;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x3092;&#x62E1;&#x5927;&#x3057;&#x3066;&#x3044;&#x308B;&#x306E;&#x3067;&#x3001;&#x9577;&#x671F;&#x7684;&#x306A;&#x518D;&#x5897;&#x6B96;&#x80FD;&#x529B;&#x3092;&#x6301;&#x3064;&#x6C7A;&#x5B9A;&#x7684;&#x306A;&#x9020;&#x8840;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x767A;&#x751F;&#x3068;&#x62E1;&#x5927;&#x306E;&#x305F;&#x3081;&#x306E;&#x65B0;&#x898F;&#x306A;&#x56E0;&#x5B50;&#x3092;&#x30C6;&#x30B9;&#x30C8;&#x3059;&#x308B;&#x306E;&#x306B;&#x7406;&#x60F3;&#x7684;&#x3067;&#x3059;&#x3002;

&#x77ED;&#x3044;&#x30EC;&#x30DD;&#x30FC;&#x30C8;&#xFF1A;&#x30D2;&#x30C8;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x304B;&#x3089;&#x9020;&#x8840;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x3068;&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x306E;&#x52B9;&#x7387;&#x7684;&#x306A;&#x751F;&#x6210;

&#xC870; &#xD608; &#xC2DC;&#xC2A4;&#xD15C;&#xC758; &#xBC30;&#xC544; &#xBC1C;&#xB2EC;&#xC744; &#xD749;&#xB0B4; &#xB0B8;&#xC5D0; &#xC758;&#xD574; &#xC870; &#xD608; &#xACC4;&#xBCF4;&#xC640; &#xC778;&#xAC04; &#xBC30;&#xC544; &#xC904;&#xAE30; &#xC138;&#xD3EC; (ESC)&#xC640; &#xC720;&#xB3C4; &#xB9CC;&#xB2A5; &#xC904;&#xAE30; &#xC138;&#xD3EC; (iPSCs)&#xC758; &#xC6D0;&#xC2DC; &#xC870; &#xD608; &#xC138;&#xD3EC;&#xC758; &#xC804;&#xC870;&#xC758; &#xC138;&#xB300;&#xB97C; &#xC704;&#xD55C; &#xCD5C;&#xC801;&#xD654; &#xB41C; &#xCCB4; &#xC678; &#xBD84;&#xD654; &#xD504;&#xB85C;&#xD1A0;&#xCF5C;&#xC744; &#xAC1C;&#xBC1C; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. Cytokines, &#xC5EC;&#xBD84;&#xC758; &#xC138;&#xD3EC; &#xB9E4;&#xD2B8;&#xB9AD;&#xC2A4; &#xAD6C;&#xC131; &#xC694;&#xC18C; &#xBC0F; &#xC791;&#xC740; &#xBD84;&#xC790;&#xB3C4; &#xCE21; &#xB450; &#xC5FD; &#xD611;&#xD68C; &#xBC0F; &#xC774;&#xB7EC;&#xD55C; &#xC694;&#xC18C;&#xC758; &#xB18D;&#xB3C4; &#xB4F1;&#xC758; &#xC694;&#xC778;&#xC5D0;&#xC11C; 7 &#xAC1C;&#xC758; &#xB2E4;&#xB978; &#xC778;&#xAC04;&#xC758; ESC &#xBC0F; iPSC &#xB77C;&#xC778;&#xC5D0;&#xC11C; &#xD14C;&#xC2A4;&#xD2B8; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC; &#xBCF4;&#xACE0; &#xCD5C;&#xB300; 84 &#xC140; % &#xC778;&#xAC04;&#xC758; CD45 + (&#xD3C9;&#xADE0; 41% &plusmn; 16%, 7 pluripotent &#xC120;&#xC5D0;&#xC11C;) &#xAE30;&#xBCF8; cd45&#xC758; &#xC0C1;&#xB2F9;&#xD55C; &#xC22B;&#xC790;&#xB97C; &#xD3EC;&#xD568; &#xD558; &#xC5EC; &#xCC28;&#xBCC4;&#xD654; &#xBB38;&#xD654;&#xC5D0;&#xC11C; &#xCC28;&#xBCC4;&#xD654; + CD34 + &#xBC0F; CD45 + CD34 + / CD38-&#xC870; &#xD608; progenitors. &#xB610;&#xD55C;, &#xC870; &#xD608; &#xC870;&#xC0C1; &#xC138;&#xD3EC; &#xC0DD;&#xC131;, &#xCF5C;&#xB85C; &#xB2C8; &#xD615;&#xC131; &#xB2E8;&#xC704; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC5D0; &#xC758;&#xD574; &#xCE21;&#xC815; &#xB41C; &#xC22B;&#xC790;&#xC5D0; &#xC2E0;&#xC120;&#xD55C; &#xD0EF; &#xD608;&#xC561; &#xB2E8; &#xC138;&#xD3EC; &#xACA9;&#xB9AC;&#xC5D0;&#xC11C; &#xC5BB;&#xC740; &#xC22B;&#xC790;&#xB97C; &#xBE44;&#xAD50; &#xD588;&#xB2E4;&#xB294; CD45 + &#xC138;&#xD3EC; &#xB2E8;&#xC704;&#xB85C; &#xB2F9;. &#xC6B0;&#xB9AC;&#xC758; &#xC811;&#xADFC; &#xBC29;&#xC2DD;&#xC744; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4; &#xB0A0;&#xC9DC;&#xC5D0; &#xBCF4;&#xACE0; &#xD558;&#xB294; &#xAC00;&#xC7A5; &#xB192;&#xC740; &#xD6A8;&#xC728;&#xC131; &#xC0AC;&#xC774; multipotent hematopoietic progenitors&#xC758; &#xACE0;&#xD6A8;&#xC728; &#xC138;&#xB300; (CD45 + CD34 +) &#xC5EC;&#xB7EC; &#xAC00;&#xC9C0; &#xC778;&#xAC04;&#xC758; ESC &#xBC0F; iPSC &#xB77C;&#xC778;&#xC5D0; &#xB2E8;&#xC77C; &#xD45C;&#xC900;&#xD654; &#xBD84;&#xD654; &#xD504;&#xB85C;&#xD1A0;&#xCF5C;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC;. &#xC6B0;&#xB9AC;&#xC758; &#xB370;&#xC774;&#xD130; &#xC774;&#xC2DD;&#xB41C; &#xC0DD;&#xC950;&#xC5D0;&#xC11C; engrafting &#xB2A5;&#xB825;&#xC744; &#xC81C;&#xD55C; &#xD558;&#xC9C0;&#xB9CC; multipotent hematopoietic &#xC7A0;&#xC7AC;&#xB825; &#xC870; &#xD608; &#xC904;&#xAE30; &#xC138;&#xD3EC; (HSC)&#xC744; &#xCCB4; &#xC678; &#xD30C;&#xC0DD;&#xB41C; &#xC804;&#xC870;&#xC758; &#xC874;&#xC7AC;&#xC5D0; &#xB300; &#xD55C; cumulating &#xAE30;&#xB85D;&#xC5D0; &#xCD94;&#xAC00;&#xD569;&#xB2C8;&#xB2E4;. &#xC774; &#xD504;&#xB85C;&#xD1A0;&#xCF5C; &#xD6A8;&#xC728;&#xC801;&#xC73C;&#xB85C; preblood &#xC720;&#xB3C4;&#xCCB4;&#xC640; &#xC870; &#xD608; progenitors &#xD655;&#xC7A5; &#xD558;&#xAE30; &#xB54C;&#xBB38;&#xC5D0; &#xC7A5;&#xAE30;&#xC801;&#xC778; repopulating &#xB2A5;&#xB825; &#xC0DD;&#xC131; &#xBC0F; &#xCD5C;&#xC885; Hscs&#xC758; &#xD655;&#xC7A5;&#xC5D0; &#xB300; &#xD55C; &#xC18C;&#xC124; &#xC694;&#xC18C;&#xB97C; &#xD14C;&#xC2A4;&#xD2B8;&#xC5D0; &#xC774;&#xC0C1;&#xC801;&#xC785;&#xB2C8;&#xB2E4;.

&#xBCF4;&#xACE0;&#xC11C;&#xC5D0; &#xB300; &#xD55C; &#xAC04;&#xB2E8;&#xD55C;: &#xC870; &#xD608; &#xC804;&#xC870;&#xC640; &#xC778;&#xAC04;&#xC758; pluripotent &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xB77C;&#xC778;&#xC5D0;&#xC11C; progenitors&#xC758; &#xD6A8;&#xC728;&#xC801;&#xC778; &#xC0DD;&#xC131;.

Al imitar el desarrollo embrionario del sistema hematopoy&#xE9;tico, hemos desarrollado una. Optimizado en el protocolo de diferenciaci&#xF3;n in vitro para la generaci&#xF3;n de los precursores de los linajes hematopoy&#xE9;ticos y c&#xE9;lulas hematopoy&#xE9;ticas primitivas de c&#xE9;lulas madre embrionarias humanas (ESC) y c&#xE9;lulas madre pluripotentes inducidas (iPSCs) Factores tales como las citoquinas, extra componentes de la matriz celular, y las mol&#xE9;culas peque&#xF1;as, as&#xED; como la asociaci&#xF3;n temporal y la concentraci&#xF3;n de estos factores fueron probados en siete diferentes humano ESC y l&#xED;neas IPSC. Se presenta la diferenciaci&#xF3;n de hasta un 84% humano CD45 + en las c&#xE9;lulas (promedio de 41% &#xB1; 16%, pasando de siete l&#xED;neas pluripotentes) a partir de la cultura de la diferenciaci&#xF3;n, incluyendo un n&#xFA;mero significativo de primitiva CD45 + / CD34 + y CD45 + / CD34 + / CD38-progenitores hematopoy&#xE9;ticos. Adem&#xE1;s, el n&#xFA;mero de c&#xE9;lulas progenitoras hematopoy&#xE9;ticas generados, medidos por ensayos de colonias de unidades que forman, fueron comparables a los n&#xFA;meros obtenidos a partir de frescas umbilicales aislados de sangre de cord&#xF3;n de c&#xE9;lulas mononucleares en una base por CD45 + c&#xE9;lulas. Nuestro enfoque muestra la generaci&#xF3;n de alta eficiencia de los progenitores hematopoy&#xE9;ticos multipotentes, con uno de los mayores eficiencias reportadas hasta la fecha (CD45 + / CD34 +) usando un &#xFA;nico protocolo estandarizado en la diferenciaci&#xF3;n de varios humana CES y las l&#xED;neas de IPSC. Nuestros datos se suman a la evidencia de acumulaci&#xF3;n de la existencia de una in vitro derivados precursor de las c&#xE9;lulas madre hematopoy&#xE9;ticas (HSC) con una capacidad limitada injertar en los ratones trasplantados, pero con un potencial hematopoy&#xE9;tica pluripotente. Debido a que este protocolo se expande de manera eficiente los precursores preblood y progenitores hematopoy&#xE9;ticos, es ideal para probar nuevos factores para la generaci&#xF3;n y la expansi&#xF3;n de las CMH definitivas a largo plazo la capacidad de repoblaci&#xF3;n.

Breve informe: la generaci&#xF3;n eficiente de precursores hematopoy&#xE9;ticos y progenitores de l&#xED;neas de c&#xE9;lulas madre humanas pluripotentes

The systematic generation of neurons from patients with neurological disorders can provide important insights into disease pathology, progression and mechanism. This review will discuss recent progress in modeling neurodegenerative and neurodevelopmental diseases using induced pluripotent stem cells (iPSCs) and highlight some of the current challenges in the field. Combined with other technologies previously used to study brain disease, iPSC modeling has the promise to influence modern medicine on several fronts: early diagnosis, drug development and effective treatment.

Induced pluripotent stem cells (iPSCs) and neurological disease modeling: progress and promises.

Psychiatric disorders, including autism spectrum disorders and schizophrenia, are extremely heritable complex genetic neurodevelopmental disorders. It is now possible to directly reprogram fibroblasts from psychiatric patients into human induced pluripotent stem cells (hiPSCs) and subsequently differentiate these disorder-specific hiPSCs into neurons. This means that researchers can generate nearly limitless quantities of live human neurons with genetic backgrounds that are known to result in psychiatric disorders, without knowing which genes are interacting to produce the disease state in each patient. With these new human-cell-based models, scientists can investigate the precise cell types that are affected in these disorders and elucidate the cellular and molecular defects that contribute to disease initiation and progression. Here, we present a short review of experiments using hiPSCs and other sophisticated in vitro approaches to study the pathways underlying psychiatric disorders.

Modeling psychiatric disorders through reprogramming.

Schizophrenia (SCZD) is a heritable developmental disorder. Although the molecular mechanism of disease remains unclear, insights into the disorder have been made through a vast array of experimental techniques. Together, magnetic resonance brain imaging, pharmacological, and post-mortem pathological studies have observed decreased brain volume, aberrant neurotransmitter signaling, reduced dendritic arborization, and impaired myelination in SCZD. Genome-wide association studies (GWAS) have identified common single nucleotide polymorphisms as well as rare copy number variants that contribute to SCZD, while mouse models of candidate SCZD genes show behavioral abnormalities and anatomical perturbations consistent with human disease. The advent of human induced pluripotent stem cells (hiPSCs) makes it possible to study SCZD using live human neurons with a genetic predisposition toward SCZD, even without knowledge of the genes interacting to produce the disease state. SCZD hiPSC neurons show cellular defects comparable to those identified in post-mortem human and mouse studies, and gene expression changes are consistent with predictions made by GWAS. SCZD hiPSC neurons represent a new tool to look beyond phenotype and begin to dissect the molecular mechanisms of SCZD.

Concise review: the promise of human induced pluripotent stem cell-based studies of schizophrenia.

Although schizophrenia affects a number of brain regions and produces a range of clinical symptoms, we believe its origins lie at the level of single neurons and simple networks. Owing to this, as well as to its high degree of heritability, we hypothesize that schizophrenia is amenable to cell-based studies in vitro. Using induced pluripotent stem cell-derived neurons and/or fibroblast-induced neurons, a limitless quantity of live human neurons can now be generated from patient skin biopsies. We predict that cell-based studies will ultimately contribute to our understanding of the molecular and cellular underpinnings of this debilitating disorder.

Modeling schizophrenia using induced pluripotent stem cell-derived and fibroblast-induced neurons.

Neural stem/progenitor cells (NSPCs) have the potential to differentiate into neurons, astrocytes, and/or oligodendrocytes. Because these cells can be expanded in culture, they represent a vast source of neural cells. With the recent discovery that patient fibroblasts can be reprogrammed directly into induced NSPCs, the regulation of NSPC fate and function, in the context of cell-based disease models and patient-specific cell-replacement therapies, warrants review.

Neural stem and progenitor cells in health and disease.

We used single-cell genomic approaches to map DNA copy number variation (CNV) in neurons obtained from human induced pluripotent stem cell (hiPSC) lines and postmortem human brains. We identified aneuploid neurons, as well as numerous subchromosomal CNVs in euploid neurons. Neurotypic hiPSC-derived neurons had larger CNVs than fibroblasts, and several large deletions were found in hiPSC-derived neurons but not in matched neural progenitor cells. Single-cell sequencing of endogenous human frontal cortex neurons revealed that 13 to 41% of neurons have at least one megabase-scale de novo CNV, that deletions are twice as common as duplications, and that a subset of neurons have highly aberrant genomes marked by multiple alterations. Our results show that mosaic CNV is abundant in human neurons.

Mosaic copy number variation in human neurons.

The immaturity of neurons differentiated from human induced pluripotent stem cells (hiPSCs) presents difficulties for modeling late-onset neurodegenerative disorders such as Parkinson's disease. Now, Miller et al. (2013) provide a strategy for inducing aging-related phenotypes in hiPSC-derived neurons, enabling in vitro study of late-onset neurodegenerative diseases.

Inducing cellular aging: enabling neurodegeneration-in-a-dish.

Schizophrenia (SZ) is a devastating complex genetic mental condition that is heterogeneous in terms of clinical etiologies, symptoms, and outcomes. Despite decades of postmortem, neuroimaging, pharmacological, and genetic studies of patients, in addition to animal models, much of the biological mechanisms that underlie the pathology of SZ remain unknown. The ability to reprogram adult somatic cells into human induced pluripotent stem cells (hiPSCs) provides a new tool that supplies live human neurons for modeling complex genetic conditions such as SZ. The purpose of this review is to discuss the technical and clinical constraints currently limiting hiPSC-based studies. We posit that reducing the clinical heterogeneity of hiPSC-based studies, by selecting subjects with common clinical manifestations or rare genetic variants, will help our ability to draw meaningful insights from the necessarily small patient cohorts that can be studied at this time.

Modeling heterogeneous patients with a clinical diagnosis of schizophrenia with induced pluripotent stem cells.

Prenatal exposure of the developing brain to various environmental challenges increases susceptibility to late onset of neuropsychiatric dysfunction; still, the underlying mechanisms remain obscure. Here we show that exposure of embryos to a variety of environmental factors such as alcohol, methylmercury, and maternal seizure activates HSF1 in cerebral cortical cells. Furthermore, Hsf1 deficiency in the mouse cortex exposed in utero to subthreshold levels of these challenges causes structural abnormalities and increases seizure susceptibility after birth. In addition, we found that human neural progenitor cells differentiated from induced pluripotent stem cells derived from schizophrenia patients show higher variability in the levels of HSF1 activation induced by environmental challenges compared to controls. We propose that HSF1 plays a crucial role in the response of brain cells to prenatal environmental insults and may be a key component in the pathogenesis of late-onset neuropsychiatric disorders.

Roles of heat shock factor 1 in neuronal response to fetal environmental risks and its relevance to brain disorders.

This study investigated human-induced pluripotent stem cell (hiPSC) -derived neurons for their ability to secrete neurotransmitters in an activity-dependent manner, the fundamental property required for chemical neurotransmission. Cultured hiPSC neurons showed KCl stimulation of activity-dependent secretion of catecholamines--dopamine (DA), norepinephrine (NE), and epinephrine (Epi)--and the peptide neurotransmitters dynorphin and enkephlain. hiPSC neurons express the biosynthetic enzymes for catecholamines and neuropeptides. Because altered neurotransmission contributes to schizophrenia (SZ), we compared SZ to control cultures of hiPSC neurons and found that SZ cases showed elevated levels of secreted DA, NE, and Epi. Consistent with increased catecholamines, the SZ neuronal cultures showed a higher percentage of tyrosine hydroxylase (TH)-positive neurons, the first enzymatic step for catecholamine biosynthesis. These findings show that hiPSC neurons possess the fundamental property of activity-dependent neurotransmitter secretion and can be advantageously utilized to examine regulation of neurotransmitter release related to brain disorders.

Human iPSC neurons display activity-dependent neurotransmitter secretion: aberrant catecholamine levels in schizophrenia neurons.

A large portion of common variant loci associated with genetic risk for schizophrenia reside within noncoding sequence of unknown function. Here, we demonstrate promoter and enhancer enrichment in schizophrenia variants associated with expression quantitative trait loci (eQTL). The enrichment is greater when functional annotations derived from the human brain are used relative to peripheral tissues. Regulatory trait concordance analysis ranked genes within schizophrenia genome-wide significant loci for a potential functional role, based on colocalization of a risk SNP, eQTL, and regulatory element sequence. We identified potential physical interactions of noncontiguous proximal and distal regulatory elements. This was verified in prefrontal cortex and -induced pluripotent stem cell-derived neurons for the L-type calcium channel (CACNA1C) risk locus. Our findings point to a functional link between schizophrenia-associated noncoding SNPs and 3D genome architecture associated with chromosomal loopings and transcriptional regulation in the brain.

A role for noncoding variation in schizophrenia.

A disruptive approach to therapeutic discovery and development is required in order to significantly improve the success rate of drug discovery for central nervous system (CNS) disorders. In this review, we first assess the key factors contributing to the frequent clinical failures for novel drugs. Second, we discuss cancer translational research paradigms that addressed key issues in drug discovery and development and have resulted in delivering drugs with significantly improved outcomes for patients. Finally, we discuss two emerging technologies that could improve the success rate of CNS therapies: human induced pluripotent stem cell (hiPSC)-based studies and multiscale biology models. Coincident with advances in cellular technologies that enable the generation of hiPSCs directly from patient blood or skin cells, together with methods to differentiate these hiPSC lines into specific neural cell types relevant to neurological disease, it is also now possible to combine data from large-scale forward genetics and post-mortem global epigenetic and expression studies in order to generate novel predictive models. The application of systems biology approaches to account for the multiscale nature of different data types, from genetic to molecular and cellular to clinical, can lead to new insights into human diseases that are emergent properties of biological networks, not the result of changes to single genes. Such studies have demonstrated the heterogeneity in etiological pathways and the need for studies on model systems that are patient-derived and thereby recapitulate neurological disease pathways with higher fidelity. In the context of two common and presumably representative neurological diseases, the neurodegenerative disease Alzheimer's Disease, and the psychiatric disorder schizophrenia, we propose the need for, and exemplify the impact of, a multiscale biology approach that can integrate panomic, clinical, imaging, and literature data in order to construct predictive disease network models that can (i) elucidate subtypes of syndromic diseases, (ii) provide insights into disease networks and targets and (iii) facilitate a novel drug screening strategy using patient-derived hiPSCs to discover novel therapeutics for CNS disorders.

Kristen J. Brennand

Department of Psychiatry,
Department of Neuroscience

Icahn School of Medicine at Mount Sinai

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

Kristen J. Brennand

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

Icahn School of Medicine at Mount Sinai

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

Department of Psychiatry,
Department of Neuroscience