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 Video 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 Video at JoVE.com

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors Video (Video) | JoVE

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.

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 ...

Research

JoVE Journal

Developmental Biology

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells Video (Video) | JoVE

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

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

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids Video (Video) | JoVE

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

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes Video (Video) | JoVE

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

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions Video (Video) | JoVE

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

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes Video (Video) | JoVE

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

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation Video (Video) | JoVE

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

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells Video (Video) | JoVE

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

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells Video (Video) | JoVE

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

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells Video (Video) | JoVE

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix Video (Video) | JoVE

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