Diese Arbeit wurde von der Yale School of Medicine and Child Health Research Award von Charles Hood Foundation finanziert.

1. Reprogrammierung von Retrovirus Reprogrammierungsfaktoren <ol><li> Humane Fibroblasten in Fibroblasten-Medium (10% FBS in DMEM mit Pen / Strep) kultiviert. </li><li> Einen Tag vor der Infektion, Platte 1x10 5 humane Fibroblasten in eine Vertiefung einer 6-Well-Platte. </li><li> Saugen Sie Medium, um tote Zellen zu entfernen und 2 ml frisches Medium Fibroblasten. In Protaminsulfat in einer Endkonzentration von 5 pg / ml. </li><li> Vorsichtig die geeignete Menge jedes GFP-exprimierenden Virus entsprechend einer Multiplizität der Infektion (MOI) 5 5. </li><li> Einen Tag nach der Infektion, entfernen Sie den Virus-Überstand, waschen, dreimal mit 2 ml PBS, dann 2 ml Fibroblasten-Medium. </li><li> Drei Tage nach der Infektion, überprüfen Sie die GFP-Fluoreszenz und füllt die gut mit 2 ml Fibroblasten-Medium. </li><li> Vier Tage nach der Infektion Platte 1x10 4 / cm 2 bestrahlt embryonalen Maus-Fibroblasten (MEFs) Fibroblasten-Feeder-Zellen in Medium auf eine10-cm-Petrischale mit 0,1% Gelatine beschichtet. Bei 37 ° C über Nacht. </li><li> Fünf Tage nach der Infektion lösen infizierten menschlichen Fibroblasten mit 1 ml 0,05% Trypsin / EDTA für 5 Minuten bei 37 ° C, und für 5 Minuten bei 200 g. Saugen Sie das Medium, und Zellen mit 10 ml Fibroblasten-Medium. Übertragen der Zellen in eine Pre-beschichteten 10-cm-Platte. </li><li> Nach 24 Stunden, ersetzen Sie das Medium mit hES-Kulturmedium (20% Knock-Out Serumersatz, DMEM/F12, 0,1 ml nicht-essentielle Aminosäuren, 4 ng / ml bFGF, Pen / Strep / Glutamat, beta-merceptoethanol). Ändern Sie das Medium täglich. ESC-ähnliche Kolonien beginnt, am Tag 20-27 nach der Infektion auftreten. </li></ol> 2. Isolierung und Expansion von IPSCs <ol><li> Unter einem Fluoreszenzmikroskop, das Fehlen der GFP-Fluoreszenz in einer Kolonie, die eine ähnliche Morphologie zeigt, HES zu überprüfen. </li><li> Mit einem 10 ul Pipette, Pick einzelnen iPSC Kolonien und legen Sie sie in eine Vertiefung einer Gelatine-und MEF-CoATed 12-Well-Platte und ergänzt mit hES-Medium. Ändern Medium täglich. </li><li> Für die Passage, waschen Sie die Platte mit 1 ml DMEM/F12, dann fügen Sie 0,5 ml Kollagenase, und inkubieren Sie für 10 Minuten bei 37 ° C </li><li> Waschen Sie die Zellen zweimal mit DMEM/F12. </li><li> 2 ml frisches Medium hESC. Die Verwendung eines Handys Heber, brechen die Kolonien in kleine Stücke und lösen verbleibenden Zellen von der Platte. </li><li> Übertragen Sie die resuspendierten Stücke von Kolonien in eine Vertiefung einer Gelatine-und MEF-beschichteten 6-Well-Platte. </li></ol> 3. Immunfluoreszenz-Analyse von pluripotenten Marker <ol><li> Die Zellen werden dreimal mit PBS und mit 4% Paraformaldehyd für 20 min bei Raumtemperatur. </li><li> Danach weitere Reinigung die Zellen dreimal mit PBS gewaschen und mit 0,2% Triton X-100 in PBS für 30 min. Permeabilisieren </li><li> Sperren nicht-spezifische Bindung durch Inkubation von Zellen mit 3% BSA in PBS für zwei Stunden. </li><li> Inkubieren der Zellen mit dem primären Antikörper über Nacht bei 4 ° C </li> &lt;li&gt; Waschen Sie die Zellen dreimal mit PBS und Inkubation der Zellen mit spezifischen sekundären Antikörper für eine Stunde bei Raumtemperatur, Fernhalten von Licht. </li><li> Die Zellen werden dreimal mit PBS und merken DAPI beim letzten Spülgang durch Inkubation bei Raumtemperatur für 5 Minuten. </li><li> Ermitteln Sie die Färbung mit einem Fluoreszenz-Mikroskop. </li></ol> 4. Quantitative Real-time PCR Assay für den pluripotenten Marker <ol><li> Isolieren der Gesamt-RNA aus den menschlichen IPSCs aus humanen Fibroblasten mit Qiagen RNeasy-Kit abgeleitet. </li><li> Synthetisieren der Erststrang-cDNA unter Verwendung von SuperScript II Reverse Transkriptase. </li><li> Führen qPCR zu Pluripotenz-Gene unter Verwendung der Primer bereits berichtet erkennen. 6 </li></ol> 5. Repräsentative Ergebnisse <ol><li> Morphologische Veränderung während der Umprogrammierung Wir infizierten menschlichen Fibroblasten BJ1 und Detroit 551 mit einem Cocktail von Retroviren tragen Oct4, Sox2, Klf4 und MYC,und konnten morphologische Veränderungen während des Programmierens (1) zu erfassen. Einundzwanzig Tage nach der Infektion, erkennen wir kleine iPSC Kolonien durch ihre hES-ähnliche Morphologie. Darüber hinaus erkennen wir IPSCs durch die GFP-Fluoreszenz. Pluripotenten Stammzellen, wie WSR und IPSCs, drücken die molekulare Maschinerie zur Unterdrückung der proviralen Genexpression 09.07. Unsere einzigartige retroviralen Vektors exprimiert GFP zusammen mit Umprogrammierung Gene durch retrovirale LTR. Somit werden Zellen, die GFP exprimieren kontinuierlich als Transgene ohne provirale Gen-Silencing auszudrücken. Getreu umprogrammiert iPSC Kolonien, die die Pluripotenz molekularen Netzwerk zu erwerben zeigen die Abwesenheit von GFP-Expression (Abbildung 2) 10. </li><li> Charakterisierung von Pluripotenz menschlicher IPSCs Wir analysierten Kolonien von Detroit-551 Fibroblasten via Immunhistochemie abgeleitet mit TRA-1-81, Tra-1-60, SSEA-4, SSEA-3, Oct4 und NANOG Antikörper ( <strong&gt; Tabelle 2). Erfolgreich umprogrammiert IPSCs exprimieren alle dieser Marker (3A). Wir haben auch analysiert Genexpression über quantitative RT-PCR-Analyse. Wir haben beobachtet, dass die Expression von Oct4, Sox2, Klf4, MYC und NANOG signifikant war im Vergleich zu den parentalen Fibroblasten-Zellen erhöht, aber angemessen mit der H9 hESCs (3B). </li></ol><img alt="1" src="/files/ftp_upload/3804/3804fig1.jpg" /> Abbildung 1. Morphologischen Veränderungen der Retrovirus-infizierten menschlichen Fibroblasten. (AD) Progressive morphologische Veränderung in Kolonien von Detroit-551 Fibroblasten mit Umprogrammierung Faktoren infiziert. Tag 5 (A), Tag 10 (B), Tag 14 (C), Tag 21 (D). Die Zellen zeigen den hES-ähnliche Morphologie nach 21 Tagen. <img alt="2" src="/files/ftp_upload/3804/3804fig2.jpg" /> Abbildung 2. Vertreter fluoreszierende GFP-Expression in Zellen, die eine Neuprogrammierung. </strong&gt; BJ und Detroit 551 Fibroblasten wurden mit Retrovirus vier Umprogrammierung Faktoren 4 infiziert und inkubiert in hES-Medium für vier Wochen. BJ-Fibroblasten (A, B) und Detroit 551 (C, D) zeigen ähnliche morphologische. Vom ersten Tag 21, beginnen GFP-negative Kolonien zu bilden, aus denen die bona fide IPSCs 10. (E, F) zeigen, verwandelt Detroit 551 Zellen, die nicht ordnungsgemäße Umprogrammierung unterzogen wurden. (A, C, E) Kolonien unter Phasenkontrast-Ansicht. (B, D) richtig Zellen, die das GFP-Silencing zeigen umprogrammiert. (F) helle GFP-Expression von transformierten Kolonie. <img alt="Abbildung 3" src="/files/ftp_upload/3804/3804fig3.jpg" /> Abbildung 3. Die Charakterisierung der menschlichen induzierten pluripotenten Stammzellen. (A) Menschliche iPS-551-K1 Zellkolonien auszudrücken Marker gemeinsam pluripotenten Zellen. DAPI-Färbung zeigt die gesamte Zellgehalt pro Feld. (B) Quantitative Echtzeit-PCR (RT-qPCR) für die Expression von Oct4, Sox2, Klf4, MYC im elterlichen fibroblast, 551-iPS-K1 IPSCs, H9 und humanen embryonalen Stammzellen (hES). Die Daten wurden gegen β-Actin Housekeeping-Gen normalisiert und aufgetragen relativ zu der Expression in den parentalen Zellen 4 Fibroblasten.

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<p class="jove_content"&gt; Wir haben nichts zu offenbaren.</p

Die Expression von vier Transkriptionsfaktoren umprogrammiert menschlichen Fibroblasten zu IPSCs. Viele Versuche wurden unternommen, um Menschen mit IPSCs nicht integrierenden oder nicht-genetische Ansätze zu einer klinisch sicheren IPSCs generieren generieren. Bisher zeigen diese Methoden extrem niedrigen Wirkungsgrad und erfordern eine weitere Optimierung, um die Reproduzierbarkeit zu verbessern 14.11. Retro-oder lentiviralen Methoden werden verwendet, um ohne weiteres abzuleiten und anzuwenden IPSCs für ...

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Concentration</td>
			<td>Company</td>
			<td>Catalogue Number</td>
		</tr>
		<tr>
			<td colspan="4">hESC medium</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>80%</td>
			<td>Invitrogen</td>
			<td>11330057</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacer</td>
			<td>20%</td>
			<td>Invitrogen</td>
			<td>10828-028</td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>2 mM</td>
			<td>Invitrogen</td>
			<td>25030081</td>
		</tr>
		<tr>
			<td>Nonessential Amino Acids (10 mM)</td>
			<td>0.1 mM</td>
			<td>Invitrogen</td>
			<td>11140050</td>
		</tr>
		<tr>
			<td>&beta;-Mercaptoethanol (14.3 M) or MTG</td>
			<td>0.1 mM</td>
			<td>Invitrogen</td>
			<td>M-6250</td>
		</tr>
		<tr>
			<td>bFGF-2 10 &mu;g/ml</td>
			<td>4 ng/ml</td>
			<td>GIBCO/BRL</td>
			<td>GF003AF</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>1%</td>
			<td>Millipore</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td colspan="4">Fiboblasts Medium</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>90%</td>
			<td>Invitrogen</td>
			<td>11965118</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>10%</td>
			<td>Invitrogen</td>
			<td>10407028</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>1%</td>
			<td>Millipore</td>
			<td>15140-122</td>
		</tr>
	</tbody>
</table>
Table 1. Culture Medium
<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Concentration</td>
			<td>Company</td>
			<td>Catalogue Number</td>
		</tr>
		<tr>
			<td colspan="4">Antibodies</td>
		</tr>
		<tr>
			<td>OCT4</td>
			<td>1:500</td>
			<td>Abcam</td>
			<td>Ab19857</td>
		</tr>
		<tr>
			<td>SSEA3</td>
			<td>1:100</td>
			<td>Milipore</td>
			<td>MAB4303</td>
		</tr>
		<tr>
			<td>SSEA4</td>
			<td>1:100</td>
			<td>BD Biosciences</td>
			<td>BD560218</td>
		</tr>
		<tr>
			<td>Tra-1-81</td>
			<td>1:100</td>
			<td>BD Biosciences</td>
			<td>BD560173</td>
		</tr>
		<tr>
			<td>Tra-1-60</td>
			<td>1:100</td>
			<td>BD Biosciences</td>
			<td>BD560174</td>
		</tr>
		<tr>
			<td>NANOG</td>
			<td>1:500</td>
			<td>Abcam</td>
			<td>Ab21624</td>
		</tr>
		<tr>
			<td>Alexa-Flur 488</td>
			<td>1:1000</td>
			<td>Invitrogen</td>
			<td>A11008</td>
		</tr>
		<tr>
			<td>Alexa-Flur 555</td>
			<td>1:1000</td>
			<td>Invitrogen</td>
			<td>A21422</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>1:5000</td>
			<td>Invitrogen</td>
			<td>D1306</td>
		</tr>
		<tr>
			<td colspan="4">Plasmids</td>
		</tr>
		<tr>
			<td>pMIG-OCT4</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>17225</td>
		</tr>
		<tr>
			<td>pMIG-SOX2</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>17226</td>
		</tr>
		<tr>
			<td>pMIG-KLF4</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>17227</td>
		</tr>
		<tr>
			<td>pMIG-MYC</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>18119</td>
		</tr>
		<tr>
			<td colspan="4">Other Materials</td>
		</tr>
		<tr>
			<td>Collagenase type IV</td>
			<td>1mg/ml</td>
			<td>Invitrogen</td>
			<td>17104019</td>
		</tr>
		<tr>
			<td>Gelatin, Porcine</td>
			<td>0.1%</td>
			<td>Sigma</td>
			<td>G 1890</td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>0.2%</td>
			<td>Sigma</td>
			<td>X100-500ML</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>4%</td>
			<td>Sigma</td>
			<td>47608</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>3%</td>
			<td>American Bioanalytical</td>
			<td>AB01800</td>
		</tr>
		<tr>
			<td>MEF feeder cells</td>
			<td>&nbsp;</td>
			<td>Millipore</td>
			<td>PMEF-N</td>
		</tr>
		<tr>
			<td>Cell Lifter</td>
			<td>&nbsp;</td>
			<td>Corning</td>
			<td>3008</td>
		</tr>
		<tr>
			<td colspan="4">Equipment</td>
		</tr>
		<tr>
			<td colspan="4">Fluorescent microscopy: inverted microscope with GFP filter</td>
		</tr>
	</tbody>
</table>
Table 2. Reagents and equipment.

reprogramming human somatic cells into induced pluripotent stem cells (ipscs) using retroviral vector with gfp

Menschliche embryonale Stammzellen (HES) sind pluripotent und einen unschätzbaren zellulären Quellen für In-vitro-Krankheit Modellierung und der regenerativen Medizin ein. Es wurde zuvor gezeigt, dass die somatischen Zellen Pluripotenz kann im Vergleich zur Expression von vier Transkriptionsfaktoren (Oct4, Sox2, Klf4 und Myc) umprogrammiert werden und werden induzierte pluripotente Stammzellen (IPSCs) 2-4. Wie hESCs, sind menschliche pluripotente IPSCs und eine potentielle Quelle für autologe Zellen. Hier beschreiben wir das Protokoll für die menschliche Fibroblasten-Zellen mit den vier Umprogrammierung Faktoren in GFP-haltigen retroviralen Backbone 4 geklont umzuprogrammieren. Mit dem folgenden Protokoll, erzeugen wir Menschen IPSCs in 3-4 Wochen unter die menschliche Kultur ESC Zustand. Menschliche iPSC Kolonien ähneln HES in der Morphologie und zeigt den Verlust der GFP-Fluoreszenz als Ergebnis von retroviralen Transgen-. iPSC Kolonien mechanisch unter einem Fluoreszenz-Mikroskopie isoliertEW verhalten sich ähnlich wie HES. In diesen Zellen erkennen wir die Expression mehrerer Gene und Pluripotenz Oberflächenmarker.

Watch this Scientific Journal Video about Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP at JoVE.com

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

Ein Verfahren für die menschliche induzierte pluripotente Stammzellen (IPSCs) über Retrovirus-vermittelte ektopische Expression von Oct4 generieren, wird Sox2, Klf4 und MYC beschrieben. Ein praktischer Weg, um menschliche iPSC Kolonien auf GFP-Expression zu identifizieren Basis wird ebenfalls diskutiert.

Reprogrammierung menschlichen Körperzellen in induzierte pluripotente Stammzellen (IPSCs) mit retroviralen Vektoren mit GFP

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

reprogramming-human-somatic-cells-into-induced-pluripotent-stem-cells

Research

JoVE Journal

Biology

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

20.3K Aufrufe.  Yale School of Medicine. Ein Verfahren für die menschliche induzierte pluripotente Stammzellen (IPSCs) über Retrovirus-vermittelte ektopische Expression von Oct4 generieren, wird Sox2, Klf4 und MYC beschrieben. Ein praktischer Weg, um menschliche iPSC Kolonien auf GFP-Expression zu identifizieren Basis wird ebenfalls diskutiert.

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

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Erzeugung von induzierten pluripotenten Stammzellen durch Reprogrammierung embryonalen Maus-Fibroblasten mit einem Vier Transcription Factor, induzierbare Doxycycline Lentivirale Transduktionssystems

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Forschung

Biologie

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

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

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

Erzeugung von induzierten pluripotenten Stammzellen durch Reprogrammierung menschlichen Fibroblasten mit dem Stemgent Menschliche TF Lentivirus Set

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

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

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

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

Transfektion und Nucleofecting menschlichen induzierten pluripotenten Stammzellen

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

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Vorbereitung von embryonalen Maus-Fibroblasten-Zellen zum Kultivieren von humanen embryonalen und induzierten pluripotenten Stammzellen

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

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, ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

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 ...

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (hiPSCs)

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (HiPSCs)  

To begin, take a flask of cultured fibroblast cells, coat a 10-centimeter cell culture plate suitable for hiPSC culture with four milliliters of cold coating solution per plate, and incubate the plate for one hour at 37 degrees Celsius. Afterward, remove the media from the fibroblasts and wash them with four milliliters of pre-warmed PBS per flask. Aspirate the PBS and associate the fibroblasts with four milliliters of Trypsin-EDTA per flask by incubating for five to seven minutes at 37 degrees Celsius.Monitor cell detachment using a phase contrast microscope, and if needed, tap against the side of the flask to detach the cells from the plastic surface. Transfer the detached cells to a 50-milliliter conical tube and wash the empty flasks with six milliliters of pre-warmed fibroblast medium. Collect and pool the remaining cells in the conical tube.Then centrifuge the cells at 200 g for five minutes at 20 degrees Celsius. Aspirate the supernatant and re-suspend the cell pellet in three milliliters of fresh, pre-warmed PBS. Count the cells, transfer 1.5 times 10 to the sixth fibroblasts into a new conical tube and fill up with PBS to the five millimeter mark.Centrifuge the tube at 200 g for five minutes at 20 degrees Celsius. Discard the supernatant, re-suspend the cell pellet in five milliliters of electroporation medium, and centrifuge again. In the meantime, aspirate the coating solution from the coated 10-centimeter cell culture plate and add seven milliliters of pre-warmed fibroblast medium.At the end of the centrifugation, discard the supernatant and re-suspend the cell pellet in electroporation medium with a concentration of 1.5 times 10 to the sixth cells in 250 microliters. Transfer the 250 microliter cell suspension to an electroporation cuvette with a four millimeter gap distance. Next, prepare a plasma transfection mix by adding four micrograms of each plasmid to a total volume of 50 microliters of electroporation medium.Transfer the transfection mix to the cuvette, mix gently by flicking, and electroporate with one pulse at 280 volts. Finally, transfer 150 microliters of the electroporated fibroblasts to the prepared 10-centimeter cell culture plate. Agitate the plate thrice in all directions to distribute the cells, and incubate overnight.Fibroblasts present in a long, spindle-like phenotype, the size of 150 to 300 nanometers. After reprogramming, colonies with 50 micrometer small hiPSCs occur displaying distinct borders. The hiPSCs are distinguished by a high nuclei to body ratio and increased proliferation rate.

Episomale Reprogrammierung von dermalen Fibroblasten zur Erzeugung human-induzierter pluripotenter Stammzellen (hiPSCs)

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

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

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

Erzeugung und Erhaltung von Primaten-induzierten pluripotenten Stammzellen aus Urin

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular ...

Charakterisierung von Oberflächenantigenen und Proliferationsfähigkeit in hiPSC-treibenden MSCs

Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are restricted by limited donation, quality variations, and biosafety, the past 10 years have seen a great rise in efforts to generate MSCs from human induced pluripotent stem cells (hiPSCs). Past and recent efforts in the differentiation of hiPSCs into MSCs have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer culture. This protocol describes these two representative methods in deriving MSC from hiPSCs. Each method presents its advantages and disadvantages, including time, cost, cell proliferation ability, the expression of MSC markers, and their capability of differentiation in vitro. This protocol demonstrates that both methods can derive mature and functional MSCs from hiPSCs. The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation, while the EB method is characterized by lower time consumption.

Autor im Rampenlicht: Fortschritte bei iPSCs und der Erforschung genetischer Krankheiten 

This protocol describes and compares two representative methods for differentiating hiPSCs into mesenchymal stromal cells (MSCs). The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation. The embryoid bodies (EBs) method is characterized by lower time consumption.

Vergleich zweier repräsentativer Methoden zur Differenzierung humaner induzierter pluripotenter Stammzellen in mesenchymale Stromazellen

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are ...