Wir bedanken uns bei Yoshiko Miyake, Sayaka Kanaami, Chihana Fujita, Miho Yamaguchi, Natsuko Henmi und Rei Ohno von der Keio University School of Medicine, um technische Unterstützung. Diese Arbeit wurde teilweise durch einen R &amp; D Systems Support-Programm, um die praktische Nutzung von Gesundheitsforschungsergebnissen zu beschleunigen, und die Autobahn-Programm für die Realisierung von regenerativen Medizin gefördert.

1. Bereiten Sie aktivierten humanen T-Zellen <ol><li> Sammeln Sie 10 ml heparinisiertes Vollblut von einem Spender durch Venenpunktion. <ol><li> Erhalten informierte Zustimmung der Spender vor der Venenpunktion. Venenpunktion müssen von qualifiziertem und geschultem Personal durchgeführt werden. </li><li> Griff Blutproben mit steriler Ausrüstung und nach entsprechender Biosicherheit Richtlinien. </li></ol></li><li> Verdünnen von 10 ml heparinisiertes Vollblut mit 10 ml D-PBS (-). </li><li> Setzen 15 ml Ficoll-Lösung (Ficoll-Paque PREMIUM) in ein neues 50 ml konischen Röhrchen. </li><li> Schicht die 20-ml Blutlösung in Schritt 1.2 auf die Ficoll-Lösung in Schritt 1.3 vorbereitet. Mit einem elektrischen Pipette, lassen Sie Blutlösung in Schritt 1.2 laufen langsam entlang der Innenwand der Röhre hergestellt. Achten Sie darauf, das Blut-Lösung und Ficoll Lösung zu mischen. </li><li> Zentrifuge bei 400xg 30 min bei RT. Stellen Sie die Schleuderverzögerungsgeschwindigkeit auf &quot;langsam&quot;. Hinweis: Nach der Zentrifugation eine weiße dünneSchicht entsteht zwischen einer oberen milchig Plasmaschicht und eine untere klare Ficoll-Schicht. Dieser weiße dünne Schicht enthält mononuklearen Zellen. </li><li> Übertragung der mononuklearen Zellschicht in ein neues 50 ml konischen Röhrchen mit einer 1-ml-Pipette. Achten Sie darauf, Ficoll-Lösung enthalten. </li><li> Hinzufügen D-PBS (-) zu den mononuklearen Zellen auf ein Gesamtvolumen von 10 ml und Zentrifugation bei 200 × g für 5 min bei RT. </li><li> Überstand verwerfen. 10 ml D-PBS (-) zu den mononuklearen Zellen und Zentrifugation bei 200 × g für 5 min bei RT. </li><li> Überstand verwerfen, 1 ml KBM502 Medium, um den gesammelten mononuklearen Zellen, und zählen Sie die Zellen mit einer Zählkammer. Mehr als 5 x 10 6 mononuklearen Zellen aus 10 ml heparinisiertem Vollblut mit angemessene Trennung unter Verwendung von Ficoll gewonnen werden. </li><li> Vorbereitung eines Anti-Mensch CD3-Antikörper-Lösung mit einer Konzentration von 10 ug / ml in D-PBS (-). Fügen Sie den Anti-Mensch-CD3-Antikörper-Lösung zu einer 6-Well-Platte, genießen die BrandungAss jede Vertiefung und inkubieren Sie die Schale bei 37 ° C in einem 5% CO 2 Inkubator für mindestens 30 min. </li><li> Entfernen Sie den Anti-Mensch-CD3-Antikörper-Lösung aus jeder Vertiefung und einmal mit D-PBS waschen (-) unmittelbar vor der Aussaat der Zellen. </li><li> Samen mononukleären Zellen, die in Schritt 1,9 bei einer Dichte von 2,5 × 10 5 Zellen / cm 2 an den Anti-Human-CD3-Antikörper-beschichtete Platten mit 6 Vertiefungen in KBM502 Medium hergestellt wurden. Bei 37 ° C in einem 5% CO 2 Inkubator für 3-7 Tage ohne Mediumwechsel, bis die T-Zellen Konfluenz erreichen. T-Zellen in dieser Zeit sind beide suspendiert und adhärenten Zellen. </li></ol> 2. Infect menschlichen T-Zellen mit SeV Vektoren <ol><li> Nach 3-7 Tagen Kultur, sammeln die aktivierten T-Zellen mit den bestehenden Medium durch Pipettieren. Übertragung auf einen 15 ml konischen Röhrchen und Zentrifugieren bei 200 × g für 5 min bei RT. </li><li> Entfernen Sie den Überstand und 1 ml frisches KBM502 Medium. Zählen Sie die Zellen und die Platte 1.5 × 10 6 Zellen (in 2 ml frisches Medium KBM502) in jede Vertiefung eines Anti-CD3-Antikörper-beschichteten 6-Well-Platte. </li><li> Auftauen SEV Lösungen OCT3 / 4-SeV / TSΔF, SOX2-SeV / TSΔF, KLF4-SeV / TSΔF und c-MYC (HNL) -SeV / TS15ΔF auf Eis. </li><li> Fügen Sie die SeV Lösungen, die OCT3 / 4-SeV / TSΔF, SOX2-SeV / TSΔF, Klf4-SeV / TSΔF und c-MYC (HNL) -SeV / TS15ΔF einzeln in die Vertiefungen, die jeweils bei einer Multiplizität der Infektion (MOI) 10. Inkubieren bei 37 ° C in einem 5% -CO 2 -Inkubator für weitere 24 hr. </li></ol> 3. Entfernen Sie SeV Vektoren von menschlichen T-Zellen <ol><li> 24 Stunden nach der Infektion, sammeln Sie die infizierten Zellen mit einer Pipette. Wenn es anhaftenden Zellen, können sie leicht abgenommen Pipettieren von ihnen mehrmals nach oben und unten ist. Übertragung auf einen 15 ml konischen Röhrchen und Zentrifugieren bei 200 × g für 5 min bei RT. </li><li> Entfernen Sie die Überstand, der die SeV-Vektoren und 2 ml frischem KBM502 Medium. Ausstrich Zellen in each der Vertiefungen. Inkubieren bei 37 ° C in einem 5% -CO 2 -Inkubator für zusätzliche 24 Std. </li></ol> 4. Reseed Cells auf einem Matrigel Schicht <ol><li> Bereiten Basalmembranmatrix Lösung durch Auftauen Matrix-Gel (finden Sie in der Materialliste für die jeweilige Marke in dieser Studie verwendet) bei 4 ° C und Auflösen von 0,2 ml in 10 ml DMEM / F12-Medium. Hinweis: Matrix-Gel (finden Sie in der Materialliste für die jeweilige Marke in dieser Studie verwendet) muss O / N bei 4 ° C aufgetaut, um vollständig aufgetaut werden. Halten Sie es auf Eis, bis kurz vor der Verwendung. </li><li> Platte 5 ml Basalmembranmatrix Lösung pro Schale in 100 mm-Schalen und lassen bei RT für mindestens 30 min vor der Aussaat Zellen. Mindestens zwei 100-mm-Schalen werden für ein Experiment benötigt wird. </li><li> Sammeln SeV-infizierten Zellen durch Pipettieren, Übertragung auf einen 15-ml konischen Röhrchen und Zentrifuge bei 200 xg für 5 Minuten bei RT. </li><li> Entfernen Sie den Überstand und 1 ml frisches KBM502 Medium. Zählen Sie die Anzahl der cells mit einer Zählkammer, entfernen Sie die Basalmembran-Matrix-Lösung von 100-mm-Schalen in Schritt 4.2 und platte 1 × 10 5 und 1 × 10 6 Zellen (in 10 ml frischem mTeSR mittel) auf einem 100-mm Basalmembranmatrix beschichtet Geschirr. Schließlich verwenden Sie die Platte, in der Kolonien werden leicht aufgenommen. </li><li> Inkubieren der Schale bei 37 ° C in einem 5% -CO 2 -Inkubator O / N. </li><li> Ändern Sie das Medium auf 10 ml Medium mTeSR Fleisch jeden zweiten Tag. Hinweis: ca. 20-30 Tage nach der Infektion, Kolonien, die sehr ähnlich sind embryonale Stammzellen (ESC) wird angezeigt. </li></ol> 5. Erweitern Sie T-Zellen abgeleiteten iPSCs <ol><li> Bereiten Basalmembranmatrix Lösung durch Auftauen Matrix-Gel (finden Sie in der Materialliste für die jeweilige Marke in dieser Studie verwendet) bei 4 ° C und Auflösen von 0,2 ml in 10 ml DMEM / F12-Medium. </li><li> Platte 1 ml der Basalmembran Matrixlösung pro Well in einer 6-Well-Platte und lassen bei RT für zuminmindestens 30 min vor Kommissionierung Kolonien. </li><li> Füllen Sie einen 96-Loch-Platte mit 20 ul mTeSR Medium pro Vertiefung. </li><li> Wählen Kolonien, die Wirtschafts- und Sozialräte unter dem Stereomikroskop mit einer 20 &amp; mgr; l-Pipette ähneln. Hinweis: Die Kolonien der WSR und iPSCs sind rund und flach, wie in 1B gezeigt. </li><li> Transfer ausgewählten Kolonien in die 96-Lochplatte mit mTeSR Medium in Schritt 5.3 gefüllt. </li><li> Fügen Sie 200 ul mTeSR Medium in jede Vertiefung pipettieren und vorsichtig nach oben und unten, um die Kolonie zu kleinen Klumpen unter dem Stereomikroskop mit einer 200 ul Pipetten brechen. Entfernen Sie die Basalmembran-Matrix-Lösung aus dem gut vorbereitet in 5.2 und legte jede Kolonie in eine Vertiefung der Basalmembran-Matrix beschichteten 6-Well-Platte mit 2 ml mTeSR Medium pro Vertiefung. </li><li> Ändern Sie den mittleren bis 2 ml mTeSR Medium Fleisch jeden zweiten Tag. </li></ol> 6. Pflegen Sie T-Zellen abgeleiteten iPSCs Die T-Zellen abgeleiteten iPSCs aufrechterhalten werden kannund gespeichert mit den gleichen Techniken wie für den menschlichen iPS-Zellen und menschlichen WSR. Sobald iPSC Kolonien heranwachsen, erweitern sie in größere Gerichte Wiederholung des gleichen Verfahrens. <ol><li> Ändern Sie den mTeSR Medium alle 1-2 Tage. </li><li> Wenn die Kolonien konfluent werden alle 5-7 Tage, Durchgang die Zellen unter Verwendung Dissoziationslösung für den menschlichen iPS-Zellen und menschlichen Wirtschafts- und Sozialräte gemäß den Anweisungen des Herstellers. </li></ol>

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We describe a protocol for generating iPSCs from human peripheral T cells in serum-free and feeder-free conditions using a combination of matrigel, mTeSR medium, and SeV vectors. For clinical applications of iPSCs, it is important to have a protocol for stably generating iPSCs and a less-invasive method for cell sampling. Although generating iPSCs with the combination of matrigel and mTeSR medium showed lower reprogramming efficiency than that with knockout serum replacement (KSR) medium and feeder-cells, this combination achieves stable generation of iPSCs from donors16. Stable iPSC generation and less-invasive cell sampling has the advantage of being able to increase the number of donors for iPSC generation. 
SeV vectors, a minus-strand RNA virus, is not integrated into the host genome. Therefore the risks of tumorigenesis can be avoided at the step of reprogramming factor induction20-24. Additionally, the feeder-free conditions, which use a combination of defined culture medium and matrigel instead of a feeder layer, make it possible to minimize the potential risks of exposure to unknown exogenous factors. The fusion of these techniques provides a less invasive and safer iPSC technology for regenerative medicine16.
Important steps in this protocol are the step of activating human T cells (Step 1) and infecting them with SeV vectors (Step 2). When no ESC-like colony is obtained in the culture dish at an optimal time after SeV infection, the following should be considered. First, the confluency of the mononuclear cells before T cell activation may not be appropriate because optimal activation of T cells is critical for SeV infection25,26. Too high a density of mononuclear cells leads to cell death, interfering with the proper activation of T cells. Too low a density of mononuclear cells also disturbs the proper activation and proliferation of T cells. Therefore, the confluency of mononuclear cells should be checked and adjusted accordingly. Second, the dosage of SeV vectors may not be sufficient. The induction efficiency of iPSC colonies depends on the dosage of the virus6. If no ESC-like colony is observed after SeV infection, the option of increasing the virus dosage up to an MOI of 15-20 should be considered. If signs of iPSC colony differentiation are observed, the frequency at which medium is changed may be increased up to every other day, or to every day when colonies are larger. 
The limitation in this protocol consists of this method not being virus free. Although SeV for cell reprogramming is commercially available with ease, users need to prepare equipment according to the appropriate biosafety level. As another method for generating integration-free iPSCs, episomal vectors have been used until now27-29. Although episomal vectors can be used with equipment with a lower level of biosafety, episomal vectors might insert into the host genome at extremely low rates. Therefore, additional checks are required to confirm the disappearance of transgenes, as when using SeV.
There is another limitation in that this technique is not absolutely free from animal-derived products. Some substrates, such as matrigel, anti-CD3 mAb, dissociation solution and SeV solution are derived from animal products and are therefore associated with a risk of transferring xenogeneic pathogens. However, the reduction of animal-derived substrates in the culture system is meaningful for the clinical application of iPSC technology due to the lower risk.

iPSCs haben beträchtliche Aufmerksamkeit als eine bahnbrechende Quelle von Zellen für die regenerative Medizin 1-3 angezogen. Bisher wurden verschiedene Methoden zur Erzeugung iPSCs wurde berichtet 4,5. Unter diesen haben iPSCs von menschlichen T-Zellen erzeugt von besonderem Interesse wegen der weniger invasiven Verfahren des Zellprobenahme 6-8 gewesen. Zusätzlich iPSCs von T-Zellen abgeleitet sind Neuanordnungen der T-Zell-Rezeptor (TCR) Gen und sind daher eine Quelle von Antigen-spezifischen T-Zellen 9,10. Daher Erzeugung T-Zell-abgeleiteten iPSCs sicher ist nützlich zur fortschreitenden regenerativen Medizin. Dieses Verfahren basiert auf dem Konzept der Verringerung der Gefahr von unvorhersehbaren Pathogenität basiert. Zum sicheren klinischen Anwendung iPSCs ist es wichtig, um das Risiko der Exposition gegenüber Pathogenen 11 zu reduzieren. Zuvor in vielen Kultursystemen pluripotenter Stammzellen haben fötalem Rinderserum und Feeder-Zellen als wesentliche Reagenzien verwendetS12. Jedoch ist die Entfernung dieser beiden Reagenzien aus dem Kultursystem bevorzugt iPSC Generation, das Risiko von unvorhersehbaren Pathogenität zu reduzieren. Zusätzlich hat dieses Verfahren den Vorteil, daß eine invasive Zellprobenahme von Patienten und mühsame Herstellung von feeder-Zellen. Da T-Zell-abgeleiteten iPSCs wurden bereits erfolgreich in Forschungs 13,14 verwendet wurde, ist dieses Verfahren ebenfalls anwendbar und nützlich für die Erzeugung krankheitsspezifische iPSCs von Patienten. Unter T Reprogrammierung Verfahren unter Verwendung des Sendai-Virus (SeV) Vektor als ein Gen Fahrzeug ist ein Verfahren, das mit hoher Effizienz iPSCs 7,16 generieren kann. Darüber hinaus, da SeV ist ein Einzelstrang-RNA-Virus und keine DNA-Phase für die Replikation benötigen, die Nutzung im iPSC Generation vermeidet das Brechen des Wirtsgenom 17-19. Daher haben wir Protokolle zur Erzeugung iPSCs aus humanen peripheren T-Zellen in serum fr festee und Feeder-freien Bedingungen mit einer Kombination von Matrigel mTeSR Medium und SeV Vektoren.

<table border="0">
	<tbody>
		<tr>
			<td>Ficoll-Paque PREMIUM</td>
			<td>GE Healthcare</td>
			<td>17-5442-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified NA/LE mouse anti-human CD3</td>
			<td>BD Pharmingen</td>
			<td>555336</td>
			<td></td>
		</tr>
		<tr>
			<td>KBM502 medium</td>
			<td>KOHJIN BIO</td>
			<td>16025020</td>
			<td>Warm in 37 &#8451; water bath before use</td>
		</tr>
		<tr>
			<td>Bovine albumin fraction V solution</td>
			<td>Gibco</td>
			<td>15260-037</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Matrigel Matrix Growth Factor Reduced</td>
			<td>BD Biosciences</td>
			<td>354230</td>
			<td>Thaw completely at 4&#8451; overnight and dilute it 50 times with Dulbecco&#39;s Modified Eagle&#39;s Medium before coating culture dishes</td>
		</tr>
		<tr>
			<td>mTeSR1 medium kit</td>
			<td>STEM CELL</td>
			<td>5850</td>
			<td>Warm at room temperature before use</td>
		</tr>
		<tr>
			<td>Dissociation Solution</td>
			<td>ReproCELL</td>
			<td>RCHETP002</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS(&#8211;)</td>
			<td>Wako</td>
			<td>045-29795</td>
			<td></td>
		</tr>
		<tr>
			<td>SeV Vector kit CytoTune-iPS ver.1.0</td>
			<td>DNAVEC</td>
			<td>DV-0303c</td>
			<td>Thaw on ice before use</td>
		</tr>
		<tr>
			<td>100-mm tissue culture dish</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture plate</td>
			<td>Falcon</td>
			<td>353078</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml Centrifuge Tube</td>
			<td>Greiner Bio-One&#160;</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml Centrifuge Tube</td>
			<td>Greiner Bio-One&#160;</td>
			<td>227261</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of induced pluripotent stem cells from human peripheral t cells using sendai virus in feeder-free conditions

Vor kurzem haben iPSCs Aufmerksamkeit als neue Quelle von Zellen für regenerative Therapien angezogen. Obwohl die ursprüngliche Verfahren zum Erzeugen iPSCs angeführten dermalen Fibroblasten durch invasive Biopsie und retrovirale genomische Insertion von Transgenen erhalten wird, hat es viele Bemühungen, diese Nachteile zu vermeiden. Menschliche periphere T-Zellen sind eine einzigartige Zellquelle zur Erzeugung von iPS-Zellen. iPSCs aus T-Zellen abgeleitet sind Neuanordnungen der T-Zell-Rezeptor (TCR) Gene und sind eine Quelle von Antigen-spezifischen T-Zellen. Zusätzlich haben die T-Zell-Rezeptor-Umlagerung in das Genom hat das Potential, einzelnen Zelllinien zu markieren und eine Unterscheidung zwischen transplantierten und Spenderzellen. Zum sicheren klinischen Anwendung iPSCs, ist es wichtig, um die Gefahr des Freilegens neu erzeugten iPSCs schädliche Mittel zu minimieren. Obwohl fötalem Rinderserum und Feeder-Zellen wurden wesentlich für pluripotente Stammzellen in Kultur ist es bevorzugt, sie aus dem Kultursystem zu entfernen, um das Risiko zu verringernunvorhersehbarer Pathogenität. Zur Lösung dieses Problems haben wir ein Protokoll zur Erzeugung iPSCs aus humanen peripheren T-Zellen unter Verwendung von Sendai-Virus, um das Risiko der Freigabe iPSCs undefinierte Pathogene reduzieren etabliert. Obwohl Umgang mit Sendai-Virus erfordert Geräte mit der entsprechenden Sicherheitsstufe, aktiviert Sendai Virus infiziert T-Zellen ohne Genom Insertion, noch mit hohem Wirkungsgrad. In diesem Protokoll zeigen wir die Erzeugung von iPSCs aus humanen peripheren T-Zellen in Feeder freien Bedingungen mit einer Kombination von aktivierten T-Zellkultur und Sendai-Virus.

<html> Unter Verwendung dieses Protokolls können Benutzer iPSCs aus humanen peripheren T-Zellen in stabiler Weise zu erzeugen. iPSC Erzeugung von T-Zellen mit SeV und Matrigel zeigte etwa 0,002%. - 0,005% der Effizienz der Reprogrammierung zwischen mehreren Geber Fälle und SeV wurden nach mehreren Passagen nicht erkannt 16 1A zeigt eine schematische Darstellung des Protokoll zur Erzeugung von T-Zellen abgeleiteten iPSCs in Feeder freien Bedingungen unter Verwendung von Matrigel und mTeSR Medium. Ca. 20-30 Tage nach Infektion mit SeV werden iPSC Kolonien von ihren ESC-Kolonie-Morphologie (Abbildung 1B) anerkannt. Immunfluoreszenzfärbung zeigte die Expression von typischen pluripotenten Zellmarker (NANOG, OCT3 / 4, SSEA4, TRA-1-60 und TRA-1-81) in T-Zell-abgeleitete iPSCs erzeugt unter Zuführung freien Bedingungen (Abbildung 1c). &quot;Breite =&quot; 700 &quot;/&gt; Figur 1 (A): Ein Schema des Protokolls für eine Neuprogrammierung T-Zellen unter Zuführung freien Bedingungen in dieser Studie. (B): Eine typische ESC-ähnliche iPSC Kolonie an Tag 27 nach der Blutentnahme unter Zuführung freien Bedingungen. (C): ALP-Färbung und Immunfluoreszenzfärbung für Pluripotenz und Oberflächenmarker (NANOG, OCT3 / 4, SSEA4, TRA-1-60 und TRA-1-81) in T-Zell-abgeleitete iPSCs erzeugt unter Zuführung freien Bedingungen. <a href="https://www.jove.com/files/ftp_upload/53225/53225fig1highres.jpg" target="_blank">Bitte Klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a>

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions at JoVE.com

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Erzeugung von induzierten pluripotenten Stammzellen aus menschlichen peripheren T-Zellen Mit Sendai Virus in Feeder-freien Bedingungen

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

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Research

JoVE Journal

Developmental Biology

11.9K Aufrufe.  Keio University School of Medicine. This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Serie: Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Erzeugung von induzierten pluripotenten Stammzellen aus Gefrorene Buffy Coats mit Non-Integration episomalen Plasmiden

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Forschung

Entwicklungsbiologie

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Generation der Integration freie Menschen induzierten pluripotenten Stammzellen Mit Haarstamm Keratinozyten

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Feeder freie Ableitung von Melanozyten aus humanen pluripotenten Stammzellen

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

Die Erzeugung von induzierten pluripotenten Stammzellen aus menschlichen Melanom-Tumor-infiltrierenden Lymphozyten

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

Induzierte pluripotente Stammzellen Erzeugung von Blutzellen mit Sendai-Virus und Zentrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Die Erzeugung von Integration freien induzierte pluripotente Stammzellen aus menschlichen peripheren einkernigen Blutzellen Mit Episomale Vektoren

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Definierte und Scalable Generierung von Leber-ähnlichen Zellen aus humanen pluripotenten Stammzellen

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

Unterscheidung von Chondrozyten aus periphere Blut-abgeleiteten menschlichen induzierten pluripotenten Stammzellen

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

Generation von standardisierten und reproduzierbaren Vorderhirn-Typ zerebralen Organellen aus menschlichen induzierten pluripotenten Stammzellen

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

Generation von 3D Haut organoide aus Cord Blood-abgeleitete induzierte pluripotente Stammzellen

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...