Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

Massachusetts Institute of Technology

University of Texas at Austin

The worldwide status of rapidly increasing demand for organ and tissue transplantation has promoted tissue engineering as a promising alternative, in particular the use of stem cells. Human embryonic stem cells (hESCs) have the advantage of differentiating to all cell types in the body and high proliferation capabilities. Considering their ability to organize into complex multi-cell-type structures during embryonic-like differentiation, hESCs can potentially provide a source of cells for tissue engineering applications and meet the growing demand for viable human tissue structures in therapeutic clinical application. This chapter describes first steps toward realizing this goal gathered from our experience in growing and differentiating hESCs in three dimensions.

Engineering three-dimensional tissue structures using stem cells.

Growing interest in using endothelial cells for therapeutic purposes has led to exploring human embryonic stem cells as a potential source for endothelial progenitor cells. Embryonic stem cells are advantageous when compared with other endothelial cell origins, due to their high proliferation capability, pluripotency, and low immunogenity. However, there are many challenges and obstacles to overcome before the vision of using embryonic endothelial progenitor cells in the clinic can be realized. Among these obstacles is the development of a productive method of isolating endothelial cells from human embryonic stem cells and elucidating their differentiation pathway. This review will focus on the endothelial potential of human embryonic stem cells that is described in current studies, with respect to the differentiation of human embryonic stem cells to endothelial cells, their isolation, and their characterization.

Endothelial potential of human embryonic stem cells.

Successful tissue engineering requires optimization of scaffold stiffness for a given application and cell type. Here, we investigated the effect of scaffold stiffness on myoblast cells, demonstrating the ability of cells to affect and to sense their mechanical microenvironment. Myoblasts were cultured on composite three-dimensional poly-lactic acid (PLLA)/poly-lactic co glycolic acid (PLGA) porous scaffolds of varied elasticity. The elasticity was controlled by changing the ratio of PLLA versus PLGA in the scaffolds. Cell organization, myotube formation, and cell viability were affected by scaffold stiffness. PLLA-containing scaffolds (100% to 25% PLLA) provided stiffness that supported myotube formation, while neat PLGA scaffold failed to support myotube formation and cell viability. Furthermore, scaffold stiffness correlated to its size/area reduction upon culturing experiments, suggesting different shrinkage degree by cell forces. Inhibition of scaffold shrinking by affixing device resulted in spacious cell organization with normal cell morphology. This may suggest that scaffold shrinkage led to cellular degeneration and shape deformation. Our results indicate that compliant scaffolds are insufficient to withstand cell forces. On the other hand, excessively firm scaffold could not lead to parallel oriented myotube organization. Hence, optimal scaffold stiffness can be tailored by PLLA/PLGA blending to direct specific stages of myoblast differentiation and organization.

Effect of scaffold stiffness on myoblast differentiation.

Les cellules souches embryonnaires humaines (CSEh) rec&egrave;lent un potentiel grand comme une ressource pour la m&eacute;decine r&eacute;g&eacute;n&eacute;rative. Avant d&#39;atteindre la pertinence th&eacute;rapeutique, les m&eacute;thodes doivent &ecirc;tre &eacute;labor&eacute;s pour contr&ocirc;ler la diff&eacute;renciation des cellules souches. Il est clair que les cellules souches peuvent r&eacute;pondre aux signaux g&eacute;n&eacute;tiques, telles que celles transmises par les acides nucl&eacute;iques, pour promouvoir la diff&eacute;renciation de la lign&eacute;e sp&eacute;cifique. Ici, nous avons d&eacute;velopp&eacute; un syst&egrave;me efficace pour livrer des siARN pour CSEh dans une matrice 3D de la culture en utilisant des mat&eacute;riaux de type lipidique. Nous montrons que non-virale ARNsi dans une 3D &eacute;chafaudages can knockdown efficacement 90 % d&#39;expression de la GFP dans GFP-CSEh. En outre, nous montrent que ce syst&egrave;me peut servir comme plate-forme pour orienter la diff&eacute;renciation CSEh. Par le biais de siARN silencing du g&egrave;ne du r&eacute;cepteur KRR, nous parvenir une diminution concomitante (60-90 %) de repr&eacute;sentant des g&egrave;nes de la couche de germe d&#39;endoderme et significative upregulation of repr&eacute;sentant des g&egrave;nes de la couche de germe du m&eacute;soderme (pli 27-90). Ceci d&eacute;montre que les siARN peuvent diriger la diff&eacute;renciation des cellules souches en bloquant les g&egrave;nes repr&eacute;sentatifs d&#39;une couche de germe et fournit &eacute;galement un moyen particuli&egrave;rement puissant pour isoler la couche de germe d&#39;endoderme du m&eacute;soderme et ectoderme. Cette capacit&eacute; d&#39;inhiber la diff&eacute;renciation des couches germinales endoderme pourrait permettre un contr&ocirc;le am&eacute;lior&eacute; au cours de la diff&eacute;renciation des CSEh aux types de la cellule souhait&eacute;e.

Diriger la diff&eacute;renciation de cellules souches embryonnaires humaines par livraison non viraux de siARN dans la culture 3D.

&#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE; (hESCs) &#x4F5C;&#x4E3A;&#x4E00;&#x79CD;&#x8D44;&#x6E90;&#x518D;&#x751F;&#x533B;&#x5B66;&#x7684;&#x5DE8;&#x5927;&#x6F5C;&#x529B;&#x3002;&#x4E4B;&#x524D;&#x5B9E;&#x73B0;&#x6CBB;&#x7597;&#x7684;&#x76F8;&#x5173;&#x6027;&#xFF0C;&#x5FC5;&#x987B;&#x5236;&#x8BA2;&#x65B9;&#x6CD5;&#xFF0C;&#x63A7;&#x5236;&#x5E72;&#x7EC6;&#x80DE;&#x5206;&#x5316;&#x3002;&#x5F88;&#x663E;&#x7136;&#x5E72;&#x7EC6;&#x80DE;&#x53EF;&#x4EE5;&#x56DE;&#x5E94;&#x9057;&#x4F20;&#x4FE1;&#x53F7;&#xFF0C;&#x5982;&#x90A3;&#x4E9B;&#x4F20;&#x6388;&#x7684;&#x6838;&#x9178;&#xFF0C;&#x4EE5;&#x4FC3;&#x8FDB;&#x7279;&#x5B9A;&#x6CBF;&#x88AD;&#x7684;&#x5206;&#x5316;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x5F00;&#x53D1;&#x4E86; siRNA &#x9001; hESCs 3D &#x6587;&#x5316;&#x77E9;&#x9635;&#x4F7F;&#x7528;&#x8102;&#x72B6;&#x6750;&#x6599;&#x4E2D;&#x6709;&#x6548;&#x7684;&#x7CFB;&#x7EDF;&#x3002;&#x6211;&#x4EEC;&#x5728; 3D &#x652F;&#x67B6;&#x53EF;&#x4EE5;&#x6709;&#x6548;&#x5730;&#x51FB;&#x5012; 90%GFP hESCs &#x7EFF;&#x8272;&#x8367;&#x5149;&#x86CB;&#x767D;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x7684;&#x663E;&#x793A;&#xFF0C;&#x975E;&#x75C5;&#x6BD2; siRNA &#x4EA4;&#x4ED8;&#x3002;&#x6211;&#x4EEC;&#x8FDB;&#x4E00;&#x6B65;&#x663E;&#x793A;&#x6B64;&#x7CFB;&#x7EDF;&#x53EF;&#x4EE5;&#x7528;&#x4F5C;&#x4E00;&#x4E2A;&#x5E73;&#x53F0;&#xFF0C;&#x7528;&#x4E8E;&#x6307;&#x5BFC; hESC &#x5206;&#x5316;&#x3002;&#x901A;&#x8FC7; siRNA &#x6C89;&#x9ED8; KDR &#x53D7;&#x4F53;&#x57FA;&#x56E0;&#x7684;&#x6211;&#x4EEC;&#x5728;&#x57FA;&#x56E0;&#x4EE3;&#x8868;&#x7684;&#x5185;&#x80DA;&#x5C42;&#x80DA;&#x5C42;&#x548C;&#x4E2D;&#x80DA;&#x5C42;&#x80DA;&#x5C42; (27-90 &#x500D;&#xFF09; &#x57FA;&#x56E0;&#x4EE3;&#x8868;&#x91CD;&#x5927;&#x4E0A;&#x8C03;&#x5B9E;&#x73B0;&#x5E76;&#x53D1;&#x4E0B;&#x8C03; (60-90%)&#x3002;&#x8FD9;&#x8868;&#x660E;&#x8BE5; siRNA &#x53EF;&#x4EE5;&#x76F4;&#x63A5;&#x901A;&#x8FC7;&#x963B;&#x6B62;&#x57FA;&#x56E0;&#x4EE3;&#x8868;&#x4E00;&#x4E2A;&#x80DA;&#x5C42;&#x5E72;&#x7EC6;&#x80DE;&#x5206;&#x5316;&#x548C;&#x8FD8;&#x63D0;&#x4F9B;&#x4E86;&#x9694;&#x79BB;&#x5185;&#x80DA;&#x5C42;&#x80DA;&#x5C42;&#x4E2D;&#x80DA;&#x5C42;&#x548C;&#x5916;&#x80DA;&#x5C42;&#x7684;&#x7279;&#x522B;&#x6709;&#x529B;&#x624B;&#x6BB5;&#x3002;&#x8FD9;&#x79CD;&#x80FD;&#x529B;&#x6765;&#x6291;&#x5236;&#x5185;&#x80DA;&#x5C42;&#x80DA;&#x5C42;&#x5206;&#x5316;&#x53EF;&#x80FD;&#x5141;&#x8BB8;&#x6539;&#x8FDB;&#x63A7;&#x5236; hESC &#x5B9A;&#x5411;&#x8BF1;&#x5BFC;&#x5206;&#x5316;&#x4E3A;&#x6240;&#x9700;&#x7684;&#x5355;&#x5143;&#x683C;&#x7C7B;&#x578B;&#x3002;

&#x5728; 3D &#x6587;&#x5316; siRNA &#x975E;&#x75C5;&#x6BD2;&#x6027;&#x4EA4;&#x4ED8;&#x7684;&#x6307;&#x5BFC;&#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x5206;&#x5316;&#x3002;

Human embryonic stem cells (hESCs) hold great potential as a resource for regenerative medicine. Before achieving therapeutic relevancy, methods must be developed to control stem cell differentiation. It is clear that stem cells can respond to genetic signals, such as those imparted by nucleic acids, to promote lineage-specific differentiation. Here we have developed an efficient system for delivering siRNA to hESCs in a 3D culture matrix using lipid-like materials. We show that non-viral siRNA delivery in a 3D scaffolds can efficiently knockdown 90% of GFP expression in GFP-hESCs. We further show that this system can be used as a platform for directing hESC differentiation. Through siRNA silencing of the KDR receptor gene, we achieve concurrent downregulation (60-90%) in genes representative of the endoderm germ layer and significant upregulation of genes representative of the mesoderm germ layer (27-90 fold). This demonstrates that siRNA can direct stem cell differentiation by blocking genes representative of one germ layer and also provides a particularly powerful means to isolate the endoderm germ layer from the mesoderm and ectoderm. This ability to inhibit endoderm germ layer differentiation could allow for improved control over hESC differentiation to desired cell types.

Directing human embryonic stem cell differentiation by non-viral delivery of siRNA in 3D culture.

Menschlicher embryonaler Stammzellen (Zellkulturen) halten gro&szlig;es Potenzial als Ressource f&uuml;r die regenerative Medizin. Vor dem therapeutischen Relevanz erlangen, m&uuml;ssen Methoden entwickelt werden, um Stammzellen Differenzierung zu steuern. Es ist klar, dass Stammzellen genetische Signale, z. B. von Nukleins&auml;uren, vermittelt Herkunfts-spezifische Differenzierung F&ouml;rderung reagieren. Hier haben wir ein effizientes System f&uuml;r die Lieferung von SiRNA f&uuml;r Zellkulturen in einer 3D Kultur-Matrix mit Lipid-&auml;hnliche Materialien entwickelt. Wir zeigen, dass nicht-viralen SiRNA-Lieferung in einem 3D Ger&uuml;sten kann effizient Niederschlag 90 % der GFP-Expression in GFP-Zellkulturen. Wir zeigen weiter, dass dieses System als Plattform f&uuml;r die Leitung der hESC Differenzierung verwendet werden kann. Durch SiRNA Unterdr&uuml;ckender im KDR-Rezeptor-Gen, erreichen wir gleichzeitige Heraufregulation (60-90 %) in Gene Vertreter der Endodermis Mikrobe-Schicht und bedeutende Hochregulation Gene Vertreter des Mesoderm Mikrobe-Schicht (Falten, 27-90). Dies zeigt, dass SiRNA kann Stammzellen Differenzierung durch die Blockierung von Genen, die repr&auml;sentativ f&uuml;r eine Mikrobe-Schicht direkt und bietet auch ein besonders wirksames Instrument, die Endodermis Mikrobe-Schicht aus dem Mesoderm und Ektoderm zu isolieren. Diese F&auml;higkeit Endodermis Mikrobe-Schicht Differenzierung zu hemmen kann f&uuml;r verbesserte Kontrolle &uuml;ber hESC Differenzierung zum gew&uuml;nschten Zelltypen erm&ouml;glichen.

Regie Differenzierung von humanen embryonalen Stammzellen durch nicht-viralen Lieferung von SiRNA in 3D Kultur.

Cellule staminali embrionali umane (hESC) tenere il grande potenziale come una risorsa per la medicina rigenerativa. Prima di raggiungere la pertinenza terapeutica, devono essere sviluppati metodi per controllare la differenziazione delle cellule staminali. &Egrave; chiaro che le cellule staminali possono rispondere ai segnali genetici, come quelli impartita dagli acidi nucleici, per promuovere la differenziazione di lineage-specifici. Qui abbiamo sviluppato un sistema efficiente per la consegna di siRNA a hESCs in una matrice di cultura 3D utilizzando materiali lipido-simile. Mostriamo che consegna non-virale siRNA in un 3D ponteggi pu&ograve; efficacemente atterramento 90% dell&#39;espressione di GFP in GFP-hESCs. Mostriamo inoltre che questo sistema pu&ograve; essere utilizzato come una piattaforma per la regia di differenziazione hESC. Attraverso siRNA silenziamento del gene del recettore KDR, raggiungiamo downregulation simultanee (60-90%) nei geni rappresentante dello strato germinale endoderma e significativo sovraregolazione del rappresentante di geni dello strato germinale mesoderma (27-90 piega). Questo dimostra che siRNA possibile indirizzare la differenziazione delle cellule staminali bloccando i geni rappresentativi di uno strato di germe e fornisce anche un mezzo particolarmente potente per isolare lo strato del germe endoderma dal mesoderma e l&#39;ectoderma. Questa capacit&agrave; di inibire la differenziazione di strato di germe endoderma potrebbe consentire per un controllo migliore hESC differenziazione per i tipi di cella desiderata.

Dirigendo la differenziazione delle cellule staminali embrionali umane dalla consegna non-virale di siRNA nella cultura 3D.

&#x30D2;&#x30C8;&#x80DA;&#x6027;&#x5E79;&#x7D30;&#x80DE; &#xFF08;hESCs&#xFF09; &#x518D;&#x751F;&#x533B;&#x7642;&#x306E;&#x305F;&#x3081;&#x306E;&#x30EA;&#x30BD;&#x30FC;&#x30B9;&#x3068;&#x3057;&#x3066;&#x5927;&#x304D;&#x306A;&#x53EF;&#x80FD;&#x6027;&#x3092;&#x4FDD;&#x6301;&#x3057;&#x307E;&#x3059;&#x3002;&#x6CBB;&#x7642;&#x95A2;&#x9023;&#x6027;&#x3092;&#x9054;&#x6210;&#x3059;&#x308B;&#x524D;&#x306B;&#x3001;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x5206;&#x5316;&#x3092;&#x5236;&#x5FA1;&#x3059;&#x308B;&#x65B9;&#x6CD5;&#x3092;&#x958B;&#x767A;&#x3059;&#x308B;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x5E79;&#x7D30;&#x80DE;&#x7CFB;&#x7D71;&#x7279;&#x7570;&#x7684;&#x306A;&#x5206;&#x5316;&#x3092;&#x4FC3;&#x9032;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x907A;&#x4F1D;&#x7684;&#x306B;&#x306A;&#x3069;&#x306E;&#x4FE1;&#x53F7;&#x305D;&#x308C;&#x3089;&#x6838;&#x9178;&#x306B;&#x3088;&#x3063;&#x3066;&#x4E0E;&#x3048;&#x3089;&#x308C;&#x308B;&#x5FDC;&#x7B54;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x306F;&#x660E;&#x3089;&#x304B;&#x3067;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x6211;&#x3005; &#x306F; siRNA hESCs &#x8102;&#x8CEA;&#x306E;&#x3088;&#x3046;&#x306A;&#x6750;&#x6599;&#x3092;&#x7528;&#x3044;&#x305F;&#x4E09;&#x6B21;&#x5143;&#x57F9;&#x990A;&#x30DE;&#x30C8;&#x30EA;&#x30C3;&#x30AF;&#x30B9;&#x5185;&#x306B;&#x914D;&#x4FE1;&#x3059;&#x308B;&#x52B9;&#x7387;&#x7684;&#x306A;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x3092;&#x958B;&#x767A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;3 D &#x8DB3;&#x5834;&#x3067;&#x304D;&#x308B;&#x52B9;&#x7387;&#x7684;&#x306B;&#x30CE;&#x30C3;&#x30AF;&#x30C0;&#x30A6;&#x30F3; 90 &#xFF05; GFP hESCs &#x306B;&#x304A;&#x3051;&#x308B; GFP &#x767A;&#x73FE;&#x306E;&#x975E;&#x30A6;&#x30A4;&#x30EB;&#x30B9; siRNA &#x30C7;&#x30EA;&#x30D0;&#x30EA;&#x30FC;&#x3092;&#x8868;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3053;&#x306E;&#x30B7;&#x30B9;&#x30C6;&#x30E0; hESC &#x5206;&#x5316;&#x3092;&#x6307;&#x793A;&#x3059;&#x308B;&#x305F;&#x3081;&#x3001;&#x30D7;&#x30E9;&#x30C3;&#x30C8;&#x30D5;&#x30A9;&#x30FC;&#x30E0;&#x3068;&#x3057;&#x3066;&#x3092;&#x4F7F;&#x7528;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;SiRNA KDR &#x53D7;&#x5BB9;&#x4F53;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x30B5;&#x30A4;&#x30EC;&#x30F3;&#x30B7;&#x30F3;&#x30B0;&#x3092;&#x901A;&#x3058;&#x3066;&#x3001;&#x6211;&#x3005; &#x4E26;&#x884C;&#x30C0;&#x30A6;&#x30F3;&#x30EC;&#x30AE;&#x30E5;&#x30EC;&#x30FC;&#x30B7;&#x30E7;&#x30F3; (60-90%) &#x907A;&#x4F1D;&#x5B50;&#x4EE3;&#x8868;&#x5185;&#x80DA;&#x8449;&#x306E;&#x80DA;&#x5C64;&#x306E;&#x6709;&#x610F;&#x306E;&#x30A2;&#x30C3;&#x30D7;&#x30EC;&#x30AE;&#x30E5;&#x30EC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x4E2D;&#x80DA;&#x8449;&#x306E;&#x80DA;&#x5C64; &#xFF08;27 90 &#x500D;&#xFF09; &#x306E;&#x907A;&#x4F1D;&#x5B50;&#x4EE3;&#x8868;&#x306E;&#x9054;&#x6210;&#x3068;&#x3002;&#x3053;&#x308C;&#x306B;&#x306F;&#x3001;&#x305D;&#x306E; siRNA &#x907A;&#x4F1D;&#x5B50;&#x306E; 1 &#x3064;&#x306E;&#x7D30;&#x83CC;&#x5C64;&#x306E;&#x4EE3;&#x8868;&#x3092;&#x30D6;&#x30ED;&#x30C3;&#x30AF;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x5E79;&#x7D30;&#x80DE;&#x5206;&#x5316;&#x3092;&#x6307;&#x793A;&#x3067;&#x304D;&#x308B;&#x3057;&#x3001;&#x5185;&#x80DA;&#x8449;&#x80DA;&#x8449;&#x3001;&#x4E2D;&#x80DA;&#x8449;&#x3068;&#x5916;&#x80DA;&#x8449;&#x304B;&#x3089;&#x5206;&#x96E2;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x7279;&#x306B;&#x5F37;&#x529B;&#x306A;&#x624B;&#x6BB5;&#x3082;&#x63D0;&#x4F9B;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x5185;&#x80DA;&#x8449;&#x80DA;&#x8449;&#x5206;&#x5316;&#x3092;&#x6291;&#x5236;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x3053;&#x306E;&#x80FD;&#x529B;&#x6539;&#x5584; hESC &#x5206;&#x5316;&#x76EE;&#x7684;&#x306E;&#x30BB;&#x30EB;&#x306E;&#x7A2E;&#x985E;&#x3092;&#x5236;&#x5FA1;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3055;&#x308C;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

SiRNA &#x4E09;&#x6B21;&#x5143;&#x57F9;&#x990A;&#x3067;&#x306E;&#x30D2;&#x30C8;&#x80DA;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x5206;&#x5316;&#x3092;&#x6F14;&#x51FA;&#x975E;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x914D;&#x9054;&#x306B;&#x3088;&#x3063;&#x3066;&#x3002;

&#xC778;&#xAC04; &#xBC30;&#xC544; &#xC904;&#xAE30; &#xC138;&#xD3EC; (hESCs) &#xC7AC;&#xC0DD; &#xC758;&#xD559;&#xC5D0; &#xB300; &#xD55C; &#xC790;&#xC6D0;&#xC73C;&#xB85C; &#xD070; &#xC7A0;&#xC7AC;&#xB825;&#xC744; &#xBCF4;&#xC720;. &#xCE58;&#xB8CC; &#xAD00;&#xB828;&#xC131;&#xC744; &#xB2EC;&#xC131; &#xD558;&#xAE30; &#xC804;&#xC5D0; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xBD84;&#xD654; &#xC81C;&#xC5B4; &#xBC29;&#xBC95;&#xC740; &#xAC1C;&#xBC1C; &#xD574;&#xC57C; &#xD569;&#xB2C8;&#xB2E4;. &#xADF8;&#xAC83;&#xC740; &#xBD84;&#xBA85; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xACC4;&#xBCF4; &#xAD00;&#xB828; &#xCC28;&#xBCC4;&#xD654; &#xD64D;&#xBCF4; &#xB4F1; &#xD575; &#xC0B0;&#xC774; &#xC218; &#xC720;&#xC804;&#xC790; &#xC2E0;&#xD638;&#xC5D0; &#xC751;&#xB2F5;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC;&#xB294; &#xC9C0;&#xC9C8; &#xAC19;&#xC740; &#xC7AC;&#xB8CC;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; 3D &#xBB38;&#xD654; &#xB9E4;&#xD2B8;&#xB9AD;&#xC2A4;&#xC5D0; Hescs&#xB97C; Sirna&#xB97C; &#xC81C;&#xACF5; &#xD558;&#xAE30; &#xC704;&#xD55C; &#xD6A8;&#xC728;&#xC801;&#xC778; &#xC2DC;&#xC2A4;&#xD15C;&#xC744; &#xAC1C;&#xBC1C; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; GFP Hescs&#xC758; GFP &#xC2DD;&#xC758; 3 &#xCC28;&#xC6D0; &#xAC74;&#xC124; &#xAE30;&#xACC4; &#xC784;&#xB300; &#xC218; &#xD6A8;&#xC728;&#xC801;&#xC73C;&#xB85C; &#xCD5C;&#xC800;&#xC758; 90%&#xC5D0;&#xC11C; &#xADF8; &#xBE44; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC131; siRNA &#xC804;&#xB2EC;&#xC744; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xB354; &#xC774;&#xC0C1;&#xC774; &#xC2DC;&#xC2A4;&#xD15C; hESC &#xCC28;&#xBCC4;&#xD654; &#xC5F0;&#xCD9C;&#xC744; &#xC704;&#xD55C; &#xD50C;&#xB7AB;&#xD3FC;&#xC73C;&#xB85C; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. KDR &#xC218;&#xC6A9; &#xCCB4; &#xC720;&#xC804;&#xC790;&#xC758; Sirna&#xB97C; &#xC785;&#xC744; &#xD1B5;&#xD574; &#xC6B0;&#xB9AC; endoderm &#xC138;&#xADE0; &#xCE35;&#xC758; &#xB300;&#xD45C; &#xC720;&#xC804;&#xC790;&#xC640; &#xC720;&#xC804;&#xC790; &#xB300;&#xD45C; mesoderm &#xC138;&#xADE0; &#xB808;&#xC774;&#xC5B4; (27-90 &#xBC30;)&#xC758; &#xC0C1;&#xB2F9;&#xD55C; upregulation &#xB3D9;&#xC2DC; downregulation (60-90%)&#xB97C; &#xB2EC;&#xC131;. &#xC774; siRNA &#xC720;&#xC804;&#xC790; &#xD55C; &#xC138;&#xADE0; &#xCE35;&#xC758; &#xB300;&#xD45C;&#xB97C; &#xCC28;&#xB2E8; &#xD558; &#xC5EC; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xBD84;&#xD654;&#xB97C; &#xC548;&#xB0B4;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; mesoderm ectoderm&#xC5D0;&#xC11C; endoderm &#xC138;&#xADE0; &#xACC4;&#xCE35;&#xC744; &#xBD84;&#xB9AC; &#xD558;&#xB294; &#xD2B9;&#xD788; &#xAC15;&#xB825;&#xD55C; &#xC218;&#xB2E8; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. Endoderm &#xC138;&#xADE0; &#xACC4;&#xCE35; &#xBD84;&#xD654;&#xB97C; &#xC5B5;&#xC81C; &#xD558;&#xB294;&#xC774; &#xAE30;&#xB2A5;&#xC740; &#xC6D0;&#xD558;&#xB294; &#xC140; &#xD615;&#xC2DD; hESC &#xCC28;&#xBCC4;&#xD654;&#xB97C; &#xD1B5;&#xD574; &#xD5A5;&#xC0C1; &#xB41C; &#xC81C;&#xC5B4;&#xC5D0; &#xB300; &#xD55C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

3 &#xCC28;&#xC6D0; &#xBB38;&#xD654;&#xC5D0; Sirna&#xC758; &#xBE44; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC131; &#xC804;&#xB2EC; &#xD558; &#xC5EC; &#xC778;&#xAC04; &#xBC30;&#xC544; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xBD84;&#xD654;&#xB97C; &#xAC10;&#xB3C5;.

C&eacute;lulas madre embrionarias humanas (hESCs) tener gran potencial como un recurso para la medicina regenerativa. Antes de alcanzar la relevancia terap&eacute;utica, se deben desarrollar m&eacute;todos para controlar la diferenciaci&oacute;n de c&eacute;lulas madre. Est&aacute; claro que las c&eacute;lulas madre puede responder a se&ntilde;ales gen&eacute;ticas, como los impartidos por los &aacute;cidos nucleicos, para promover la diferenciaci&oacute;n del linaje espec&iacute;fico. Aqu&iacute; hemos desarrollado un sistema eficaz para la entrega de siRNA a hESCs en una matriz de cultura 3D utilizando materiales de l&iacute;pidos. Mostramos entrega no viral siRNA en andamios 3D puede eficientemente precipitaci&oacute;n 90% de la expresi&oacute;n de GFP en GFP-hESCs. Adem&aacute;s, demostramos que este sistema puede ser utilizado como una plataforma para dirigir la diferenciaci&oacute;n de c&eacute;lulas. A trav&eacute;s de siRNA silenciamiento del gen del receptor KDR, logramos downregulation concurrente (60-90%) en representante de genes de la capa de germen del endodermo y upregulation significativo de representante de genes de la capa del germen del mesodermo (27-90 veces). Esto demuestra siRNA puede dirigir la diferenciaci&oacute;n de c&eacute;lulas madre mediante el bloqueo de genes representativos de una capa de germen y tambi&eacute;n proporciona un medio particularmente poderoso para aislar la capa del germen del endodermo del ectodermo y mesodermo. Esta capacidad de inhibir la diferenciaci&oacute;n de endodermo capa germinal podr&iacute;a permitir mejor control sobre la diferenciaci&oacute;n de c&eacute;lulas a tipos celulares deseados.

Dirigir la diferenciaci&oacute;n de c&eacute;lulas madre embrionarias humanas por la no entrega de siRNA en cultura 3D.

Forze meccaniche sono fondamentali per l&#39;embriogenesi, in particolare, nella fase di gastrulazione lineage-specifica, al che l&#39;embrione si trasforma da una semplice palla sferica di cellule di un organismo multi-layered, contenenti correttamente organizzata endoderma, mesoderma ed ectoderma strati germinali. Diversi report hanno proposto che tale regia e movimenti coordinati dei collettivi di grandi cellule sono guidati da cellular responses to cell deformazioni e le forze generate delle cellule. Per comprendere meglio questi cambiamenti ambientali-indotta delle cellule, noi abbiamo modellato il processo di formazione di strato di germe di coltura di cellule staminali embrionali umane (hESC) su tre dimensionale ponteggi (3D) con rigidit&agrave; al modello che trovato in strati germinali specifici. Mostriamo che la differenziazione per ogni strato del germe &egrave; stato promosso da una soglia differente rigidit&agrave; dei ponteggi, che ricorda le forze esercitate durante il processo di gastrulazione. I risultati suggeriscono che tridimensionale (3D) tre ponteggi possono riassumere gli stimoli meccanici necessari per dirigere hESC differenziazione e che questi stimoli possono giocare un ruolo significativo nel determinare il destino hESC.

L&#39;influenza di elasticit&agrave; impalcatura su strato di germe specifica delle cellule staminali embrionali umane.

&#x673A;&#x68B0;&#x90E8;&#x961F;&#x81F3;&#x5173;&#x91CD;&#x8981;&#x80DA;&#x80CE;&#x53D1;&#x751F;&#xFF0C;&#x5177;&#x4F53;&#x800C;&#x8A00;&#xFF0C;&#x5728;&#x6CBF;&#x88AD;&#x89C4;&#x683C; gastrulation &#x9636;&#x6BB5;&#xFF0C;&#x4E8E;&#x662F;&#x80DA;&#x80CE;&#x4ECE;&#x7B80;&#x5355;&#x7684;&#x7403;&#x9762;&#x7403;&#x7684;&#x5355;&#x5143;&#x683C;&#x8F6C;&#x6362;&#x4E3A;&#x4E00;&#x4E2A;&#x591A;&#x5C42;&#x6B21;&#x7684;&#x6709;&#x673A;&#x4F53;&#xFF0C;&#x5305;&#x542B;&#x6B63;&#x786E;&#x7EC4;&#x7EC7;&#x5185;&#x80DA;&#x5C42;&#x3001; &#x4E2D;&#x80DA;&#x5C42;&#x3001; &#x5C42;&#x548C;&#x5916;&#x80DA;&#x5C42;&#x80DA;&#x5C42;&#x3002;&#x8FD9;&#x79CD;&#x6307;&#x793A;&#x548C;&#x5927;&#x7EC6;&#x80DE;&#x96C6;&#x4F53;&#x7684;&#x534F;&#x8C03;&#x4E00;&#x81F4;&#x7684;&#x52A8;&#x4F5C;&#x7531;&#x7EC6;&#x80DE;&#x5E94;&#x7B54;&#x53CD;&#x5E94;&#x7EC6;&#x80DE;&#x53D8;&#x5F62;&#x548C;&#x7EC6;&#x80DE;&#x751F;&#x6210;&#x90E8;&#x961F;&#x63D0;&#x51FA;&#x51E0;&#x4E2A;&#x62A5;&#x544A;&#x3002;&#x66F4;&#x597D;&#x5730;&#x7406;&#x89E3;&#x8FD9;&#x4E9B;&#x73AF;&#x5883;&#x8BF1;&#x5BFC;&#x7EC6;&#x80DE;&#x53D8;&#x5316;&#xFF0C;&#x6211;&#x4EEC;&#x6709;&#x4EFF;&#x7167;&#x80DA;&#x5C42;&#x5F62;&#x6210;&#x8FC7;&#x7A0B;&#x7684;&#x57F9;&#x517B;&#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE; (hESCs) &#x4E09;&#x4E2A;&#x4E09;&#x7EF4; (3D) &#x652F;&#x67B6;&#x521A;&#x5EA6;&#x7684;&#x8BBE;&#x8BA1;&#x53EF;&#x4EE5;&#x4F7F;&#x7279;&#x5B9A;&#x7684;&#x80DA;&#x5C42;&#x4E2D;&#x53D1;&#x73B0;&#x7684;&#x6A21;&#x578B;&#x3002;&#x6211;&#x4EEC;&#x5C55;&#x793A;&#x652F;&#x67B6;&#x4E0D;&#x540C;&#x521A;&#x5EA6;&#x9608;&#x503C;&#x5F97;&#x5230;&#x63D0;&#x62D4;&#xFF0C;&#x5411;&#x6BCF;&#x4E2A;&#x80DA;&#x5C42;&#x5206;&#x5316;&#x3001; &#x8BA9;&#x4EBA;&#x60F3;&#x8D77;&#x90E8;&#x961F;&#x7684;&#x65BD;&#x52A0;&#x5728; gastrulation &#x8FC7;&#x7A0B;&#x4E2D;&#x3002;&#x6574;&#x4F53;&#x7ED3;&#x679C;&#x8868;&#x660E;&#x4E09;&#x4E2A;&#x4E09;&#x7EF4; (3D) &#x652F;&#x67B6;&#x53EF;&#x4EE5;&#x91CD;&#x8FF0;&#x6307;&#x6325; hESC &#x5206;&#x5316;&#x4E3A;&#x6240;&#x9700;&#x7684;&#x673A;&#x68B0;&#x523A;&#x6FC0;&#xFF0C;&#x8FD9;&#x4E9B;&#x523A;&#x6FC0;&#x53EF;&#x4EE5;&#x53D1;&#x6325;&#x91CD;&#x8981;&#x7684;&#x4F5C;&#x7528;&#xFF0C;&#x5728;&#x786E;&#x5B9A; hESC &#x7684;&#x547D;&#x8FD0;&#x3002;

&#x5BF9;&#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x7684;&#x80DA;&#x5C42;&#x89C4;&#x8303;&#x811A;&#x624B;&#x67B6;&#x5F39;&#x6027;&#x7684;&#x5F71;&#x54CD;&#x3002;

Mechanical forces are critical to embryogenesis, specifically, in the lineage-specification gastrulation phase, whereupon the embryo is transformed from a simple spherical ball of cells to a multi-layered organism, containing properly organized endoderm, mesoderm, and ectoderm germ layers. Several reports have proposed that such directed and coordinated movements of large cell collectives are driven by cellular responses to cell deformations and cell-generated forces. To better understand these environmental-induced cell changes, we have modeled the germ layer formation process by culturing human embryonic stem cells (hESCs) on three dimensional (3D) scaffolds with stiffness engineered to model that found in specific germ layers. We show that differentiation to each germ layer was promoted by a different stiffness threshold of the scaffolds, reminiscent of the forces exerted during the gastrulation process. The overall results suggest that three dimensional (3D) scaffolds can recapitulate the mechanical stimuli required for directing hESC differentiation and that these stimuli can play a significant role in determining hESC fate.

The influence of scaffold elasticity on germ layer specification of human embryonic stem cells.

Les forces m&eacute;caniques sont essentiels &agrave; l&#39;embryogen&egrave;se, plus pr&eacute;cis&eacute;ment, dans la phase de gastrulation lineage-sp&eacute;cification, apr&egrave;s quoi l&#39;embryon se transforme d&#39;une simple boule sph&eacute;rique de cellules d&#39;un organisme multi-couches, contenant correctement organis&eacute; couches embryonnaires ectoderme, m&eacute;soderme et endoderme. Plusieurs rapports ont propos&eacute; que ces r&eacute;alis&eacute; et mouvements coordonn&eacute;s des collectifs de grandes cellules reposent sur les r&eacute;ponses cellulaires aux d&eacute;formations de la cellule et les forces g&eacute;n&eacute;r&eacute;es par cellule. Pour mieux comprendre ces changements environnementaux induits par les cellules, nous avons mod&eacute;lis&eacute; le processus de formation de couche de germe en cultivant des cellules souches embryonnaires humaines (CSEh) sur trois les &eacute;chafaudages tridimensionnels (3D) avec raideur machin&eacute; pour mod&egrave;le celle trouv&eacute;e dans les couches de germe sp&eacute;cifiques. Nous montrons que la diff&eacute;renciation de chaque couche de germe est promue par un seuil diff&eacute;rent de rigidit&eacute; des &eacute;chafaudages, qui rappelle les forces exerc&eacute;es au cours du processus de gastrulation. L&#39;ensemble des r&eacute;sultats sugg&egrave;rent que les trois dimensions (3D) &eacute;chafaudages peuvent r&eacute;capituler les stimuli m&eacute;caniques requises pour diriger la diff&eacute;renciation CSEh et que ces stimuli peut jouer un r&ocirc;le important dans la d&eacute;termination sort de CSEh.

L&#39;influence de l&#39;&eacute;lasticit&eacute; de l&#39;&eacute;chafaudage sur la sp&eacute;cification de la couche de germe des cellules souches embryonnaires humaines.

Mechanischen Kr&auml;fte sind entscheidend f&uuml;r die Embryogenese, woraufhin der Embryo in eine vielschichtige Organismus, aus einem einfachen sph&auml;rischen Ball Zellen transformiert wird organisiert insbesondere in der Phase der Herkunfts-Spezifikation Gastrulation, richtig mit Endoderm, Mesoderm und Ektoderm Keim Schichten. Mehrere Berichte haben vorgeschlagen, dass solche leitete und koordinierte Bewegungen gro&szlig;zelliges kollektive werden von zellul&auml;ren Reaktionen auf Zelle Verformungen und Zelle erzeugten Kr&auml;fte angetrieben. Um diese Umwelt-induced Zelle Ver&auml;nderungen besser zu verstehen, haben wir den Mikrobe-Schicht-Bildung-Prozess modelliert durch Z&uuml;chtung menschlichen embryonalen Stammzellen (Zellkulturen) auf drei dimensionale (3D)-Ger&uuml;sten mit Steifigkeit konstruiert, Modell, das in bestimmten Keim-Schichten gefunden. Wir zeigen, dass die Differenzierung jeder Keim-Schicht wurde bef&ouml;rdert durch eine unterschiedliche Steifigkeit-Schwelle von den Ger&uuml;sten, erinnert an die Kr&auml;fte ausge&uuml;bt, w&auml;hrend des Gastrulation-Prozess. Die Gesamtergebnisse deuten darauf hin, dass drei dimensionale (3D)-Ger&uuml;sten die mechanischen Reize, die f&uuml;r die Leitung der hESC Differenzierung erforderlich rekapitulieren k&ouml;nnen und dass diese Reize eine bedeutende Rolle bei der Bestimmung hESC Schicksal spielen k&ouml;nnen.

Der Einfluss der Ger&uuml;st-Elastizit&auml;t auf Germ Layer Spezifikation von menschlichen embryonalen Stammzellen.

&#x80DA;&#x7D30;&#x80DE;&#x304B;&#x3089;&#x30B7;&#x30F3;&#x30D7;&#x30EB;&#x306A;&#x306E;&#x7403;&#x5F62;&#x306E;&#x7403;&#x306F;&#x591A;&#x5C64;&#x6709;&#x6A5F;&#x4F53;&#x306B;&#x5909;&#x63DB;&#x3055;&#x308C;&#x308B;&#x3068;&#x3059;&#x3050;&#x6A5F;&#x68B0;&#x529B;&#x306F;&#x80DA;&#x767A;&#x751F;&#x306B;&#x91CD;&#x8981;&#x3067;&#x3059;&#x5177;&#x4F53;&#x7684;&#x306B;&#x306F;&#x3001;&#x30EA;&#x30CD;&#x30FC;&#x30B8;&#x30E5;&#x4ED5;&#x69D8;&#x539F;&#x8178;&#x9665;&#x5165;&#x30D5;&#x30A7;&#x30FC;&#x30BA;&#x3067;&#x306F;&#x3001;&#x6B63;&#x3057;&#x304F;&#x3092;&#x542B;&#x3080;&#x5185;&#x80DA;&#x8449;&#x3001;&#x4E2D;&#x80DA;&#x8449;&#x3001;&#x304A;&#x3088;&#x3073;&#x5916;&#x80DA;&#x8449;&#x306E;&#x80DA;&#x5C64;&#x7D44;&#x7E54;&#x3002;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x30EC;&#x30DD;&#x30FC;&#x30C8;&#x306F;&#x3001;&#x3053;&#x306E;&#x3088;&#x3046;&#x306A;&#x76E3;&#x7763;&#x3057;&#x3001;&#x5354;&#x8ABF;&#x904B;&#x52D5;&#x5927;&#x7D30;&#x80DE;&#x306E;&#x96C6;&#x56E3;&#x306E;&#x7D30;&#x80DE;&#x5FDC;&#x7B54;&#x306B;&#x30BB;&#x30EB;&#x5909;&#x5F62;&#x3068;&#x30BB;&#x30EB;&#x751F;&#x6210;&#x529B;&#x306B;&#x3088;&#x3063;&#x3066;&#x99C6;&#x52D5;&#x3055;&#x308C;&#x308B;&#x63D0;&#x6848;&#x3057;&#x3066;&#x3044;&#x308B;.&#x3053;&#x308C;&#x3089;&#x74B0;&#x5883;&#x306B;&#x3088;&#x308B;&#x7D30;&#x80DE;&#x306E;&#x5909;&#x5316;&#x3092;&#x3088;&#x308A;&#x3088;&#x304F;&#x7406;&#x89E3;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x6211;&#x3005; &#x80DA;&#x82BD;&#x5C64;&#x5F62;&#x6210;&#x904E;&#x7A0B;&#x30D2;&#x30C8;&#x80DA;&#x6027;&#x5E79;&#x7D30;&#x80DE; &#xFF08;hESCs&#xFF09; &#x990A;&#x6B96;&#x306B;&#x3088;&#x308B; 3 &#x6B21;&#x5143; &#xFF08;3 D&#xFF09; &#x8DB3;&#x5834;&#x3067;&#x7279;&#x5B9A;&#x306E;&#x80DA;&#x82BD;&#x5C64;&#x306B;&#x30E2;&#x30C7;&#x30EB;&#x306B;&#x8A2D;&#x8A08;&#x525B;&#x6027;&#x3092;&#x6709;&#x3059;&#x308B;&#x30E2;&#x30C7;&#x30EB;&#x3068;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x8DB3;&#x5834;&#x306E;&#x7570;&#x306A;&#x308B;&#x525B;&#x6027;&#x306E;&#x3057;&#x304D;&#x3044;&#x5024;&#x306B;&#x3088;&#x3063;&#x3066;&#x5404;&#x80DA;&#x8449;&#x3078;&#x306E;&#x5206;&#x5316;&#x304C;&#x4FC3;&#x9032;&#x3055;&#x308C;&#x305F;, &#x539F;&#x8178;&#x9665;&#x5165;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x4E2D;&#x306B;&#x9023;&#x60F3;&#x3055;&#x305B;&#x308B;&#x529B;&#x306E;&#x884C;&#x4F7F;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x5168;&#x4F53;&#x7684;&#x306A;&#x7D50;&#x679C;&#x3067;&#x306F;&#x3001;3 &#x6B21;&#x5143; (3 D) &#x8DB3;&#x5834; hESC &#x5206;&#x5316;&#x3092;&#x6307;&#x793A;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x5FC5;&#x8981;&#x306A;&#x529B;&#x5B66;&#x523A;&#x6FC0;&#x3092;&#x8981;&#x7D04;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x523A;&#x6FC0;&#x306E;&#x305F;&#x3081;&#x306B;&#x60AA;&#x540D;&#x9AD8;&#x304F;&#x904B;&#x547D;&#x3092;&#x6C7A;&#x5B9A;&#x3059;&#x308B;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x518D;&#x751F;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x30D2;&#x30C8;&#x80DA;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x80DA;&#x82BD;&#x5C64;&#x4ED5;&#x69D8;&#x8DB3;&#x5834;&#x5F3E;&#x6027;&#x306E;&#x5F71;&#x97FF;

&#xAE30;&#xACC4;&#xC801;&#xC778; &#xD798; embryogenesis, &#xC911;&#xC694; &#xD55C; &#xD2B9;&#xD788; &#xD608;&#xD1B5; &#xC0AC;&#xC591; gastrulation &#xB2E8;&#xACC4;&#xC5D0;&#xC11C; &#xBC30;&#xC544; &#xC138;&#xD3EC;&#xC758; &#xAC04;&#xB2E8;&#xD55C; &#xAD6C;&#xD615; &#xBCFC;&#xC5D0;&#xC11C; &#xB2E4;&#xCE35;&#xB41C; &#xC720;&#xAE30; &#xCCB4; &#xBCC0;&#xD658; &#xB429;&#xB2C8;&#xB2E4; &#xADF8; &#xC704;&#xC5D0; &#xC81C;&#xB300;&#xB85C; &#xD3EC;&#xD568; &#xB41C; &#xAD6C;&#xC131; &#xB429;&#xB2C8;&#xB2E4; endoderm mesoderm, &#xBC0F; ectoderm &#xC138;&#xADE0; &#xB808;&#xC774;&#xC5B4;. &#xC5EC;&#xB7EC; &#xBCF4;&#xACE0;&#xC11C; &#xAC19;&#xC740; &#xAC10;&#xB3C5;&#xACFC; &#xD070; &#xC138;&#xD3EC; &#xC9D1;&#xB2E8;&#xC758; &#xC870;&#xC815;&#xB41C; &#xC6C0;&#xC9C1;&#xC784; &#xC140;&#xB8F0;&#xB7EC; &#xC751;&#xB2F5; &#xC138;&#xD3EC; &#xBCC0;&#xD615; &#xBC0F; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xC0DD;&#xC131; &#xB41C; &#xC138;&#xB825;&#xC5D0; &#xC758;&#xD574; &#xAD6C;&#xB3D9; &#xB429;&#xB2C8;&#xB2E4; &#xC81C;&#xC548; &#xD588;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xD658;&#xACBD; &#xC720;&#xB3C4; &#xC138;&#xD3EC; &#xBCC0;&#xD654;&#xB97C; &#xC774;&#xD574; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC6B0;&#xB9AC;&#xB294; &#xBAA8;&#xB378;&#xB85C; &#xC138;&#xADE0; &#xCE35; &#xD615;&#xC131; &#xACFC;&#xC815; &#xC778;&#xAC04; &#xBC30;&#xC544; &#xC904;&#xAE30; &#xC138;&#xD3EC; (hESCs)&#xB97C; &#xBC30;&#xC591; &#xD558; &#xC5EC; 3 &#xCC28;&#xC6D0; (3D) &#xAC74;&#xC124; &#xAE30;&#xACC4; &#xC784;&#xB300; &#xD2B9;&#xC815; &#xC138;&#xADE0; &#xCE35;&#xC5D0; &#xBAA8;&#xB378;&#xC744; &#xC124;&#xACC4; &#xD558;&#xB294; &#xAC15;&#xC131;&#xACFC;. &#xC6B0;&#xB9AC;&#xB294; &#xAC01; &#xC138;&#xADE0; &#xCE35;&#xC744; &#xCC28;&#xBCC4;&#xD654;&#xB294; &#xC7A5;&#xBE44;&#xC758; &#xB2E4;&#xB978; &#xAC15;&#xC131; &#xC784;&#xACC4;&#xAC12;&#xC5D0; &#xC758;&#xD574; &#xC2B9;&#xC9C4; &#xD588;&#xB2E4;, gastrulation &#xACFC;&#xC815; &#xC911;&#xAC00; &#xD574;&#xC9C0;&#xB294; &#xC138;&#xB825;&#xC758; &#xC5F0;&#xC0C1;&#xC744; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. &#xC804;&#xBC18;&#xC801;&#xC778; &#xACB0;&#xACFC; 3 &#xCC28;&#xC6D0; (3D) &#xAC74;&#xC124; &#xAE30;&#xACC4; &#xC784;&#xB300; hESC &#xCC28;&#xBCC4;&#xD654; &#xAC10;&#xB3C5;&#xC5D0; &#xD544;&#xC694;&#xD55C; &#xAE30;&#xACC4;&#xC801; &#xC790;&#xADF9;&#xC744; &#xC815;&#xB9AC; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xBC0F; &#xC774;&#xB7EC;&#xD55C; &#xC790;&#xADF9; hESC &#xC6B4;&#xBA85;&#xC744; &#xACB0;&#xC815; &#xD558;&#xB294; &#xB370; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xD560; &#xC218;&#xB294; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xC778;&#xAC04; &#xBC30;&#xC544; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC758; &#xC138;&#xADE0; &#xB808;&#xC774;&#xC5B4; &#xC0AC;&#xC591;&#xC5D0; &#xBE44; &#xACC4; &#xD0C4;&#xC131;&#xC758; &#xC601;&#xD5A5;.

Fuerzas mec&aacute;nicas son fundamentales para la embriog&eacute;nesis, espec&iacute;ficamente, en la fase de gastrulaci&oacute;n de linaje-especificaci&oacute;n, con lo cual el embri&oacute;n se transforma de una simple bola esf&eacute;rica de c&eacute;lulas en un organismo varias capas, que contienen adecuadamente organizado capas germinales de ectodermo, mesodermo y endodermo. Varios informes han propuesto que tal dirigidos y coordinados movimientos de colectivos de c&eacute;lulas grandes son conducidos por respuestas celulares a las deformaciones de la c&eacute;lula y las fuerzas generadas por la c&eacute;lula. Para entender mejor estos cambios celular inducida por el medio ambiente, hemos modelado el proceso de formaci&oacute;n de la capa del germen por cultivo de c&eacute;lulas madre embrionarias humanas (hESCs) en tres dimensiones andamios de (3D) con rigidez dirigido al modelo que se encuentra en capas de germen espec&iacute;ficas. Demostramos que la diferenciaci&oacute;n a cada capa del germen fue promovida por un umbral diferente de la rigidez de los andamios, que recuerdan a las fuerzas ejercidas durante el proceso de gastrulaci&oacute;n. Los resultados sugieren que las tres dimensiones (3D) andamios pueden recapitular los est&iacute;mulos mec&aacute;nicos necesarios para dirigir la diferenciaci&oacute;n de c&eacute;lulas y que estos est&iacute;mulos pueden desempe&ntilde;ar un papel importante en determinar el destino de hESC.

La influencia de la elasticidad del andamio en la especificaci&oacute;n de la capa germinal de c&eacute;lulas madre embrionarias humanas.

Concerns over neurotoxicity have impeded the development of sustained release formulations providing prolonged duration local anesthesia (PDLA) from a single injection, for which there is an urgent clinical need. Here, we have used toxicogenomics to investigate whether nerve injury occurred during week-long continuous sciatic nerve blockade by microspheres containing bupivacaine, tetrodotoxin, and dexamethasone (TBD). Animals treated with amitriptyline solution (our positive control for local anesthetic-associated nerve injury) developed irreversible nerve blockade, had severely abnormal nerve histology, and the expression of hundreds of genes was altered in the dorsal root ganglia at 4 and 7 days after injection. In marked contrast, TBD-treated nerves reverted to normal function, were normal histologically and there were changes in the expression of a small number of genes. Toxicogenomic studies have great potential in delineating patterns of gene expression associated with specific patterns of tissue injury (e.g. amitriptyline neurotoxicity), and in identifying related changes in gene expression upon exposure to a drug, biomaterial, or drug delivery system.

Toxicogenomic analysis of a sustained release local anesthetic delivery system.

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations, including their reliance on exogenous materials or electrical fields, which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30-80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to 11 cell types, including embryonic stem cells and immune cells. When used for the delivery of transcription factors, the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed, its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications.

A vector-free microfluidic platform for intracellular delivery.

Reactive oxygen species (ROS) scavengers such as beta-mercaptoethanol (BME) and monothiol glycerol (MTG) are extensively used in stem cell research to prevent cellular oxidative stress. However, how these antioxidant supplements impact stem cell cardiac differentiation, a process regulated by redox-signaling remains unknown. In this study, we found that removal of BME from the conventional high-glucose, serum-based differentiation medium improved cardiac differentiation efficiency by 2-3 fold. BME and MTG treatments during differentiation significantly reduced mRNA expression of cardiac progenitor markers (NKX2.5 and ISL1) as well as sarcomeric markers (MLC2A, MLC2V, TNNI3, MYH6 and MYH7), suggesting reduced cardiomyogenesis by BME or MTG. Moreover, BME and MTG altered the expression ratios between the sarcomeric isoforms. In particular, TNNI3 to TNNI1 ratio and MLC2V to MLC2A ratio were significantly lower in BME or MTG treated cells than untreated cells, implying altered cardiomyocyte phenotype and maturity. Lastly, BME and MTG treatments resulted in less frequent beating, slower contraction and relaxation velocities than untreated cells. Interestingly, none of the above-mentioned effects was observed with Trolox, a non-thiol based antioxidant, despite its strong antioxidant activity. This work demonstrates that commonly used antioxidant supplements may cause considerable changes to cellular redox state and the outcome of differentiation.

Commonly used thiol-containing antioxidants reduce cardiac differentiation and alter gene expression ratios of sarcomeric isoforms.

Human pluripotent stem cell-derived cardiomyocytes represent a promising cell source for cardiac repair. However, their clinical translation is hindered by limited evidence for functional benefits in primate models, potential risks for arrhythmias, and teratoma formation. A recent study by Liu et al. (2018) makes significant progress on these critical issues.

Moving iPSC-Derived Cardiomyocytes Forward to Treat Myocardial Infarction.

Anisotropic biomaterials can affect cell function by driving cell alignment, which is critical for cardiac engineered tissues. Recent work, however, has shown that pluripotent stem cell-derived cardiomyocytes may self-align over long periods of time. To determine how the degree of biomaterial substrate anisotropy impacts differentiating cardiomyocyte structure and function, we differentiated mESCs to cardiomyocytes on non-aligned, semi-aligned, and aligned fibrous substrates and evaluated cell alignment, contractile displacement, and calcium transient synchronicity over time. Although cardiomyocyte gene expression was not affected by fiber alignment, we observed gradient- and threshold-based differences in cardiomyocyte alignment and function. Cardiomyocyte alignment increased with the degree of fiber alignment in a gradient-based manner at early time points and in a threshold-based manner at later time points. Calcium transient synchronization tightly followed cardiomyocyte alignment behavior, allowing highly anisotropic biomaterials to drive calcium transient synchronization within 8 days, while such synchronized cardiomyocyte behavior required 20 days of culture on non-aligned biomaterials. In contrast, cardiomyocyte contractile displacement had no directional preference on day 8 yet became anisotropic in the direction of fiber alignment on aligned fibers by day 20. Biomaterial anisotropy impact on differentiating cardiomyocyte structure and function is temporally-dependent.

Janet Zoldan

David H. Koch Institute for Integrative Cancer Research

Massachusetts Institute of Technology

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Janet Zoldan

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}David H. Koch Institute for Integrative Cancer Research

Massachusetts Institute of Technology

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

David H. Koch Institute for Integrative Cancer Research