Die Autoren möchten die folgenden Zuschüsse aus dem NIH für die Unterstützung dieser Forschung zu bestätigen: K08DK082783 (AR), P30DK56465 (BTS), U01HL099993 (BTS), T32HL00715036 (SKS) und K12HL0806406 (SKS); und die JP McCarthy Foundation (AR).

Eine schriftliche Einverständniserklärung, wie sie durch die Institutional Review Boards des Fred Hutchinson Cancer Research Center und die Kinder &quot;s Hospital von Philadelphia genehmigt wurde von Patienten vor dem Sammeln peripheren Blutproben erhalten. Alle institutionellen Richtlinien festgestellt. Alle Tierversuche einschließlich MEF Erzeugung und Teratom Bildung wurden von der Institutional Animal Care und Use Committee genehmigt. 1. Ficoll Separation von PBMCs und Ausbau der Erythroblasten - Tag 0 <ol><li> Verdünnte peripheren Blut 1: 1 mit Dulbecco-Phosphat-gepufferter Saline (DPBS). In einem 15 ml Rundbodenpolystyrolröhrchen vorsichtig Schicht 7 ml des verdünnten Blutes auf 3 ml Raumtemperatur Ficoll-Hypaque. </li><li> Zentrifugieren Sie die Probe bei 400 g für 30 min. </li><li> Die PBMCs werden in die Grenzfläche zwischen dem Plasma und dem Ficoll-Hypaque, als trübe weiße Schicht gesehen niedergelassen haben. Mit einer sterilen Transferpipette, sammeln die PBMCs in einem 15 ml konischen Röhrchen. Bringen Sie die Lautstärke auf 10 ml mit DPBS. </li><li> Mit einem Hämazytometer, zähle die PBMCs. </li><li> Pipette 2 × 10 6 PBMCs in einen separaten 15 ml konischen Röhrchen und Zentrifuge bei 300 xg für 5 min. Spin-down die restlichen Zellen und Einfrieren bei einer Konzentration von 2 × 10 6 PBMC / ml in 90% fötalem Rinderserum (FBS) / 10% Dimethylsulfoxid (DMSO). </li><li> Bereiten Expansion Medium: QBSF-60 Serum freiem Medium (vor Licht schützen) + Penicillin / Streptomycin (1%) + Ascorbinsäure (50 ng / ml) + Stammzellfaktor (SCF, 50 ng / ml) + Interleukin 3 (IL 3, 10 ng / ml) + Erythropoietin (EPO, 2 U / ml) + Insulin-ähnlichen Wachstumsfaktor 1 (IGF-1, 40 ng / ml) + Dexamethason (1 uM; vor Licht schützen; auftauen einem anderen aliquoten Teil jedes Medium ändern). </li><li> Resuspendieren 2 × 10 6 PBMCs in 2 ml frisch zubereitete Expansionsmedium und in eine Vertiefung einer 12-Well-Gewebekulturplatte legen und inkubieren in einem 37 ° C befeuchteten Inkubator mit einer Atmosphäre von 5% O 2, 5% CO 2 und 90% N 2. </li><li> An Tag 3 und Tag 6, ersetzen Medien. <ol><li> Sammeln Sie die Zellen und waschen die gut mit 2 ml QBSF-60, um alle verbleibenden Zellen zu sammeln. </li><li> Zentrifugieren der Zellsuspension bei 300 xg für 5 min und saugen Sie den Überstand. </li><li> Die Zellen in 2 ml neue Expansionsmedium und bringt sie auf den gleichen 12-Well-Platte. </li></ol></li></ol> 2. Reprogrammierung Zellen durch Nucleofection - Tag 9 <ol><li> Pipettieren Sie die Umprogrammierung Plasmide in ein steriles 1,5 ml Eppendorf-Röhrchen (Tabelle 1). </li><li> Mischen Sie die Nukleofektion Lösung mit Supplement (vom Hersteller mitgeliefert) und Pipette in ein steriles 1,5 ml Eppendorf-Röhrchen. </li><li> Pipette 2 ml des Expansionsmediums in eine Vertiefung einer 12-Well-Platte und in einem befeuchteten Inkubator bei 37ºC (5% O 2, 5% CO 2 und 90% N 2) für die Äquilibrierung vor der Zugabe von Zellen. </li><li> Sammeln Sie diekultivierten Zellen und waschen Sie die gut mit 2 ml QBSF-60, um die verbleibenden Zellen zu sammeln. </li><li> Zentrifugieren Sie die Zellen bei 300 xg für 5 min und saugen Sie den Überstand. </li><li> Die Zellen werden durch Resuspendieren in 5 ml DPBS und Zentrifugieren bei 300 × g für 5 min. </li><li> Saugen Sie den Überstand. </li><li> Zellpellet in 100 ul Nukleofektion Lösung + Beilage, wobei darauf zu Erzeugen von Blasen zu vermeiden. </li><li> Pipettieren der Zellsuspension in die Röhre, die die Neuprogrammierung Plasmide, Pipette auf und ab sofort, und pipettieren die Probe in den unteren Bereich des Nukleofektion Küvette. </li><li> Setzen Sie die Küvette in die Nukleofektion Maschine und Programm ausführen T-019. </li><li> Übertragen Sie 100 ul der vorgewärmten Expansionsmedium (von der ausgleichenden Platte in Schritt 2.3) in die Nukleofektion Küvette. </li><li> Sofort sammeln Sie das gesamte Probe und Einzahlung in den Brunnen der vorgewärmten Expansionsmedium. Vermeiden Sie das Sammeln der weiß, schwimm toten Zelltrümmer. </li&gt; <li> Die Platte wird in einem befeuchteten Inkubator bei 37ºC (5% O 2, 5% CO 2 und 90% N 2) zu wachsen. </li></ol> 3. Überzug Feeder-Zellen - Tag 10 oder 11 <ol><li> Am Tag 10, Platte Feeder-Zellen. <ol><li> Coat ein 6-Well-Gewebekulturplatte mit Gelatine. <ol><li> Pipette 1 m von 0,1% Gelatine in Hanks gepufferter Salzlösung (HBSS) in jede Vertiefung der Platte. </li><li> Die Platte wird in einem befeuchteten 37 ° C Inkubator für mindestens 5 min. </li><li> Unmittelbar vor dem Gebrauch absaugen Gelatinelösung von der Platte. </li></ol></li><li> Auftauen einer Ampulle von 2 × 10 6 embryonalen Maus-Fibroblasten mit 3000 cGy bestrahlt, in 10 ml vorgewärmtem MEF Medien (Dulbecco Modified Eagle Medium (DMEM) + FBS (10%) + Penicillin / Streptomycin (1%)). </li><li> Zentrifugieren Sie die Zellen bei 300 xg für 5 min und saugen Sie den Überstand. Die Zellen in 12 ml MEF Medien und Pipette 2 ml inin jeder Vertiefung der mit Gelatine beschichtete 6-Well-Platte. Die Platte wird in einem befeuchteten 37 ° C-Inkubator (5% O 2, 5% CO 2 und 90% N 2), bis 12. Tag. </li></ol></li></ol> 4. Plattierzellen auf Feeder-Zellen und ändern Medium - Tag 12 <ol><li> Sammeln Sie die nucleofected Zellen in einem 15 ml konischen Röhrchen und sammeln Sie alle restlichen Zellen durch Waschen der gut mit 2 ml QBSF-60. Zentrifugieren Sie die Probe bei 300 xg für 5 min. </li><li> Bereiten Reprogrammierung Medium: Iscove modifiziertem Dulbecco-Medium (IMDM) + FBS (10%) + nicht-tierischen L-Glutamin (1 mM) + nicht-essentiellen Aminosäuren (NEAA, 1%) + Penicillin / Streptomycin (1%) + β Mercaptoethanol (0,1 mm) + basischen Fibroblasten-Wachstumsfaktor (bFGF, fügte frisch bei Fütterung, 10 ng / ml) + Ascorbinsäure (hinzugefügt frisch bei Fütterung, 50 ug / ml). </li><li> Saugen Sie den Überstand und resuspendieren in 12 ml Reprogrammierung Medium. Pipette 2 ml pro Vertiefung der Zellsuspension inder 6-Well-Platte von Feederzellen (wie in Abschnitt 3 hergestellt). Zentrifugieren Sie die Platte bei 50 g für 30 Minuten bei Raumtemperatur. </li><li> Die Platte wird in einem befeuchteten Inkubator bei 37ºC (5% O 2, 5% CO 2 und 90% N 2) zu wachsen. Ändern Sie die Umprogrammierung Medium jeden zweiten Tag, bis iPSC Kolonien beginnen erscheinen (1-2 Wochen). </li><li> Bereiten hESC Medium: DMEM / F12 1: 1 + Knockout Serum Replacement (20%) + Penicillin / Streptomycin (1%) + NEAA (1%) + nicht-tierischen L-Glutamin (1 mM) + Natriumpyruvat (1%) + Natriumbicarbonat (0,12%) + β-Mercaptoethanol (0,1 mM) + bFGF (aufgenommen bei frischen Zufuhr, 10 ng / ml) </li><li> Bereiten HDAC-Inhibitoren: Natriumbutyrat (hinzugefügt frisch bei Fütterung, 100 uM), suberanilohydroxamic Säure (SAHA, fügte frisch bei Fütterung, 200 Nm). Sobald iPSC Kolonien erscheinen, beginnen Fütterung der Zellen mit hES Medium + HDAC-Inhibitoren, wechselnde Medium jeden Tag. HINWEIS: Sodium Butyrat ist ein kleines Molekül, das in Kunststoffröhrchen adsorbiert ist, therefore speichern sie in einer Borosilikatglas Rohr bei 4 ° C. </li></ol> 5. Isola iPSC Clones <ol><li> Sobald iPSC Kolonien groß genug sind, um isoliert werden (20-25 Zellen), holen sie mit einem P20-Pipette. </li><li> Bereiten HDAC-Inhibitoren: Natriumbutyrat (hinzugefügt frisch bei Fütterung, 100 uM), Sacha (hinzugefügt frisch bei Fütterung, 200 nM) </li><li> Legen Sie die isolierte Kolonien auf iMEFs in einzelnen 35 mm Schalen mit hESC Medium + HDAC-Inhibitoren + Cloning &amp; Recovery Supplement. </li><li> Der Tag nach der Ernte die Kolonien, ändern Sie das Medium, um hESC Medium + HDAC-Inhibitoren. </li><li> Ändern Sie das Medium täglich bis iPSC Kolonien sind bereit für das Passagieren. </li><li> Entfernen HDAC-Inhibitoren aus dem Medium einmal Kolonien Stabilisierung bei Passage 2-3. </li></ol>

<ol>
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Die Autoren erklären, dass sie keine konkurrierenden finanziellen Interessen.

Für eine erfolgreiche iPSC Generation, wenn Sie dieses Protokoll, gibt es mehrere wichtige Einschränkungen, die berücksichtigt werden sollten. Während der Erythroblasten Ausbaustufe sollte die Medienwechsel Zeitplan strikt eingehalten werden, da Abweichungen können zu einer ineffizienten Stimulation des Zielvorläuferzellpopulation und einer niedrigeren Effizienz iPSC Generation führen. Es ist wichtig, neue Expansionsmedium mit frischem Dexamethason mit jedem Medienwechsel zu machen; und die QBSF-60 Basismedium un...

iPS-Zellen werden aus somatischen Geweben über ektopische Expression von einem minimalen Satz von Pluripotenz-Gene abgeleitet. Diese Technik wurde ursprünglich von retroviralen Transduktion von humanen Fibroblasten mit OCT4, SOX2, KLF4 und cMYC, die besonders in den pluripotenten Zustand 1 ausgedrückt werden demonstriert. Diese transient exprimiert &quot;Reprogrammierungsfaktoren&quot; alter epigenetische Landschaft und Genexpressionsprofil der Zielzelle analog zu menschlichen embryonalen Stammzellen 2. Einmal erstellt, können iPS-Zellen potenziell in jede Gewebeart zur weiteren Untersuchung differenziert werden. So halten sie versprechen für den Einsatz in der regenerativen Medizin, Krankheitsmodelle und Gentherapieanwendungen. , Stören das Genom mit der Integration von Viren hat jedoch das Potenzial, um die endogene Genexpression beeinflussen zelluläre Phänotyp zu ändern und letztendlich vorspannen wissenschaftlichen Ergebnissen. Darüber hinaus können Zufalls virale Integrationen zu schädlichen Zell e führenUSWIRKUNGEN, einschließlich der Möglichkeit der malignen Transformation 3 oder Wieder Expression der Transgene onkogenen 4. Zukünftige klinische Anwendungen benötigen nicht-integrierenden iPSC Generation. Episomen, die zusätzliche chromosomale zirkuläre DNA-Moleküle sind, bieten eine Strategie, um kostengünstige, Integration freien IPSCs 5 zu erzeugen. Die Kombination von in Tabelle 1 gezeigt episomalen Vektoren auszudrücken das Reprogrammierungsfaktoren OCT4, SOX2, KLF4, MycL und LIN28A. Die pCXLE_hOCT3 / 4-shp53-F-Plasmid enthält auch eine p53 shRNA für temporäre Unterdrückung der TP53 an zelluläre Reprogrammierung 6 verbessern. Die replikationsdefizienten pCXWB-EBNA1-Vektor fördert Amplifikation Reprogrammierungsfaktoren und erhöhte Effizienz Neuprogrammierung durch eine vorübergehende Erhöhung der Expression EBNA1 7. Die pCXLE_EGFP Plasmid kann zum Nukleofektion Gemisch zum Zwecke der dete hinzugefügt werdenrmining die Transfektionseffizienz oder zur Zellsortierung. Mit Ausnahme von p-CXWB EBNA1, die episomalen Plasmiden in diesem Protokoll verwendet werden, enthalten den Epstein-Barr-Virus Ursprung der viralen Replikation und EBNA1-Gen, das die Replikation und Teilung des Episom während der Teilung der Wirtszelle 8 zu vermitteln. Die Episomen spontan verlor aufeinander iPSC Expansion 7. Subklonierung und Charakterisierung von iPS-Zellen mit episomalen Vektor Verlust, die aus dem Verlust von eGFP Expression geschlossen werden kann, kann vollständig Integration kostenlos IPSCs für zukünftige klinische Anwendungen führen. Inhärente dem Verfahren iPSC Generation Unterdrückung von linienspezifischen Genen und Reaktivierung von Pluripotenz-assoziierten Genen. Regulation der Genexpression erfolgt auf mehreren Ebenen innerhalb des Ringes, einschließlich Modifikationen an DNA und Chromatin Transkriptionsfaktoren erlauben, regulatorische DNA-Elemente, und die RNA-Polymerase den Zugang zu ZielGene. Umbau des epigenetischen Landschaft über weltweite Chromatinmodifikationen ist eine Schlüsselkomponente erneut Ausdruck der Pluripotenz genetische Programm. Eine spezifische Chromatin Modifikation, bei der Regulation der Genexpression wichtig ist, ist die Acetylierung von Histonen in bestimmten Lysinreste, die den Zugriff ermöglicht, um Gene durch die verringerte Spannung der Histon-DNA Spule Ziel. HDAC-Inhibitoren sind kleine Moleküle, die gezeigt haben, um iPSC Umprogrammierung und hESC Selbsterneuerung 9,10 erweitern, die wahrscheinlich auf die Unterstützung der acetylierten Zustand 11. Die nachfolgend beschriebene Protokoll aus einer früheren Veröffentlichung mit Integration Lentiviren 12 angepasst, bietet eine Schritt-für-Schritt-Methode zur optimierten iPSC Generation aus dem peripheren Blut mit Episomen und HDAC-Inhibitoren. Die HDAC-Inhibitor-Konzentrationen werden dabei die Hälfte derjenigen von Ware et al. 9 und routinemäßig in eine 2-fache Zunahme in vollständig umprogrammiert iPSC Kolonien über Standard führtenepisomaler Umprogrammierung Protokolle ohne HDAC-Inhibitoren. Dieses Niveau der Umprogrammierung ist auf Augenhöhe mit der Effizienz beobachten wir mit lentiviralen Methoden. Mit diesem Protokoll haben wir effizient erzeugt iPSC von Einzelpersonen so alt wie 87 Jahre alt.

<table border="0" cellpadding="0" cellspacing="0" width="1100">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Name of Reagent/ Equipment</td>
			<td style="width:151px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:503px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Ficoll-Paque PLUS</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">45-001-750</td>
			<td>Store at 4&#176;C. Warm to room temperature before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">DPBS</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">14190-250</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">RPMI 1640</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11875-093</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">fetal bovine serum (FBS)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">10437028</td>
			<td>Store at -20&#176;C until needed. Thaw, aliquot, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">dimethyl sulfoxide (DMSO)</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">154938</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">QBSF-60 serum-free medium</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">50-983-234</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">penicillin/streptomycin</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">15140122</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Cell Line Nucleofector Kit V</td>
			<td style="width:151px;">Lonza</td>
			<td style="width:119px;">VCA-1003</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">2% gelatin solution</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1393</td>
			<td>Store at 4&#176;C, liquify in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Hank&#39;s Balanced Saline Solution (HBSS)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">14175-103</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:329px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11965-092</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">12440-061</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">L-glutamine, 200 mM</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">25030-081</td>
			<td>Aliquot, freeze at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">non-essential amino acids (NEAA), 100X</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11140-050</td>
			<td>Store at 4&#176;C, away from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">DMEM/Ham&#39;s F12, 1:1</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">SH30023.02</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">KnockOut Serum Replacement</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">10828-028</td>
			<td>Aliquot, freeze at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">sodium pyruvate, 100 mM</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11360-070</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">sodium bicarbonate, 7.5%</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">25080094</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">basic fibroblast growth factor (bFGF)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">PHG0263</td>
			<td>Make 10 mg/mL stock in DPBS, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">sodium butyrate&#160;</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">B5887-1G</td>
			<td>Make 2000X stock (400 mM) in DPBS, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">hES Cell Cloning &#38; Recovery Supplement</td>
			<td style="width:151px;">Stemgent</td>
			<td style="width:119px;">01-0014-500</td>
			<td>Store at -20&#176;C until needed. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">ESC-qualified BD matrigel</td>
			<td style="width:151px;">BD Biosciences</td>
			<td style="width:119px;">35-4277</td>
			<td>Thaw overnight on ice at 4&#176;C, aliquot into pre-chilled tubes using pre-chilled pipette tips. Store at -20&#176;C until needed. Thaw at 4&#176;C, use immediately.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">StemSpan SFEM&#160;</td>
			<td style="width:151px;">STEMCELL Technologies</td>
			<td style="width:119px;">9650</td>
			<td>Aliquot, freeze at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">ascorbic acid, powdered</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4403-100MG</td>
			<td>Make 5 mg/mL stock in DPBS, sterile filter, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">recombinant human stem cell factor (SCF)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">255-SC-010</td>
			<td>Make 100 &#956;g/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">recombinant human interleukin 3 (IL-3)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">203-IL-010</td>
			<td>Make 100 &#956;g/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">erythropoietin (EPO)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">287-TC-500</td>
			<td>Make 1000 U/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">recombinant human insulin-like growth factor 1 (IGF-1)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">291-G1-200</td>
			<td>Make 100 &#956;g/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">&#946;-mercaptoethanol</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7522</td>
			<td>Make 1000X stock (100 mM) in DPBS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">dexamethasone</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">D4902-25MG</td>
			<td>Make 50X stock (50 &#956;M) in DPBS, sterile filter, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">SAHA (vorinostat)</td>
			<td style="width:151px;">Cayman Chemical</td>
			<td style="width:119px;">149647-78-9</td>
			<td>Make 2000X stock (400 mM) in DPBS, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">12 well tissue culture plate</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">08-772-29</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">15 mL conical tube</td>
			<td style="width:151px;">Sarstedt</td>
			<td style="width:119px;">62553002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">1.5 mL Eppendorf tube</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">6 well tissue culture plate</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">08-772-1B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">35 mm tisue culture plates</td>
			<td style="width:151px;">BD Biosciences</td>
			<td style="width:119px;">353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">10 mL disposable serological pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-675-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">5 mL disposable serological pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-675-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">2 mL disposable serological pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-675-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">20 &#956;L pipette tips, barrier tips</td>
			<td style="width:151px;">Genessee</td>
			<td style="width:119px;">24-404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">glass Pasteur pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-678-20D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pipette aid</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-681-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-hOCT3/4-shp53-F</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-hSK</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-hUL</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-EGFP</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27082</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXWB-EBNA1</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">37624</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient ips cell generation from blood using episomes and hdac inhibitors

Diese Handschrift zeigt ein Protokoll für die effiziente Erstellung Integration freien menschlichen induzierten pluripotenten Stammzellen (iPS-Zellen) aus dem peripheren Blut mit episomalen Plasmiden und Histon-Deacetylase (HDAC) Inhibitoren. Die Vorteile dieses Ansatzes sind: (1) die Verwendung einer minimalen Menge von peripherem Blut als Ausgangsmaterial; (2) nonintegrating Umprogrammierung Vektoren; (3) eine kostengünstige Methode zur Erzeugung von Vektor freien iPSCs; (4) einen einzigen Transfektion; und (5) die Verwendung von kleinen Molekülen an epigenetische Neuprogrammierung zu erleichtern. Kurz gesagt, werden periphere mononukleäre Blutzellen (PBMCs) von Routine Phlebotomie Proben isoliert und dann in einem definierten Wachstumsfaktoren kultiviert, um eine hoch proliferativen Erythrozytenvorläuferzellpopulation, die bemerkenswert zugänglich Umprogrammierung ergeben. Nonintegrating, nontransmissible episomalen Plasmiden OCT4, SOX2, KLF4, MycL, LIN28A und eine p53 short hairpin (sh) RNA sind Einfühüber eine einzige Nukleofektion in den abgeleiteten Erythroblasten ced. Kotransfektion Episom das Enhanced Green Fluorescent Protein exprimiert (eGFP) ermöglicht die einfache Identifizierung von transfizierten Zellen. Eine separate replikationsdefizienten Plasmid, Epstein-Barr nuclear antigen 1 (EBNA1) ebenfalls zu dem Reaktionsgemisch für eine erhöhte Expression von Proteinen episomal zugegeben. Die transfizierten Zellen werden dann auf eine Schicht aus bestrahlten embryonalen Maus-Fibroblasten (iMEFs) zum fort Umprogrammierung plattiert. Sobald iPSC artige Kolonien erscheinen etwa zwölf Tage nach Nukleofektion, sind HDAC-Inhibitoren zu dem Medium gegeben, um epigenetische Umbau erleichtert. Wir haben gefunden, dass der Einschluss von HDAC-Inhibitoren routine erhöht die Entwicklung vollständig reprogrammiert iPSC Kolonien das 2-fache. Sobald iPSC Kolonien zeigen typischen menschlichen embryonalen Stammzellen (hES) Morphologie, sie sanft zu einzelnen IMEF beschichteten Gewebekulturplatten für das weitere Wachstum und die Expansion übertragen werden.

Drei Tage nach Nukleofektion und vor dem Ausplattieren der Zellen auf nucleofected iMEFs sollte die Effizienz der erfolgreichen Nukleofektion durch Fluoreszenzmikroskopie für eGFP geschätzt werden. Figur 1 zeigt einen typischen Nukleofektion Experiment mit etwa 5-10% der Gesamtzellpopulation exprimiert eGFP. Reprogrammed iPSC Kolonien beginnen, etwa zwei Wochen nach Nukleofektion erscheinen. Die Kolonien sind in der Regel kreisförmig mit gut definierten Grenzen und könne...

Watch this Scientific Journal Video about Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors at JoVE.com

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

Hier beschreiben wir ein Protokoll zur Erzeugung von menschlichen induzierten pluripotenten Stammzellen aus dem peripheren Blut mit Hilfe eines Episoms basierend Neuprogrammierung Strategie und Histon-Deacetylase-Inhibitoren.

Effiziente iPS Zellgeneration aus Blut Mit Episomen und HDAC-Inhibitoren

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

efficient-ips-cell-generation-from-blood-using-episomes-hdac

Research

JoVE Journal

Biology

12.7K Aufrufe.  Fred Hutchinson Cancer Research Center.  Hier beschreiben wir ein Protokoll zur Erzeugung von menschlichen induzierten pluripotenten Stammzellen aus dem peripheren Blut mit Hilfe eines Episoms basierend Neuprogrammierung Strategie und Histon-Deacetylase-Inhibitoren. 

Serie: Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

Blutentnahme aus der amerikanischen Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Forschung

Biologie

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Generierung von Einzel-Zellsuspensionen aus Maus Nervengewebe

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolierung und Kultivierung von pulmonaler Endothelzellen aus neugeborenen Mäusen

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generation der Mäuse aus induzierten pluripotenten Stammzellen

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

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.

Generation of Human induzierten pluripotenten Stammzellen aus dem peripheren Blut mit dem STEMCCA Lentivirusvektor

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.

Schnelle und effiziente Erzeugung von Neuronen aus menschlichen pluripotenten Stammzellen in einem Multititre Platte Format

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

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

Isometrische und exzentrische Krafterzeugung Beurteilung der Skelettmuskulatur Isoliert von Mausmodellen von Muskeldystrophien

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Schnelle und effiziente Verwendung von Zebrafisch-Genotypisierung PCR mit Hochauflösende Schmelzanalyse

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Isolierung von Blutgefäß abgeleiteten multi Vorläufer von menschlichen Skelettmuskel

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

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.

Effiziente Erzeugung Menschen induzierten pluripotenten Stammzellen aus menschlichen Körperzellen mit 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 ...