Name: generation of integration-free induced pluripotent stem cells from human peripheral blood mononuclear cells using episomal vectors
Uploaded: 2017-01-01T15:00:00
Description: Watch this Scientific Journal Video about Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors at JoVE.com

This work was supported by the Ministry of Science and Technology of China (2015CB964902, 2013CB966902 and 2012CB966601), the National Natural Science Foundation of China (81500148, 81570164 and 81421002), the Loma Linda University School of Medicine GCAT grant (2015), and Telemedicine and Advanced Technology Research Center (W81XWH-08-1-0697).

All of the human PB samples were obtained from anonymous adult donors with no identification information available from Tianjin Blood Center with approval of the local research ethics committee.
1. Endo-free Plasmid Preparation
<ol>
	<li>Use a commercial Plasmid Purification Maxi Kit to extract episomal vectors from E. coli according to manufacturer&#39;s protocol. For the final step, substitute TE buffer with endotoxin-free sterile water to dissolve the DNA pellet.</li>
	<li>Measur...

<ol>
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Acquiring blood samples from healthy donors or patients is convenient and noninvasive, making it an attractive cell source for basic research and clinical cell therapy. Here we have described a protocol for highly efficient generation of integration-free iPSCs from peripheral blood samples. This reproducible and affordable approach should benefit the iPSC field.
We have reported that there are two critical factors responsible for the highly efficient PB reprogramming30. One is equim...

After forced expression of several transcription factors (i.e. OCT4, SOX2, MYC and KLF4), somatic cells can be reprogrammed to induced Pluripotent Stem Cells (iPSCs), which hold great promise for applications in regenerative medicine and cell replacement therapy1-3. To date, diverse methods have been developed to increase the success rate of reprogramming4-7. Viral vectors-induced reprogramming is widely used for efficient generation of iPSCs, because viral integration leads to a high-level, stable expression of the reprogramming factors. However, permanent integration of the vector DNA into the cell genome may induce insertional mutagenesis5. In addition, insufficient inactivation of reprogramming factors may disturb iPSCs differentiation8. As such, the use of iPSCs without integration of reprogramming factors is imperative, especially for use in cell therapy applications.
Episomal Vectors (EVs) are widely used in the generation of integration-free iPSCs. The most commonly used EV is a plasmid containing two elements, origin of viral replication (oriP) and EB Nuclear Antigen 1 (EBNA1), from the Epstein-Barr (EB) virus9. The oriP element promotes plasmid replication in mammalian cells, while the EBNA1 element tethers the oriP-containing plasmid DNA to the chromosomal DNA that allows for the partitioning of the episome during division of the host cell. In comparison to other integration-free approaches, including Sendai Virus (SV) and RNA transfection, EVs possess multiple advantages5,6,10. As plasmid DNA, EVs can be readily produced and modified in house, making them extremely affordable. In addition, reprogramming with EV is a less labor-intensive process since a single transfection with EVs is sufficient for iPSC generation, whereas several RNA transfections are necessary for successful reprogramming.
Dermal fibroblasts have been used in many reprogramming studies. However, skin biopsy is not only an invasive and painful process, but also time-consuming for expanding cells to sufficient quantities for reprogramming. Of greater concern, skin cells of adult donors have often been exposed to long-term UV light radiation, which may lead to mutations associated with tumors, thus limiting the applications for iPSCs derived from skin fibroblasts11,12. Recently, it has been reported that normal human skin cells accumulate somatic mutations and multiple cancer genes, including most of the key drivers of cutaneous squamous cell carcinomas, are under strong positive selection13.
In contrast to skin fibroblasts, peripheral blood (PB) cells are a preferable source of cells for reprogramming&#160;because 1) blood cells can be easily obtained through a minimally invasive process, 2) peripheral blood cells are the progeny of hematopoietic stem cells residing in bone marrow, thus protected from harmful radiation. Peripheral blood mononuclear cells (PB MNCs) can be collected in an hour from the buffy coat layer following a simple gradient centrifugation using Ficoll-Hypaque (1.077 g/mL). The obtained PB MNCs are composed of lymphocytes, monocytes and a few Hematopoietic Progenitor Cells (HPCs) 14. Although human T lymphocytes are one of the major cell types in PB, mature T cells contain rearrangements of the T cell receptor (TCR) genes and lack an intact genome thus limiting their potential for applications15,16. However, rejuvenation of T cells via iPSC generation may have potential applications in Chimeric Antigen Receptor (CAR) T-cell therapy 17-19. In comparison, HPCs have an intact genome and are readily reprogrammable. Although only 0.01 - 0.1% cells in peripheral circulation are HPCs, these cells can be&#160;expanded ex vivo&#160;in erythroid medium that favors proliferation of erythroid progenitor cells14.
In our previous study, we used the factor BCL-XL in addition to the Yamanaka factors (OCT4, SOX2, MYC and KLF4), which resulted&#160;in a 10x increase in PB reprogramming efficency20. BCL-XL, also known as BCL2L1, is a potent inhibitor of cell death, by inhibiting activation of caspases21,22. But, BCL-XL may also play an important role in maintaining pluripotency21,22. Recently, we have further optimized our EV reprogramming system by separately expressing MYC and KLF4 with two vectors, which leads to an approximately 100x increase in reprogramming efficiency23. Using this method, the reprogramming efficiency, defined by colony number divided by starting cell number at transfection, is 0.2&#160;- 0.5% from healthy donors. As follows, we describe the detailed experimental procedure for generating integration-free iPSCs from PB.

<table border="0" cellpadding="0" cellspacing="0" width="1666">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Hematopoietic Stem Cell Expansion Medium</td>
			<td style="width:263px;">Sigma</td>
			<td style="width:136px;">S0192</td>
			<td style="width:723px;">Store at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Human stem cell factor (SCF)</td>
			<td style="width:263px;">Peprotech</td>
			<td style="width:136px;">300-07</td>
			<td>Store at -20 or -80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Interleukin-3 (IL-3)</td>
			<td style="width:263px;">Peprotech</td>
			<td style="width:136px;">AF-200-03</td>
			<td>Store at -20 or -80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Eryrthropoietin (EPO)</td>
			<td style="width:263px;">Peprotech</td>
			<td style="width:136px;">100-64</td>
			<td>Store at -20 or -80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Insulin growth factor-1 (IGF-1)</td>
			<td style="width:263px;">Peprotech</td>
			<td style="width:136px;">100-11</td>
			<td>Store at -20 or -80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Dexamethasone</td>
			<td style="width:263px;">Sigma</td>
			<td style="width:136px;">D4902</td>
			<td>Store at -20 or -80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">1-thioglycerol (MTG)</td>
			<td style="width:263px;">Sigma</td>
			<td style="width:136px;">M6145</td>
			<td>Store at -20 or -80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">DMEM/F12 medium</td>
			<td style="width:263px;">Gibco</td>
			<td style="width:136px;">112660-012</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">L-glutamine (100x)</td>
			<td style="width:263px;">Gibco</td>
			<td style="width:136px;">25030-081</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Penicillin/Streptomycin (100x)</td>
			<td style="width:263px;">Gibco</td>
			<td style="width:136px;">15140-122</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Non-essential amino acids solution (100x)</td>
			<td style="width:263px;">Gibco</td>
			<td style="width:136px;">11140-050</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Fibroblast growth factor 2 (FGF2)</td>
			<td style="width:263px;">Peprotech</td>
			<td style="width:136px;">100-18B</td>
			<td>Store at -20 or -80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">ITS (100x)</td>
			<td style="width:263px;">Gibco</td>
			<td style="width:136px;">41400-045</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Ascorbic acid</td>
			<td style="width:263px;">Sigma</td>
			<td style="width:136px;">49752</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">DMEM (high glucose) medium</td>
			<td style="width:263px;">Thermo</td>
			<td style="width:136px;">SH30243.01B</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">FBS</td>
			<td style="width:263px;">Hyclone</td>
			<td style="width:136px;">SV30087.01</td>
			<td>Store at -40 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Ficoll</td>
			<td style="width:263px;">GE Healthcare, SIGMA</td>
			<td style="width:136px;">17-5442-02</td>
			<td>Store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Trehalose&#160;</td>
			<td style="width:263px;">Sigma</td>
			<td style="width:136px;">T9531</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">DMSO</td>
			<td style="width:263px;">Sigma</td>
			<td style="width:136px;">D2650</td>
			<td>Store at RT, protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Endofree Plasmid Maxi Kit(10)</td>
			<td style="width:263px;">Qiagen</td>
			<td style="width:136px;">12362</td>
			<td>Store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">IMDM</td>
			<td style="width:263px;">Gibco</td>
			<td style="width:136px;">21056-023</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Human CD34+ Cell Nucleofection Kit</td>
			<td style="width:263px;">Lonza</td>
			<td style="width:136px;">VPA-1003</td>
			<td>Store at RT, nucleofection buffer and supplement buffer should be stored at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Sodium butyrate</td>
			<td style="width:263px;">Sigma</td>
			<td style="width:136px;">B5887</td>
			<td>Store at -20 or -80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">ROCK inhibitor Y27632</td>
			<td style="width:263px;">STEMGENT</td>
			<td style="width:136px;">04-0012-10</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Essential 8 basal medium (E8)</td>
			<td style="width:263px;">Gibco</td>
			<td style="width:136px;">A15169-01</td>
			<td>Store at 4 &#176;C, the supplement should be stored at -20 or -80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Matrigel</td>
			<td style="width:263px;">BD</td>
			<td style="width:136px;">354277</td>
			<td>Store at -20 or -80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">2x EasyTaq PCR SuperMix(+dye)</td>
			<td style="width:263px;">TransGen Biotech</td>
			<td style="width:136px;">AS111</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Cell detachment solution</td>
			<td style="width:263px;">STEMGENT</td>
			<td style="width:136px;">01-0006</td>
			<td>Store at -20 &#176;C, Accutase as a cell detachment solution to obtain a single cell suspension</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">DAPI</td>
			<td style="width:263px;">Sigma</td>
			<td style="width:136px;">D9542-1MG</td>
			<td>Store at 4 &#176;C or -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Anti-nanog-AF488</td>
			<td style="width:263px;">BD</td>
			<td style="width:136px;">560791</td>
			<td style="width:723px;">Store at 4 &#176;C, primary antibody used for Immunofluorescence, dilute 1/100 when use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Anti-OCT4</td>
			<td style="width:263px;">abcam</td>
			<td style="width:136px;">ab19857</td>
			<td style="width:723px;">Store at 4 &#176;C, primary antibody used for Immunofluorescence, dilute 1/100 when use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">AF488 donkey anti-mouse IgG</td>
			<td style="width:263px;">Invitrogen</td>
			<td style="width:136px;">A21202</td>
			<td style="width:723px;">Store at 4 &#176;C, secondary antibody used for Immunofluorescence,&#160; dilute 1/500 when use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">PE anti-human TRA-1-60-R Antibody</td>
			<td style="width:263px;">Biolegend</td>
			<td style="width:136px;">330610</td>
			<td style="width:723px;">Store at 4 &#176;C, antibody used for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">eFluor 570-conjugated anti-SSEA4</td>
			<td style="width:263px;">eBioscience</td>
			<td style="width:136px;">41-8843</td>
			<td>Store at 4 &#176;C, antibody used for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Isotype antibody</td>
			<td style="width:263px;">eBioscience</td>
			<td style="width:136px;">11-4011</td>
			<td>Store at 4 &#176;C, antibody used for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Alkaline Phosphatase Detection Kit</td>
			<td style="width:263px;">SiDanSai</td>
			<td style="width:136px;">1102-100</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Genomic DNA Extraction Kit</td>
			<td style="width:263px;">TIANGEN</td>
			<td style="width:136px;">DP304-02</td>
			<td>Store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Trypan Blue solution</td>
			<td style="width:263px;">Sigma</td>
			<td style="width:136px;">T8154</td>
			<td>Store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Flow cytometry cell analyzer</td>
			<td style="width:263px;">BD</td>
			<td style="width:136px;">LSRII</td>
			<td>for flow cytometry analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Spinning disk confocal microscope (SDC)</td>
			<td style="width:263px;">PerkinElmer</td>
			<td style="width:136px;">UltraVIEW VOX</td>
			<td>for confocal imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Nucleofection device</td>
			<td style="width:263px;">Lonza</td>
			<td style="width:136px;">Nucleofector 2b</td>
			<td>for the nucleofection of PB MNC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">ImageQuant LAS-4010</td>
			<td style="width:263px;">GE</td>
			<td style="width:136px;"></td>
			<td>take photo of AP staining in bulk</td>
		</tr>
	</tbody>
</table>

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.

Using this protocol, we can obtain hundreds of colonies from 1 x 105 nucleofected PB MNCs (Figures 1A and 1B). The reprogramming efficiency is approximately 0.2 - 0.5% and the colonies express pluripotency markers (Figures 1C and 1D). iPSCs generated using the described protocol are integration-free and have the ability to form teratoma composing the 3 germ layers (Figures 1E and 1F</st...

Watch this Scientific Journal Video about Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors at JoVE.com

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

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.

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

The overall goal of this procedure is to generate iPS cells from adult human peripheral blood mononuclear cells using non-integrating episomal vectors. This protocol can help scientists in the stem cell field who wish to generate iPS cells for disease monitoring, drug screening, and regular radio medicine. Our reprogramming protocol is very simple, yet highly efficient.Following this procedure, one can easily master the technique of blood cell reprogramming. The quality of the plasma is as important for successful reprogramming. Contaminants may decrease reprogramming efficiency, therefore we recommend using Endofree kits to purify episomal plasmids.iPS cells are more fragile than other cells. Preparing cells for more than one during passing gene may induce massive cell deaths, in particular at early passages. To begin this procedure, prepare all the required culture media according to the accompanying manuscript.Then combine 10 mLs of fresh PB sample and ten mLs of PBS buffer into a 50 mL tube and mix well. Bring PBS to room temperature or 37 degrees Celsius prior to use. Next, add 10 mL of ficoll to the bottom of the tube.This process should be carried out slowly to ensure that the ficoll layer does not mix with the PB.Centrifuge the mixture at 400 x g for 30 minutes with low acceleration and deceleration. After centrifugation, the PB MNCs are in the white layer located between the PB plasma and ficoll. Slowly aspirate 10 mL of PB plasma without disturbing the buff ficoll layer.Then carefully harvest the white layer into a new 50 mL tube. The total volume of the collected cells from the white layer should be about three to six mL. After that, add PBS to bring the total volume to 30 mL and mix well.Then centrifuge the sample at 400 x g for ten minutes. After ten minutes, remove the supernatant and resuspend the cell pellet in 20 mL of culture medium. Mix well and centrifuge the sample at 400 x g for five minutes to remove the majority of the platelets.Subsequently, remove the supernatant and resuspend the cell pellet in one to two mL of IMDM. The cells may be used for immediate culture or frozen down for later use. For cryopreservation, add an equal amount of cryopreservation medium.Aliquot the cells into cryovials and transfer them to a 80 degrees Celsius freezer immediately. In this procedure, prepare five mL of IMDM medium in a 15 mL tube. Quickly thaw the frozen PB MNCs in a 37 degrees Celsius water bath before transferring them to the tube with IMDM medium.Centrifuge the sample at 400 x g for five to ten minutes. Afterward, remove the supernatant and resuspend the cell pellet in erythroid medium. Then count the cells under a microscope using a hemocytometer.Subsequently, culture PB MNCs in a non-tissue culture treated six well plate with two mL of medium per well at 37 degrees Celsius in a 5%Carbon Dioxide, humidified incubator. One day three and day five, add one mL of fresh erythroid medium directly in each well without changing the medium. On day six, harvest PB MNCs for nucleofection.One day before nucleofection, precoat the tissue culture treated six well plate with 0.1%gelatin at 37 degrees Celsius for 20 minutes. Thaw the inactivated MEF feeder cells in a 37 degrees Celsius water bath and immediately transfer them to a 15 mL tube containing five mL of MEF medium. Next, centrifuge them at 400 x g for five minutes and remove the supernatant.Then remove the gelatin from the six well plate and resuspend the cell pellet in MEF medium. Afterward, seed the inactivated MEF cells in two mL of MEF medium in each well of a gelatin pre-treated six well plate and culture them at 37 degrees Celsius in a 5%carbon dioxide humidified incubator. On the day of nucleofection, aspirate the MEF medium and replace it with one mL of erythroid medium.Then pre-equilibrate the culture plate for 10-30 minutes at 37 degrees Celsius in a 5%carbon dioxide humidified incubator. Add two micrograms PEV OCT4 to Sox2, one microgram PEV MYC, one microgram PEV KLF4, and 0.5 micrograms PEV Bcl-XL in a 1.5 mL sterile tube. Heat the tube at 50 degrees Celsius for five minutes to prevent contamination and then cool it down to room temperature.Afterward, add 57 microliters of nucleofection buffer and 13 microliters of supplement buffer to the sample. Harvest two times ten to the sixth cultured PBMNCs to a five mL tube by centrifugation at 200 x g for seven minutes. After that, remove the supernatant and add the plasmid and nucleofection buffer mix to the cell pellet.Then mix well by flicking the tube with a finger. Next, transfer the DNA in cell suspension to the cuvette provided by the kit and cap the cuvette. Select program U-008 on the nucleofection device.Subsequently, insert the cuvette into the holder and press OK.Afterward, take the cuvette out of the holder. Add 0.5-1 mL of prewarmed erythroid medium to each cuvette and transfer the cells to the pre-equilibrated MEF plate immediately. For some samples, you may get thousands of colonies from one mil of blood.If you wish to pick single colony for further culture you may need to seed one extra well with only 1 million cells. Right before placing the hypoxia chamber into the incubator, gently rock the chamber several times to create an even distribution of cells in the culture wells. Then transfer the plate to a hypoxia chamber.Flush the chamber with a mixed gas for one to two minutes at a rate of 20 Liters per minute. Afterward, seal the chamber and culture the sample at 37 degrees Celsius. On day two post-nucleofection, add two mL of iPSC medium directly into each well.On day four post-nucleofection, remove three mL of medium and add two mL of fresh iPSC medium. At this stage, most of the live cells have attached to the feeder layer. After day six post nucleofection, change the medium every two days by leaving 500 microliters of spent medium and adding two mL of fresh E8 medium supplemented with 0.25 millimolars sodium butyrate until days 14-18.Shown here is a schematic of the protocol for reprogramming peripheral blood cells. This image shows the AP standing in bowl, and this one shows a typical ESC-like iPSC colony on day 14 after PB MNCs nucleofection. Here are representative FACS diagrams of iPSCs at passage five expressing TRA-1-60 or SSEA4.Here are the representative confocal images of iPSC colonies expressing NANOG and OCT4. The images here show the H&E staining of terratoma, comprising all three germ layers. After watching this video, you should have a good understanding of how to generate integration free iPS cells from human peripheral blood mononuclear cells.Once mastered, one can generate large quantities of iPS cells in three weeks, so hands on time will be less than three hours. We have developed a simple, episomal vector system for efficient blood cell reprogramming. We believe this affordable system can be easily adopted, in particular in small labs with limited resources.Don't forget that working with human blood and plasmas can be hazardous, and precautions must be taken such as wearing gloves and lab coats while performing this procedure.

generation-integration-free-induced-pluripotent-stem-cells-from-human

Research

JoVE Journal

Developmental Biology

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.

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

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.

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

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

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

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.

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

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.

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.

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

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.

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.

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 Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

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.

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 ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...