These studies were funded in part by NIH UO1HL107443-01 Award to GJM and GM.

1. Isolation and Expansion of Peripheral Blood Mononuclear Cells (PBMCs)
DAY 0
<ol>
 <li>Draw 4 ml of peripheral blood into a BD Vacutainer CPT Cell Preparation Tube with sodium citrate. Invert the tube 8 to 10 times and centrifuge at 1,800 x g for 30 min at room temperature. Ideally, this step should be done within 2 hr of collection.</li>
 <li>Collect the mononuclear cells (MCs) by pipetting the buffy coat (cell layer between gel barrier and plasma) into a sterile 15 ml conical centrifuge tube. Bring total volume to 10 ml with sterile phosphate-buffered saline (PBS), invert several times and centrifuge at 300 x g for 15 min.</li>
 <li>Resuspend the cells in 10 ml of sterile PBS and perform cell count. Transfer 1 to 2x106 cells into a sterile 15 ml conical centrifuge tube and centrifuge at 300 x g for 10 min.</li>
 <li>Resuspend the cells in 2 ml of expansion medium (EM) (QBSF-60 Stem Cell Medium containing 50 &mu;g/ml Ascorbic Acid, 50 ng/ml SCF, 10 ng/ml IL-3, 2 U/ml EPO, 40 ng/ml IGF-1, 1 &mu;M Dexamethasone and 100 &mu;g/ml primocin or 1% Pen/Strep) and transfer to one well of a 12-well plate. Incubate the cells in a 37 &deg;C, 5% CO2 incubator.</li>
 <li>Centrifuge remaining cells at 300 x g for 10 min and freeze ~2x106 cells/vial in FBS containing 10% DMSO.</li>
 <li>To start the protocol using frozen PBMCs, thaw 1 vial of cells into 10 ml of QBSF medium and centrifuge at 300 x g for 10 min. Resuspend the cells in 2 ml of EM and transfer to one well of a 12-well plate. Incubate the cells in a 37 &deg;C, 5% CO2 incubator.</li>
</ol>
DAY 3 and DAY 6
<ol start="17">
 <li>Transfer the cells to a sterile 15 ml conical tube and wash the well once with 1 ml of QBSF-60 Stem Cell Medium to collect adherent cells.</li>
 <li>Centrifuge the cells at 300 x g for 10 min.</li>
 <li>Resuspend the cells in 2 ml of EM and transfer to one well of a 12-well plate. Incubate the cells in a 37 &deg;C, 5% CO2 incubator.</li>
</ol>
2. Transduction of PBMCs with STEMCCA Lentivirus
DAY 9
<ol>
 <li>Transfer the cells to a sterile 15 ml conical tube and wash the well once with 1 ml of QBSF-60 Stem Cell Medium to collect adherent cells.</li>
 <li>Centrifuge the cells at 300 x g for 10 min.</li>
 <li>Resuspend the cells in 1 ml of fresh EM containing 5 &mu;g/ml of polybrene and STEMCCA lentivirus (MOI=1 to 10) and transfer to one well of a 12-well plate. (The protocol describing production of lentiviral particles can be found at <a href="http://www.bumc.bu.edu/stemcells/protocols/" target="_blank">http://www.bumc.bu.edu/stemcells/protocols/</a>)</li>
 <li>Spin the plate at 2,250 rpm at 25 &deg;C for 90 min.</li>
 <li>After spin, add an additional 1 ml of fresh EM containing 5 &mu;g/ml of polybrene for a total of 2 ml of medium and incubate the plate in a 37 &deg;C, 5% CO2 incubator.</li>
</ol>
DAY 10
<ol start="16">
 <li>Transfer the cells to a sterile 15 ml conical tube and wash the well once with 1 ml of QBSF-60 Stem Cell Medium to collect adherent cells.</li>
 <li>Centrifuge the cells at 300 x g for 10 min.</li>
 <li>Resuspend the cells in 2 ml of EM and transfer to 1 well of a 12-well plate. Incubate the cells in a 37 &deg;C, 5% CO2 incubator.</li>
</ol>
3. Plating Transduced Cells onto MEFs
DAY 11
<ol>
 <li>Coat the wells of a 6-well plate with 0.1% gelatin and plate inactivated mouse embryonic fibroblasts (MEFs) at 2x105 cells/well in MEF medium (IMDM containing 10% FBS, 1% Non-Essential Amino Acids, 100 &mu;M &beta;-mercaptoethanol, 2 mM L-Glutamine, 100 &mu;g/ml primocin or 1% Pen/Strep). Prepare 3 wells per infection.</li>
</ol>
DAY 12
<ol start="2">
 <li>Transfer the cells to a sterile 15 ml conical tube and wash the well once with 1 ml of QBSF-60 Stem Cell Medium to collect adherent cells.</li>
 <li>Resuspend the cells in 3 ml of MEF medium containing 10 ng/ml bFGF, and Ascorbic Acid and growth factors at the same concentrations as used in EM medium (50 &mu;g/ml Ascorbic Acid, 50 ng/ml SCF, 10 ng/ml IL-3, 2 U/ml EPO, 40 ng/ml IGF-1, 1 &mu;M Dexamethasone).</li>
 <li>Plate 1 ml of cells per well of a 6-well plate containing MEFs. Add 1.5 ml of MEF media with bFGF, Ascorbic Acid, and growth factors for a total of 2.5 ml of media/well.</li>
 <li>Spin the plate at 500 rpm at 25 &deg;C for 30 min. Incubate the plate in a 37 &deg;C, 5% CO2 incubator.</li>
</ol>
DAY 14
<ol start="6">
 <li>Feed cells every other day with 2.5 ml of MEF media containing 10 ng/ml bFGF and 50 &mu;g/ml Ascorbic Acid (no growth factors). Aspirate and discard floating cells with each feed.</li>
 <li>Add additional MEFs as needed (usually once a week).</li>
</ol>
~ DAY 20
<ol start="8">
 <li>Once small colonies appear, feed cells daily with 2 ml of human embryonic stem cell (hESC) medium (DMEM/F12 containing 20% Knockout Serum Replacement, 1% Non-Essential Amino Acids, 100 &mu;M &beta;-mercaptoethanol, 2 mM L-Glutamine, 100 &mu;g/ml primocin or 1% Pen/Strep and 10 ng/ml bFGF).</li>
</ol>
4. Picking and Expansion of iPSC Clones
~ DAY 30 - 40
<ol>
 <li>Pick each colony in individual wells of a 12-well plate pre-seeded with inactivated MEFs containing 1 ml/well of hESC medium plus 10 &mu;M of Stemolecule Y27632 (ROCK inhibitor).</li>
 <li>Feed cells daily thereafter with 1 ml of hESC medium only (no ROCK inhibitor).</li>
</ol>
5. Immunofluorescence Staining for Pluripotency Markers
<ol>
 <li>Staining was performed using the kit "ES Cell Marker Sample Kit" from Millipore and following the manufacturer's protocol.</li>
</ol>
6. Excision of Integrated STEMCCA Vector
<ol>
 <li>Excision of reprogramming cassette is achieved utilizing a Cre-IRES-Puro expressing vector following transfection and brief Puromycin selection as described6.</li>
</ol>

<ol>
	<li>Lowry, W. E., Richter, L., Yachechko, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+induced+pluripotent+stem+cells+from+dermal+fibroblasts.">Generation of human induced pluripotent stem cells from dermal fibroblasts.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2883-2888 (2008).</li><li>Park, I. H., Zhao, R., West, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+human+somatic+cells+to+pluripotency+with+defined+factors.">Reprogramming of human somatic cells to pluripotency with defined factors.</a> Nature. 451, 141-146 (2008).</li><li>Takahashi, K., Tanabe, K., Ohnuki, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Yu, J., Vodyanik, M. A., Smuga-Otto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (2007).</li><li>Stadtfeld, M., Hochedlinger, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotency:+history,+mechanisms,+and+applications.">Induced pluripotency: history, mechanisms, and applications.</a> Genes Dev. 24, 2239-2263 (2010).</li><li>Somers, A., Jean, J. C., Sommer, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+transgene-free+lung+disease-specific+human+induced+pluripotent+stem+cells+using+a+single+excisable+lentiviral+stem+cell+cassette.">Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette.</a> Stem Cells. 28, 1728-1740 (2010).</li><li>Loh, Y. H., Hartung, O., Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+T+cells+from+human+peripheral+blood.">Reprogramming of T cells from human peripheral blood.</a> Cell Stem Cell. 7, 15-19 (2010).</li><li>Seki, T., Yuasa, S., Oda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+induced+pluripotent+stem+cells+from+human+terminally+differentiated+circulating+T+cells.">Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells.</a> Cell Stem Cell. 7, 11-14 (2010).</li><li>Staerk, J., Dawlaty, M. M., Gao, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+human+peripheral+blood+cells+to+induced+pluripotent+stem+cells.">Reprogramming of human peripheral blood cells to induced pluripotent stem cells.</a> Cell Stem Cell. 7, 20-24 (2010).</li><li>Chou, B. K., Mali, P., Huang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+human+iPS+cell+derivation+by+a+non-integrating+plasmid+from+blood+cells+with+unique+epigenetic+and+gene+expression+signatures.">Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures.</a> Cell Res. 21, 518-529 (2011).</li><li>vanden Akker, E., Satchwell, T. J., Pellegrin, S., Daniels, G., Toye, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+majority+of+the+in+vitro+erythroid+expansion+potential+resides+in+CD34(-)+cells,+outweighing+the+contribution+of+CD34(+)+cells+and+significantly+increasing+the+erythroblast+yield+from+peripheral+blood+samples.">The majority of the in vitro erythroid expansion potential resides in CD34(-) cells, outweighing the contribution of CD34(+) cells and significantly increasing the erythroblast yield from peripheral blood samples.</a> Haematologica. 95, 1594-1598 (2010).</li></ol>

No conflicts of interest declared.

We herein describe the use of the STEMCCA lentiviral vector to generate human iPSCs from mononuclear cells isolated from a few milliliters of freshly collected peripheral blood. The protocol can also be used to reprogram frozen PBMCs (obtained directly from the buffy coat), a detail of significant practical implications when utilizing donor cells acquired from a distant location. Before the induction of reprogramming, isolated PBMCs must undergo a critical expansion step that renders a healthy proliferating population of...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>BD Vacutainer CPT Cell Preparation Tube with sodium citrate</td>
 <td>BD Biosciences</td>
 <td>362760</td>
 <td></td>
 </tr>
 <tr>
 <td>QBSF-60 Stem Cell Medium </td>
 <td>Quality Biological</td>
 <td>160-204-101</td>
 <td></td>
 </tr>
 <tr>
 <td>IMDM</td>
 <td>Invitrogen</td>
 <td>12440</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM/F12</td>
 <td>Invitrogen</td>
 <td>11330</td>
 <td></td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Atlanta Biologicals</td>
 <td>S10250</td>
 <td></td>
 </tr>
 <tr>
 <td>Knockout Serum Replacement</td>
 <td>Invitrogen</td>
 <td>10828</td>
 <td></td>
 </tr>
 <tr>
 <td>Primocin</td>
 <td>Invivogen</td>
 <td>ant-pm-2</td>
 <td></td>
 </tr>
 <tr>
 <td>Pen/Strep</td>
 <td>Invitrogen</td>
 <td>15140</td>
 <td></td>
 </tr>
 <tr>
 <td>L-Glutamine</td>
 <td>Invitrogen</td>
 <td>25030</td>
 <td></td>
 </tr>
 <tr>
 <td>Non-Essential Amino Acids</td>
 <td>Invitrogen</td>
 <td>11140</td>
 <td></td>
 </tr>
 <tr>
 <td>&beta;-mercaptoethanol</td>
 <td>MP Biomedicals</td>
 <td>190242</td>
 <td></td>
 </tr>
 <tr>
 <td>Ascorbic Acid</td>
 <td>Sigma</td>
 <td>A4544</td>
 <td></td>
 </tr>
 <tr>
 <td>IGF-1</td>
 <td>R&amp;D Systems</td>
 <td>291-G1</td>
 <td></td>
 </tr>
 <tr>
 <td>IL-3</td>
 <td>R&amp;D Systems</td>
 <td>203-IL</td>
 <td></td>
 </tr>
 <tr>
 <td>SCF</td>
 <td>R&amp;D Systems</td>
 <td>255-SC</td>
 <td></td>
 </tr>
 <tr>
 <td>EPO</td>
 <td>R&amp;D Systems</td>
 <td>286-EP</td>
 <td></td>
 </tr>
 <tr>
 <td>Dexamethasone</td>
 <td>Sigma</td>
 <td>D4902</td>
 <td></td>
 </tr>
 <tr>
 <td>Polybrene</td>
 <td>Sigma</td>
 <td>H-9268</td>
 <td></td>
 </tr>
 <tr>
 <td>bFGF</td>
 <td>R&amp;D Systems</td>
 <td>233-FB</td>
 <td></td>
 </tr>
 <tr>
 <td>Stemolecule Y27632</td>
 <td>Stemgent</td>
 <td>04-0012</td>
 <td></td>
 </tr>
 <tr>
 <td>ES Cell Marker Sample Kit</td>
 <td>Millipore</td>
 <td>SCR002</td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

We demonstrate a simple and effective protocol for the generation of human iPSCs from PBMCs using a single lentiviral vector. Figure 1A shows a schematic representation of the protocol. The blood is collected into a BD Vacutainer CPT Cell Preparation Tube with sodium citrate, and after centrifugation, mononuclear cells can be collected from the interface between the polyester gel and the plasma (buffy coat) (Figure 1B). The isolated PBMCs are then expanded in culture for 9 days. ...

Watch this Scientific Journal Video about Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector at JoVE.com

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

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.

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

generation-human-induced-pluripotent-stem-cells-from-peripheral-blood

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26.4K Görüntüleme.  Boston University School of Medicine. Burada tek bir lentiviral yeniden programlanması vektörü kullanılarak periferal kan 3-4 ml insan iPSCs üretimi için basit ve etkili bir protokol göstermektedir. Hazır kan hücrelerinin yeniden programlanması daha geniş bir araştırma toplum için erişilebilir kılarak IPSC teknoloji kullanımı hızlandırmak için vaat ediyor.

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

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.

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

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.

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.

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

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

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

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

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

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

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

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

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

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

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.

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

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.

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

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

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

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

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

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

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

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

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 ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.