We thank Yoshiko Miyake, Sayaka Kanaami, Chihana Fujita, Miho Yamaguchi, Natsuko Henmi, and Rei Ohno from the Keio University School of Medicine for technical assistance. This work was partly funded by an R&#38;D Systems support program to accelerate the practical use of health research outcomes, and the Highway Program for the Realization of Regenerative Medicine.

1. Prepare Activated Human T Cells
<ol>
	<li>Collect 10 ml heparinized whole blood from a donor by venipuncture.
	<ol>
		<li>Obtain informed consent of donors in advance of venipuncture. Venipuncture must be done by qualified and trained personnel.</li>
		<li>Handle obtained blood samples with sterile equipment and following appropriate biosafety guidelines.</li>
	</ol>
	</li>
	<li>Dilute 10 ml heparinized whole blood with 10 ml D-PBS(&#8211;).</li>
	<li>Put 15 ml Ficoll solution (Ficoll-Paque PREMIUM) into a new 50-ml conical tube.</li>
	<li>Layer the 20-ml blood solution prepared in Step 1.2 onto the Ficoll solution in Step 1.3. Using an electric pipette, let blood solution prepared in Step 1.2 run slowly along the inside wall of tube. Be careful not to mix the blood solution and Ficoll solution.</li>
	<li>Centrifuge at 400 &#215; g for 30 min at RT. Set the centrifugal deceleration speed to &#8220;slow&#8221;. 
	Note: After centrifugation, a white thin layer emerges between an upper milky plasma layer and a lower clear Ficoll layer. This white thin layer contains mononuclear cells.</li>
	<li>Transfer the mononuclear cell layer to a new 50-ml conical tube with a 1-ml pipette. Be careful not to include Ficoll solution.</li>
	<li>Add D-PBS(&#8211;) to the mononuclear cells to a total volume of 10 ml, and centrifuge at 200 &#215; g for 5 min at RT.</li>
	<li>Discard the supernatant. Add 10 ml of D-PBS(&#8211;) to the mononuclear cells, and centrifuge at 200 &#215; g for 5 min at RT.</li>
	<li>Discard the supernatant, add 1 ml of KBM502 medium to the collected mononuclear cells, and count the cells using a hemocytometer. Over 5 &#215; 106 mononuclear cells can be obtained from 10 ml heparinized whole blood with appropriate separation using Ficoll.</li>
	<li>Prepare an anti-human CD3 antibody solution at a concentration of 10 &#181;g/ml in D-PBS(&#8211;). Add the anti-human CD3 antibody solution to a 6-well plate, soak the surface of each well, and incubate the dish at 37 &#176;C in a 5% CO2 incubator for at least 30 min.</li>
	<li>Remove the anti-human CD3 antibody solution from each well and wash once with D-PBS(&#8211;) just prior to seeding the cells.</li>
	<li>Seed mononuclear cells that were prepared in Step 1.9 at a density of 2.5 &#215; 105 cells/cm2 on the anti-human CD3 antibody-coated 6-well plates in KBM502 medium. Incubate at 37 &#176;C in a 5%-CO2 incubator for 3&#8211;7 days without medium change until the T cells reach confluency. T cells in this period are both suspended and adhering cells.</li>
</ol>
2. Infect Human T Cells with SeV Vectors
<ol>
	<li>After 3&#8211;7 days culture, collect the activated T cells with existing medium by pipetting. Transfer them to a 15-ml conical tube and centrifuge at 200 &#215; g for 5 min at RT.</li>
	<li>Remove the supernatant and add 1 ml fresh KBM502 medium. Count the cells and plate 1.5 &#215; 106 cells (in 2 ml fresh KBM502 medium) into each well of an anti-CD3 antibody-coated 6-well plate.</li>
	<li>Thaw the SeV solutions of OCT3/4-SeV/TS&#916;F, SOX2-SeV/TS&#916;F, KLF4-SeV/TS&#916;F, and c-MYC (HNL)-SeV/TS15&#916;F on ice.</li>
	<li>Add the SeV solutions containing OCT3/4-SeV/TS&#916;F, SOX2-SeV/TS&#916;F, KLF4-SeV/TS&#916;F, and c-MYC (HNL)-SeV/TS15&#916;F individually to the wells, each at a multiplicity of infection (MOI) of 10. Incubate at 37 &#176;C in a 5%-CO2 incubator for a further 24 hr.</li>
</ol>
3. Remove SeV Vectors from Human T Cells
<ol>
	<li>24 hr after infection, collect the infected cells with a pipette. If there are adhering cells, they can easily be detached pipetting them repeatedly up and down. Transfer them to a 15-ml conical tube, and centrifuge at 200 &#215; g for 5 min at RT.</li>
	<li>Remove the supernatant containing the SeV vectors, and add 2 ml fresh KBM502 medium. Replate cells in each of the wells. Incubate at 37 &#176;C in a 5%-CO2 incubator for an additional 24 hr.</li>
</ol>
4. Reseed Cells on a Matrigel Layer
<ol>
	<li>Prepare basement membrane matrix solution by thawing matrix gel (please see the Materials List for the specific brand used in this study) at 4 &#176;C and dissolving 0.2 ml in 10 ml of DMEM/F12 medium. 
	Note: Matrix gel (please see the Materials List for the specific brand used in this study) needs to thaw O/N at 4 &#176;C to be completely thawed. Keep it on ice until just before use.</li>
	<li>Plate 5 ml basement membrane matrix solution per dish into 100-mm dishes and leave at RT for at least 30 min before seeding cells. At least two 100-mm dishes are needed for one experiment.</li>
	<li>Collect SeV-infected cells by pipetting, transfer them to a 15-ml conical tube, and centrifuge at 200 &#215; g for 5 min at RT.</li>
	<li>Remove the supernatant and add 1 ml fresh KBM502 medium. Count the number of cells using a hemocytometer, remove the basement membrane matrix solution from 100-mm dishes prepared in Step 4.2 and plate 1 &#215; 105 and 1 &#215; 106 cells (in 10 ml fresh mTeSR medium) on a 100-mm basement membrane matrix-coated dishes. Finally, use the plate in which colonies are easily picked.</li>
	<li>Incubate the dish at 37 &#176;C in a 5%-CO2 incubator O/N.</li>
	<li>Change the medium to 10 ml flesh mTeSR medium every other day. 
	Note: Approximately 20&#8211;30 days after infection, colonies that closely resemble embryonic stem cells (ESCs) will appear.</li>
</ol>
5. Expand T Cell-derived iPSCs
<ol>
	<li>Prepare basement membrane matrix solution by thawing matrix gel (please see the Materials List for the specific brand used in this study) at 4 &#176;C and dissolving 0.2 ml in 10 ml of DMEM/F12 medium.</li>
	<li>Plate 1 ml of the basement membrane matrix solution per well in a 6-well plate and leave at RT for at least 30 min before picking colonies.</li>
	<li>Fill a 96-well plate with 20 &#181;l of mTeSR medium per well.</li>
	<li>Select colonies that resemble ESCs under the stereomicroscope using a 20 &#181;l pipette. 
	Note: The colonies of ESCs and iPSCs are round and flat as shown in Figure 1B.</li>
	<li>Transfer selected colonies into the 96-well plate filled with mTeSR medium in Step 5.3.</li>
	<li>Add 200 &#181;l of mTeSR medium to each well and carefully pipette up and down to break the colony into small clumps under the stereomicroscope using a 200 &#181;l pipette. Remove the basement membrane matrix solution from the well prepared in 5.2 and put each colony into one well of the basement membrane matrix-coated 6-well plate with 2 ml of mTeSR medium per well.</li>
	<li>Change the medium to 2 ml flesh mTeSR medium every other day.</li>
</ol>
6. Maintain T Cell-derived iPSCs
The T cell-derived iPSCs can be maintained and stored using the same techniques as for human iPSCs and human ESCs. Once iPSC colonies grow up, expand them into larger dishes by repeating the same procedure.
<ol>
	<li>Change the mTeSR medium every 1&#8211;2 days.</li>
	<li>When the colonies become confluent every 5&#8211;7 days, passage the cells using dissociation solution for human iPSCs and human ESCs according to the manufacturer&#8217;s instructions.</li>
</ol>

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The authors have no competing or conflicting interests to disclose.

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

iPSCs have attracted considerable attention as a groundbreaking source of cells for regenerative medicine1-3. To date, diverse methods for generating iPSCs have been reported4,5. Among these, iPSCs generated from human T cells have been of particular interest because of the less-invasive method of cell sampling6-8. Additionally, iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) gene and are thus a source of antigen-specific T cells9,10. Therefore, generating T cell derived iPSCs safely is useful for progressing regenerative medicine.
This method is based on the concept of reducing the risk of unpredictable pathogenicity. For safe clinical application of iPSCs it is important to reduce the risk of exposure to pathogens11. Previously in many culture systems of pluripotent stem cells, fetal bovine serum and feeder cells have been used as essential reagents12. However, removal of both these reagents from the culture system is preferable for iPSC generation to reduce the risk of unpredictable pathogenicity.
Additionally, this method has the advantage of avoiding invasive cell sampling from patients and laborious preparation of feeder cells. Because T cell derived iPSCs have already been used successfully in disease research13,14, this method is also applicable and useful for generating disease-specific iPSCs from patients.
Among T cell reprogramming methods, using the Sendai virus (SeV) vector as a gene vehicle is a method that can generate iPSCs with high efficiency7,16. Additionally, because SeV is a single-stranded RNA virus and does not need a DNA phase for replication, its use in iPSC generation avoids breaking the host genome17-19. Therefore, we have established protocols for generating iPSCs from human peripheral T cells in serum-free and feeder-free conditions using a combination of matrigel, mTeSR medium, and SeV vectors.

<table><tbody><tr><td>Ficoll-Paque PREMIUM</td><td>GE Healthcare</td><td>17-5442-02</td><td></td></tr><tr><td>Purified NA/LE mouse anti-human CD3</td><td>BD Pharmingen</td><td>555336</td><td></td></tr><tr><td>Bovine albumin fraction V solution</td><td>Gibco</td><td>15260-037</td><td></td></tr><tr><td>mTeSR1 medium kit</td><td>STEM CELL</td><td>5850</td><td>Warm at room temperature before use</td></tr><tr><td>Dissociation Solution</td><td>ReproCELL</td><td>RCHETP002</td><td></td></tr><tr><td>D-PBS(&amp;ndash;)</td><td>Wako</td><td>045-29795</td><td></td></tr><tr><td>SeV Vector kit CytoTune-iPS ver.1.0</td><td>DNAVEC</td><td>DV-0303c</td><td>Thaw on ice before use</td></tr><tr><td>100-mm tissue culture dish</td><td>Falcon</td><td>353003</td><td></td></tr><tr><td>96-well tissue culture plate</td><td>Falcon</td><td>353078</td><td></td></tr><tr><td>6-well tissue culture plate</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>15&amp;#160;ml Centrifuge Tube</td><td>Greiner Bio-One</td><td>188271</td><td></td></tr><tr><td>50&amp;#160;ml Centrifuge Tube</td><td>Greiner Bio-One</td><td>227261</td><td></td></tr></tbody></table>

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

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.

Using this protocol, users are able to generate iPSCs from human peripheral T cells stably. iPSC generation from T cells with SeV and matrigel showed approximately 0.002% &#8211; 0.005% of efficiency of cell reprogramming in between several donor cases and SeV were not detected after several passages16. Figure 1A shows a schematic of the protocol for generating T cell-derived iPSCs in feeder-free conditions using matrigel and mTeSR medium. Around 20&#8211;30 days after infection with SeV, iPSC colonies are recognized by their ESC colony-like morphology (Figure 1B). Immunofluorescence staining revealed expression of typical pluripotent cell markers (NANOG, OCT3/4, SSEA4, TRA-1-60, and TRA-1-81) in T cell-derived iPSCs generated under feeder-free conditions (Figure 1C).
<img alt="Figure 1" src="/files/ftp_upload/53225/53225fig1highres.jpg" width="700" /> 
Figure 1. (A): A schematic of the protocol for reprogramming T cells under feeder-free conditions in this study. (B): A typical ESC-like iPSC colony on day 27 after blood sampling under feeder-free conditions. (C): ALP staining and immunofluorescence staining for pluripotency and surface markers (NANOG, OCT3/4, SSEA4, TRA-1-60, and TRA-1-81) in T cell-derived iPSCs generated under feeder-free conditions. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53225/53225fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

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

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

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

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

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

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

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

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

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 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 ...

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

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

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

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

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 ...

Biology

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

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

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

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

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 ...