ד&quot;ר חי נתמכה על ידי מלגה RCUK, ד&quot;ר המערבית נתמכה על ידי מחלקת כירורגיה, ד&quot;ר Medine נתמך על ידי מענק מהקרן Core BHF, מר בלטסאר Lucendo-Villarin נתמכה על ידי דוקטור MRC Studenship. ד&quot;ר ג&#39;ואו נתמכה על ידי מלגה מהממשלה הסינית.

<ol>
	<li>Asgari, S., Pournasr, B., Salekdeh, G. H., Ghodsizadeh, A., Ott, M., Baharvand, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells:+a+new+era+for+hepatology.">Induced pluripotent stem cells: a new era for hepatology.</a> J. Hepatol. 53, 738-751 (2010).</li><li>Hay, D. C., Pernagallo, S., Diaz-Mochon, J. J., Medine, C. N., Greenhough, S., Hannoun, Z., Schrader, J., Black, J. R., Fletcher, J., Dalgetty, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbiased+Screening+of+Polymer+Libraries+to+Define+Novel+Substrates+for+Functional+Hepatocytes+with+Inducible+Drug+Metabolism.">Unbiased Screening of Polymer Libraries to Define Novel Substrates for Functional Hepatocytes with Inducible Drug Metabolism.</a> Stem Cell Research. 6, 92-101 (2011).</li><li>Payne, C. M., Samuel, K., Pryde, A., King, J., Brownstein, D., Schrader, J., Medine, C. N., Forbes, S. J., Iredale, J. P., Newsome, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+of+Functional+Hepatocyte+Like+Cells+in+Immune+Compromised+Mice.">Persistence of Functional Hepatocyte Like Cells in Immune Compromised Mice.</a> Liver International. 31, 254-262 (2011).</li><li>Greenhough, S., Medine, C., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+Stem+Cell+Derived+Hepatocyte+Like+Cells+and+their+Potential+in+Toxicity+Screening.">Pluripotent Stem Cell Derived Hepatocyte Like Cells and their Potential in Toxicity Screening.</a> Toxicology. 278, 250-255 (2010).</li><li>Medine, C. N., Greenhough, S., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Stem+Cell+Derived+Hepatic+Endoderm+in+Human+Drug+Discovery.">The Role of Stem Cell Derived Hepatic Endoderm in Human Drug Discovery.</a> Biochemical Society Transactions. 38, 1033-1036 (2010).</li><li>Hannoun, Z., Fletcher, J., Greenhough, S., Medine, C. N., Samuel, K., Sharma, R., Pryde, A., Black, J. R., Ross, J. A., Wilmut, I., Iredale, J. P., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Comparison+between+Conditioned+Media+and+Serum+Free+Media+in+Human+Embryonic+Stem+Cell+Culture+and+Differentiation.">The Comparison between Conditioned Media and Serum Free Media in Human Embryonic Stem Cell Culture and Differentiation.</a> Cellular Reprogramming. 12, 133-140 (2010).</li><li>Dalgetty, D. M., Medine, C., Iredale, J. P., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+Future+Challenges+in+Stem+Cell-Derived+Liver+Technologies.">Progress and Future Challenges in Stem Cell-Derived Liver Technologies.</a> American Journal of Physiology - Gastrointestinal and Liver Physiology. 297, 241-248 (2009).</li><li>Hay, D. C., Fletcher, J., Payne, C., Terrace, J. D., Gallagher, R. C. J., Snoeys, J., Black, J., Wojtacha, D., Samuel, K., Hannoun, Z., Pryde, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Efficient+Differentiation+of+hESCs+to+Functional+Hepatic+Endoderm+Requires+ActivinA+and+Wnt3a+Signalling.">Highly Efficient Differentiation of hESCs to Functional Hepatic Endoderm Requires ActivinA and Wnt3a Signalling.</a> Proceedings of the National Academy of Sciences. 105, 12301-12306 (2008).</li><li>Fletcher, J., Cui, W., Samuels, K., Black, J. R., Currie, I. S., Terrace, J. D., Payne, C., Filippi, C., Newsome, P., Forbes, S. J., Ross, J. A., Iredale, J. P., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Inhibitory+Role+of+Stromal+Cell+Mesenchyme+on+Human+Embryonic+Stem+Cell+Hepatocyte+Differentiation+is+Overcome+by+Wnt3a+Treatment.">The Inhibitory Role of Stromal Cell Mesenchyme on Human Embryonic Stem Cell Hepatocyte Differentiation is Overcome by Wnt3a Treatment.</a> Cloning and Stem Cells. 10, 331-340 (2008).</li></ol>

אין ניגודי אינטרסים הכריז.

פיתחנו מודל פשוט, הומוגנית לשעתקו מאוד במבחנה כדי לייצר רמות להרחבה של HLCs האדם. המודל שלנו קיבל תוקף על ידי מספר מעבדות פעולה חיצוניים. אנו שגרתי לאפיין בתאי גזע שמקורם HLCs באמצעות ארגז הכלים שלנו לבית של סמנים התפתחותי מסוים מבחני תפקודי כבד (שרובם זמינים מסחרית)...

Matrigel לוחות ציפוי צלוחיות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Matrigel (10 מ&quot;ל, BD Biosciences, בריטניה); חנות ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> KO-DMEM (500 מ&quot;ל, Gibco, Invitrogen, בריטניה); חנות ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> תרבות רקמות צלחות (6 טוב, 12 טוב, קורנינג, בריטניה) </li><li style=";text-align:right;direction:rtl"> תרבות רקמות צפחת (25 CM2 פרקו, קורנינג, בריטניה) </li></ol> hESC תחזוקה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עכבר בינוני פיברובלסטים עובריים מותנה (MEF-CM) (100 מ&quot;ל, R &amp; D מערכות, ארה&quot;ב); חנות ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> פתרון BSA (50 מ&quot;ל, סיגמא אולדריץ&#39;, בריטניה); חנות ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> צמיחה האדם הבסיסיות גורם פיברובלסטים (100 מיקרוגרם, Peprotech, ארה&quot;ב); חנות ב -20 ° C. </li></ol> HESCs Passaging עם collagenase <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> גם בקבוק ומחוברות או hESCs. </li><li style=";text-align:right;direction:rtl"> Matrigel בארות מצופה צלוחיות או על פי הצורך. </li><li style=";text-align:right;direction:rtl"> Phospate שנאגרו מלוחים (-MgCl 2,-CaCl 2) (500 מ&quot;ל, Gibco, Invitrogen, בריטניה); לאחסן בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> Collagenase IV (1 גרם, Gibco, Invitrעוגן, בריטניה); חנות ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> עכבר בינוני פיברובלסטים עובריים מותנה (MEF-CM) (100 מ&quot;ל, R &amp; D מערכות, ארה&quot;ב). </li><li style=";text-align:right;direction:rtl"> צמיחה האדם הבסיסיות גורם פיברובלסטים (100 מיקרוגרם, Peprotech, ארה&quot;ב). </li></ol> דיפרנציאציה של hESCs האנדודרם אל הכבד <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> RPMI 1640 (500 מ&quot;ל, Gibco, Invitrogen, בריטניה); חנות ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> B27 מוסף (10 מ&quot;ל, Gibco, Invitrogen, בריטניה); חנות ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> Activin (2 מיקרוגרם, Peprotech, ארה&quot;ב); חנות ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> רקומביננטי עכבר Wnt3a (2 מיקרוגרם, R &amp; D מערכות, ארה&quot;ב); חנות ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> KO-DMEM (500 מ&quot;ל, Gibco, Invitrogen, בריטניה); חנות ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> KO-SR (500 מ&quot;ל, Gibco, Invitrogen, בריטניה); חנות ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> ללא חומצות אמינו חיוניות (100 מ&quot;ל, Gibco, Invitrogen, בריטניה); חנות ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> β-Mercaptoethanol (10 מ&quot;ל, Gibco, Invitrogen, בריטניה); חנות ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> DMSO (סיגמא אולדריץ&#39;, בריטניה); לאחסן בטמפרטורת החדר </li><li style=";text-align:right;direction:rtl"> ליבוביץ L-15 תרבות בינוני (500 מ&quot;ל, Sigmאולדריץ&#39;, בריטניה); חנות ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> Tryptose פוספט מרק (100 מ&quot;ל, סיגמא אולדריץ&#39;, בריטניה); חנות ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> Foetal שור בסרום, חום מומת (500 מ&quot;ל, Gibco, Invitrogen, בריטניה); חנות ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> 21-hemisuccinate (100 מ&quot;ג, סיגמא אולדריץ&#39;, בריטניה) הידרוקורטיזון: חנות ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> אינסולין (הלבלב שור) (100 מ&quot;ג, סיגמא אולדריץ&#39;, בריטניה); חנות ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> L-גלוטמין (100 מ&quot;ל, Gibco, Invitrogen, בריטניה); חנות ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> חומצה אסקורבית (25 גרם, סיגמא אולדריץ&#39;, בריטניה); חנות ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> האדם HGF (10 מיקרוגרם, Peprotech, ארה&quot;ב); חנות ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> רקומביננטי Oncostatin M האדם (OSM) (50 מיקרוגרם, R &amp; D מערכות, ארה&quot;ב); חנות ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> מזרק מונעת יחידת מסנן 0.22 מיקרומטר (Millipore, בריטניה) </li></ol> אפיון האנדודרם נגזר hESC כבדית Immunostaining <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> פוספט חיץ מלוחים (-MgCl 2,-CaCl 2) (500 מ&quot;ל, Gibco, Invitrogen, בריטניה); stoמחדש בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> PBST, PBS מורכב עם 0.1% tween 20 (Sigma-Aldrich, בריטניה). </li><li style=";text-align:right;direction:rtl"> Paraformaldehyde (PFA) (Sigma-Aldrich, בריטניה) מורכבת ב-PBS, חנות -20 ° C. </li><li style=";text-align:right;direction:rtl"> גליצרול (Sigma-Aldrich, בריטניה), לאחסן בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> טריס Base (Sigma-Aldrich, בריטניה), לאחסן בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> אתנול </li><li style=";text-align:right;direction:rtl"> סרום (עבד Serotech, בריטניה), לאחסן ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> נוגדן משניים, fluorophores אלקסה (בדיקות מולקולריות, Invitrogen, בריטניה). </li><li style=";text-align:right;direction:rtl"> MOWIOL 4-88 (Polysciences Inc, ארה&quot;ב) מורכב ב טריס HCL וגליצרול כמו לכל הוראות היצרנים. DAPI (פירס, תרמו פישר סיינטיפיק, בריטניה) הוא הוסיף הפתרון MOWIOL בדילול 1:1000. </li></ol><table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td colspan="2"> ראשי נוגדנים </td><td></td><td></td></tr><tr><td> Antigen * </td><td> סוג </td><td> ספק </td><td> מהילה </td></tr><tr><td> Alb </td><td> חד שבטיים עכבר </td><td> סיגמא אולדריץ&#39; </td><td> 1 / 5<html> 00 </td></tr><tr><td> E-cadherin </td><td> חד שבטיים עכבר </td><td> Millipore </td><td> 1 / 100 </td></tr><tr><td> α-fetoprotein </td><td> חד שבטיים עכבר </td><td> סיגמא </td><td> 1 / 500 </td></tr><tr><td> SSEA-4 FITC </td><td> חד שבטיים עכבר </td><td> Biolegend </td><td> 1 / 100 </td></tr><tr><td> IgG </td><td> חד שבטיים עכבר </td><td> DAKO </td><td> 1 / 500 </td></tr><tr><td colspan="2"> משני נוגדנים </td><td></td><td></td></tr><tr><td> אנטי עכבר FITC המצומד </td><td> חד שבטיים עיזים </td><td> Invitrogen </td><td> 1 / 400 </td></tr></tbody></table> טבלה 2. הנוגדנים המשמשים hESC נגזר immunostaining הכבד האנדודרם, בריכוזים השתמשו, המינים התפתחו וחברות שהם שנרכשו. ניתוח פונקציונלי של האנדודרם הכבד Normalisation (לכל חלבון מ&quot;ג) ss = &quot;jove_step&quot;&gt; מבחני ציטוכרום P450 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> P4-Glo CYP3A4, CYP1A2, ערכות ו luminometer (Promega, ארה&quot;ב). </li><li value="1" style=";text-align:right;direction:rtl"> לבן קרקעית שטוחה 96 גם assay צלחת (BD Biosciences, בריטניה). </li><li style=";text-align:right;direction:rtl"> BCA Assay Kit (פירס, תרמו פישר סיינטיפיק, בריטניה). </li><li style=";text-align:right;direction:rtl"> Assay שקוף 96 צלחת גם (Iwaki, בריטניה) </li></ol>

robust generation of hepatocyte-like cells from human embryonic stem cell populations

למרות ההתקדמות רעילות התרופה דוגמנות אדם, תרכובות רבים נכשלים במהלך הניסויים הקליניים, בשל תופעות לוואי צפויות. העלות של מחקרים קליניים הם משמעותיים, ולכן חיוני יותר מסכי הרעלים חזוי מפותחים לפרוס בשלב מוקדם של פיתוח התרופה (Greenhough et al 2010). Hepatocytes האדם מייצגים את המודל הנוכחי זהב סטנדרטי להערכת רעילות התרופה, אבל הם משאב מוגבל כי התערוכה פונקציה משתנה. לכן, השימוש של שורות תאים ורקמות הנציח במודלים של בעלי חיים מועסקים באופן קבוע בשל השפע שלהם. בעוד שני מקורות מידע, הם מוגבלים על ידי פונקציה עניים, השתנות המינים ו / או חוסר יציבות של תרבות (Dalgetty et al 2009). בתאי גזע pluripotent (PSCs) הם מקור חלופה אטרקטיבית של hepatocyte אדם כמו תאים (HLCs) (Medine et al 2010). PSCs מסוגלים התחדשות עצמית והבחנה לכל סוגי תאים סומטיים נמצא המבוגר ובכך מייצגיםמקור פוטנציאלי בלתי נדלה של תאים מובחנים. פיתחנו הליך פשוט, יעיל ביותר, מקובל אוטומציה התשואות HLCs אדם תפקודית (Hay et al 2008; פלטשר et al 2008; חנון et al 2010; פיין et al 2011 ו Hay et al 2011). אנו מאמינים כי הטכנולוגיה שלנו יוביל לייצור להרחבה של HLCs עבור גילוי תרופות, מחלות דוגמנות, בניית חוץ הגשמי מכשירים סלולריים ואולי טיפולים להשתלה מבוסס.

Watch this Scientific Journal Video about Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations at JoVE.com

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

מאמר זה יתמקד דור האנדודרם הכבד האנושי אנושי אוכלוסיות תאים גזע עובריים.

הדור יציבה של hepatocyte דמויי תאים של אדם אוכלוסיות תאים גזע עובריים

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

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47.7K Views.  University of Edinburgh. מאמר זה יתמקד דור האנדודרם הכבד האנושי אנושי אוכלוסיות תאים גזע עובריים.

Video: Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

רביה של האדם גזע עובריים (ES) תאים

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

גזירת בתאי גזע hematopoietic מ Murine בתאי גזע עובריים

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

גזירת בתאי גזע עובריים אנושיים על ידי Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

הכרומטין immunoprecipitation מן בתאי גזע עובריים אנושיים

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

הדור של האדם CD40 בתאי B המופעל

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

מקפיא הפשרה בתאי גזע עובריים אנושיים

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

התמיינות של תאים עובריים לתוך מבשרי Oligodendrocyte

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

הגורל מיפוי בתאי גזע עובריים אנושיים על ידי גיבוש תפלצת

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

גזירת יעיל של מבשרי לב cardiomyocytes האדם מן האדם תאים pluripotent גזע עובריים עם אינדוקציה מולקולה קטנה

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

הסרת פיברובלסטים מהירה מתרבויות צפיפות גבוהות אדם תאי גזע עובריות

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...