תמיכה לKKB, MJB, JLH וKln סופק על ידי המכון בקליפורניה הרגנרציה רפואה, נאמנויות תכנית חוקרי ביו צדקת Pew, אסתר ב &#39;קרן המשפחה או&#39; קיף וקרן משפחת שפירא. KKB הוא דונלד א &#39;והדלייה ב קסטר קרן הפקולטה Scholar.

<ol>
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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPS+cells+produce+viable+mice+through+tetraploid+complementation.">iPS cells produce viable mice through tetraploid complementation.</a> Nature. 461, 86-90 (2009).</li><li>Kang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viable+mice+produced+from+three-factor+induced+pluripotent+stem+(iPS)+cells+through+tetraploid+complementation.">Viable mice produced from three-factor induced pluripotent stem (iPS) cells through tetraploid complementation.</a> Cell Res. 21, 546-549 (2011).</li><li>Zhao, X. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viable+Fertile+Mice+Generated+from+Fully+Pluripotent+iPS+Cells+Derived+from+Adult+Somatic+Cells.">Viable Fertile Mice Generated from Fully Pluripotent iPS Cells Derived from Adult Somatic Cells.</a> Stem Cell Reviews and Reports. 6, 390-397 (2010).</li><li>Han, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tbx3+improves+the+germ-line+competency+of+induced+pluripotent+stem+cells.">Tbx3 improves the germ-line competency of induced pluripotent stem cells.</a> Nature. 463, 1096-1100 (2010).</li><li>Stadtfeld, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascorbic+acid+prevents+loss+of+Dlk1-Dio3+imprinting+and+facilitates+generation+of+all-iPS+cell+mice+from+terminally+differentiated+B+cells.">Ascorbic acid prevents loss of Dlk1-Dio3 imprinting and facilitates generation of all-iPS cell mice from terminally differentiated B cells.</a> Nat. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+and+direct+comparison+of+the+dimerizer+and+reverse+tet+transcriptional+control+systems.">Optimization and direct comparison of the dimerizer and reverse tet transcriptional control systems.</a> The Journal of Gene Medicine. 4, 258-270 (2002).</li><li>Huangfu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+by+defined+factors+is+greatly+improved+by+small-molecule+compounds.">Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds.</a> Nature. 26, 795-797 (2008).</li><li>Li, X. -. y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passage+number+affects+the+pluripotency+of+mouse+embryonic+stem+cells+as+judged+by+tetraploid+embryo+aggregation.">Passage number affects the pluripotency of mouse embryonic stem cells as judged by tetraploid embryo aggregation.</a> Cell and Tissue Research. 327, 607-614 (2007).</li><li>George, S. H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+and+adult+phenotyping+directly+from+mutant+embryonic+stem+cells.">Developmental and adult phenotyping directly from mutant embryonic stem cells.</a> Proceedings of the National Academy of Sciences. 104, 4455-4460 (2007).</li><li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the mouse embryo: A laboratory manual. , (2003).</li><li>Kirak, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transnuclear+Mice+with+Pre-defined+T+Cell+Receptor+Specificities+Against+Toxoplasma+gondii+Obtained+Via+SCNT.">Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT.</a> J. Vis. Exp. (43), e2168 (2010).</li></ol>

אין ניגודי האינטרסים הכריזו.

עכברים שהניבו מקווי iPSC באמצעות מבחני TEC מספקים מבחן פונקציונלי מחמיר לpluripotency של קו iPSC. בדיקה זו עשויה להיות שימושית כדי להעריך את היעילות היחסית של שיטות שונות תכנות מחדש או לזהות קווי iPSC שעשויה להיות שימושיים ביותר ליצירת תאים מסוגים מסוימים במבחנה. עכברים שנוצרו מiPSCs ניתן להשתמש בקפדנות על מנת לבחון את היציבות לטווח ארוך וtumorigenicity של רקמות iPSC נגזרות. פרוטוקול זה יהיה שימושי לחוקרים המעוניינים לייצר קווי iPSC pluripotent מלא או עכברי iPSC או להשוות את התועלת היחסית של שיטות שונות תכנות מחדש. את המנגנונים ששולטים הדור וזיהוי של iPSCs pluripotent מלא יישארו הבין היטב וזה אפשרי כי כמה שורות iPSC הופקו באמצעות שיטה זו לא עוברת את מבחן TEC. גורמים רבים עשויים להשתנות בין ניסויים, כולל רקע גנטי, כייל lentiviral, דפוסים של i lentiviralnsertion, פרמטרי מחזור התא של האוכלוסייה התורמת, הבדלים בין מעבדה בשלבים שונים של הליך TEC ונטיות משתנות של iPSCs לנמל סטיות גנטיות או epigenetic. הטוב ביותר כדי להבטיח הצלחה, אנו דואגים להקמת רמות המתאימות של ביטוי גנים בניסויי lentiviral גזירת iPSC ידי בדיקת דילולים נגיפיים על השליטה MEFs כדי להבטיח שכל נגיף מרוכז מספיק כדי לייצר ביטוי לגילוי גן לפחות 80% ואידיאליים של 100% MEFs. זה מאפשר לנו לזהות את הקווים עם מספר עותקים של lentiviruses השונה תוך הגבלת הרעילות לMEFs ולייצר מושבות בלי בארות הצפיפות. יצוין כי פרוטוקולים מרובים אחרים הוכחו לייצר iPSCs עם פוטנציאל ההתפתחותי מלא, תוך שימוש במספר שיטות ומקורות של תאים תורמים טוענות כי נתיבים מרובים לpluripotency מלא עשויים להתקיים 1,8-13,15. בשלב הנוכחי, iPSC עם זאת, אין סמן ביולוגי סופי של pluripotent מלאזוהה ולכן בדיקת TEC נותרת מבחן תקן הזהב של אם קו iPSC יכול לייצר את כל שושלות תאים באורגניזם.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>DMEM (high glucose)</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ES cell qualified FBS</td>
			<td>Invitrogen</td>
			<td>104392-024</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Invitrogen</td>
			<td>16140-071</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&beta;-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>Sigma M7522</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.1% Gelatin</td>
			<td>Millipore</td>
			<td>ES006-B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids</td>
			<td>Invitrogen</td>
			<td>11140</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Invitrogen</td>
			<td>11150-059</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ESGRO (murine LIF)</td>
			<td>Millipore</td>
			<td>ESG1106</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Valproic Acid</td>
			<td>Sigma</td>
			<td>P4543</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher</td>
			<td>BP231-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS Ca2+/Mg2+</td>
			<td>Invitrogen</td>
			<td>14040-133</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS Ca2+/Mg2+ free</td>
			<td>Invitrogen</td>
			<td>14190-144</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pregnant mare serum gonadotropin, for superovulation, freeze-dried, 2,000 IU</td>
			<td>Harbor-UCLA Research Institute</td>
			<td>n/a</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chorionic gonadotropin, human</td>
			<td>Sigma</td>
			<td>C1063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FHM medium with Hyaluronidase</td>
			<td>Millipore</td>
			<td>MR-056-F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KSOM-1/2 AA medium</td>
			<td>Millipore</td>
			<td>MR-106-D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FHM</td>
			<td>Millipore</td>
			<td>MR-024-D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Water, for embryo transfer, embryo tested</td>
			<td>Sigma</td>
			<td>W1503</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mineral oil, embryo tested</td>
			<td>Sigma</td>
			<td>M5310</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C7902</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M2773</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D-Mannitol</td>
			<td>Sigma</td>
			<td>M4125</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA), embryo tested</td>
			<td>Sigma</td>
			<td>A3311</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse embryonic fibroblasts, non-irradiated</td>
			<td>Millipore</td>
			<td>PMEF-CFL</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>
			Media and buffers used in this protocol
			HEK293T growth medium. 90% DMEM, 10% FBS, 100 U/ml penicillin and 10 mg/ml streptomycin. Exclude penicillin and streptomycin from HEK media used on day of transfection. HEK medium can be stored at 4 &deg;C for up to 1 month.
			2x HBS. 42 mM Hepes, 274 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4&middot;7H2O, 12 mM Dextrose. pH to 7.1 +/- 0.1. pH is critical! Sterile filter and store at 4 &deg;C.
			Mouse embryonic fibroblast (MEF) growth medium (also for use with feeders). 70% DMEM, 20% Medium 199, 10% FBS, 100 U/ml penicillin and 10 mg/ml streptomycin. Store at 4 &deg;C for up to 1 month.
			ESC growth medium. 85% DMEM,15% ES cell qualified FBS, 1x Glutamax, 0.1 mM non-essential amino acids, 0.1 mM &beta;-mercaptoethanol, 1,000 U/ml ESGRO, 100 U/ml penicillin and 10 mg/ml streptomycin. ESC media can be stored at 4 &deg;C for up to three weeks.
			Electrofusion medium. 0.3 M Mannitol, 0.1 mM MgSO4, 50 mM CaCl2, and 3% BSA in embryo tested water. Store at 4 &deg;C for up to 3 months.</td>
		</tr>
	</tbody>
</table>

generation of mice derived from induced pluripotent stem cells

ייצור תאי גזע pluripotent מושרים (iPSCs) מתאים הסומטיים מספק אמצעי ליצירת כלים רבי ערך למחקר בסיסי ויכול גם לייצר מקור של תאי חולו מותאם לטיפולי התחדשות. iPSCs יכול להיות שנוצר תוך שימוש בפרוטוקולים מרובים ונובע ממקורות סלולריים מרובים. נוצר רגע, iPSCs נבדק תוך שימוש במגוון של מבחנים כוללים immunostaining לסמני pluripotency, דור משלוש שכבות ניבטות בגופי embryoid וteratomas, השוואות של ביטוי גנים בתאי גזע עובריים (ESCs) וייצור של עכברי chimeric עם או בלי תרומת germline 2 . חשוב לציין, קווי iPSC שעוברים בדיקות אלה עדיין משתנים וביכולת שלהם לייצר סוגי תאים מובחנים שונים 2. זה עשה את זה קשה לקבוע שפרוטוקולי iPSC גזירה, מקורות תא תורם או שיטות בחירה הם שימושיים ביותר ליישומים שונים. המבחן המחמיר ביותרלשאלה אם קו תאי גזע יש פוטנציאל ההתפתחותי מספיק כדי לייצר את כל הרקמות דרושות להישרדותו של אורגניזם (מכונה pluripotency המלא) הוא complementation העובר tetraploid (TEC) 3-5. מבחינה טכנית, TEC כרוך electrofusion של עוברי שני תאים לייצר (4n) עוברי 1 תאי tetraploid שניתן בתרבית במבחנה לשלב הבלסטוציסט 6. (2n) תאי גזע pluripotent (דיפלואידי ESCs או iPSCs למשל) לאחר מכן, מוזרקים לתוך חלל blastocoel של הבלסטוציסט tetraploid והועברו לנקבת נמען להריון (ראה תרשים 1). מרכיב tetraploid של העובר השלים תורם באופן כמעט בלעדי לרקמות extraembryonic (שליה, צק חלמון), ואילו תאים דיפלואידיים מהווים את העובר תקין, וכתוצאה מכך עובר נגזר לחלוטין משורת תאי הגזע מוזרקת. לאחרונה, דיווח הגזירה של קווי iPSC שreproducibly לייצר עכברים בוגריםדרך 1 TEC. קווי iPSC אלו מעוררים גורי קיימא עם יעילות של 5-13%, אשר ניתן להשוות ESCs 3,4,7 וגבוה מזה שדווח עבור רוב קווי iPSC אחרים 8-12. דיווחים אלו מראים כי תכנות מחדש ישיר יכול לייצר iPSCs pluripotent שתואם באופן מלא את הפוטנציאל שלהם בESCs התפתחותית והיעילות של יצירת גורים בבדיקות TEC. נכון לעכשיו, לא ברור מה מבדיל בין iPSCs pluripotent מלא וקווים פחות עוצמתיות 13-15. זה גם לא ברור אילו שיטות תכנות מחדש תהיינה לייצר שורות אלה עם היעילות הגבוהה ביותר. כאן אנו מתארים שיטה אחת שמייצרת iPSCs pluripotent מלא ועכברים &quot;כל iPSC&quot;, שעשוי להיות מועילים עבור חוקרים המבקשים להשוות pluripotency של קווי iPSC או להקים את השקילות של שיטות שונות תכנות מחדש.

In step 3, "Derivation of iPSCs from MEFs", one should observe morphological heterogeneity and immature iPSC colony formation starting 4-5 days after doxycycline/VPA addition and mature colonies between 7-10 days (Figure 2). The production of one-cell tetraploid embryos in step 5 is highly efficient (Figure 3). We routinely observe up to 95% of treated two-cell embryos successfully fuse to produce tetraploid one-cell embryos. The protocol followed to inject iPSCs into tetraploid blastocysts (Step 6, Figure 4) is similar to the protocol for injection of ESCs into diploid blastocysts to generate chimeric mice, and can be performed by an experienced microinjectionist. The number of live pups born depends on the cell line (Table 1).
<table border="1">
	<tbody>
		<tr>
			<td colspan="5">MEF preprogramming efficiency 0.01-0.03%</td>
		</tr>
		<tr>
			<td colspan="5">Efficiency of iPSC mouse production by TEC</td>
		</tr>
		<tr valign="top">
			<td>Name</td>
			<td>Description</td>
			<td>Blastocysts injected</td>
			<td>Live Newborn</td>
			<td>Live Adult</td>
		</tr>
		<tr valign="top">
			<td>iMZ-21</td>
			<td>iPSC</td>
			<td>867</td>
			<td>53 (6.1%)</td>
			<td>19 (2.2%)</td>
		</tr>
		<tr valign="top">
			<td>iMZ-9</td>
			<td>iPSC</td>
			<td>195</td>
			<td>7 (3.6%)</td>
			<td>4 (2.1%)</td>
		</tr>
		<tr valign="top">
			<td>iMZ-11</td>
			<td>iPSC</td>
			<td>338</td>
			<td>1(0.3%)</td>
			<td>0 (0%)</td>
		</tr>
	</tbody>
</table>
Table 1. Representative Results.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/4003/4003fig1highres.jpg" src="/files/ftp_upload/4003/4003fig1.jpg" /> 
Figure 1. Schematic of experimental design. Top left: Production of tetraploid blastocysts. Fertilized two-cell embryos from albino mice are electrofused to generate tetraploid one-cell embryos, which are cultured in vitro to the blastocyst stage. Bottom left: Reprogramming. Mouse embryonic fibroblasts are transduced with lentiviral particles encoding Oct4, Sox2, Klf4 and c-Myc and the reverse tetracycline transactivating protein, rtTAM2.2. Addition of doxycycline results in transgene expression and the initiation of reprogramming to iPSCs. Right: Production of iPSC mice. iPSCs derived from pigmented mice are injected into the blastocoel of tetraploid blastocysts and then surgically implanted into pseudo-pregnant recipient mice. Newborn iPSC mice are delivered by Caesarian section and cross-fostered. <a href="https://www.jove.com/files/ftp_upload/4003/4003fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/4003/4003fig2highres.jpg" src="/files/ftp_upload/4003/4003fig2.jpg" /> 
Figure 2. Morphological changes associated with reprogramming. From left to right: Examples of the morphological progression from fibroblasts to iPSC colonies during the course of a reprogramming experiment. <a href="https://www.jove.com/files/ftp_upload/4003/4003fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3" src="/files/ftp_upload/4003/4003fig3.jpg" /> 
Figure 3. Production of tetraploid embryos. Diploid two-cell embryos are subjected to an electric pulse resulting in blastomere fusion and generation of one-cell tetraploid embryos.
<img alt="Figure 4" src="/files/ftp_upload/4003/4003fig4.jpg" /> 
Figure 4. Production of iPSC mice. Left: iPSCs are injected into the blastocoel of a tetraploid blastocyst. Middle: Newborn iPSC mice are distinguished by pigmented eyes. Right: iPSC mouse at three weeks post-delivery.

Watch this Scientific Journal Video about Generation of Mice Derived from Induced Pluripotent Stem Cells at JoVE.com

Generation of Mice Derived from Induced Pluripotent Stem Cells

תאי גזע pluripotent מושרה קווים מניבים (iPSC) מייצרים שורות של פוטנציאל ההתפתחותי שונים גם כאשר הם עוברים בדיקות תקן לpluripotency. כאן אנו מתארים פרוטוקול לייצר עכברים נגזרים לחלוטין מiPSCs, המגדיר את קווי iPSC כבעל pluripotency המלא<sup&gt; 1</sup&gt;.

דור של עכברים שמקורם בתאי גזע pluripotent מושרים

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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21.2K Views.  The Scripps Research Institute. תאי גזע pluripotent מושרה קווים מניבים (iPSC) מייצרים שורות של פוטנציאל ההתפתחותי שונים גם כאשר הם עוברים בדיקות תקן לpluripotency. כאן אנו מתארים פרוטוקול לייצר עכברים נגזרים לחלוטין מiPSCs, המגדיר את קווי iPSC כבעל pluripotency המלא 1.

Video: Generation of Mice Derived from Induced Pluripotent Stem Cells

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

הדור של גזע תאים מושרה pluripotent על ידי תכנות מחדש Fibroblasts האדם עם Lentivirus Stemgent TF האדם הגדר

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Transfecting וNucleofecting תאי גזע אנושיים pluripotent מושרים

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

בחירה בידוד מושבות של בתאי גזע pluripotent מושרה לתכנות מחדש של Fibroblasts למבוגרים

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

תכנות מחדש האדם תאים סומטיים תוך המושרה בתאי גזע pluripotent (iPSCs) באמצעות וקטור retroviral עם GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

דור של תאים אנושיים מושרים pluripotent גזע מדם היקפי באמצעות STEMCCA lentiviral הווקטור

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

דור מהיר ויעיל של נוירונים מתאי גזע pluripotent אדם בפורמט פלייט Multititre

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

דור של cardiomyocytes אדם: פרוטוקול בידול מתאי גזע מושרה pluripotent אדם ללא מזין

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

תאי גזע יעיל דור מושרה אדם פלוריפוטנטיים מתאים הסומטיים של אדם עם סנדאי וירוס

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a homogenous population of cells, the iPSC-CMs generated by current differentiation protocols represent a mixture of cells with ventricular-, atrial-, and nodal-like phenotypes, which complicates phenotypic analyses. Here, a method to optically record action potentials specifically from ventricular-like iPSC-CMs is presented. This is achieved by lentiviral transduction with a construct in which a genetically-encoded voltage indicator is under the control of a ventricular-specific promoter element. When iPSC-CMs are transduced with this construct, the voltage sensor is expressed exclusively in ventricular-like cells, enabling subtype-specific optical membrane potential recordings using time-lapse fluorescence microscopy.

Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein.

סוג משנה ספציפי פוטנציאל הפעולה אופטי הקלטות-אנוש המושרה Cardiomyocytes חדרית נגזר תאי גזע Pluripotent

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a ...

Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular mechanisms of disease, development, and evolution. To make primate induced pluripotent stem cells (iPSCs) more accessible, this paper presents a non-invasive method to generate human and non-human primate iPSCs from urine-derived cells, and their maintenance using a feeder-free culturing method.
The urine can be sampled from a non-sterile environment (e.g., the cage of the animal) and treated with a broad-spectrum antibiotic cocktail during primary cell culture to reduce contamination efficiently. After propagation of the urine-derived cells, iPSCs are generated by a modified transduction method of a commercially available Sendai virus vector system. First&#160;iPSC colonies may already be visible after 5 days, and can be picked after 10 days at the earliest. Routine clump passaging with enzyme-free dissociation buffer supports pluripotency of the generated iPSCs for more than 50 passages.

The present protocol describes a method to isolate, expand, and reprogram human and non-human primate urine-derived cells to induced pluripotent stem cells (iPSCs), as well as instructions for feeder-free maintenance of the newly generated iPSCs.