We thank Dr. Shinya Yamanaka (Kyoto University) for providing iPS-MEF-Ng-20D-17 cell line, Dr. Dario Vignali (St. Jude Children's Research Hospital) for supporting the OT1-2A&bull;pMig II construct, Dr. Juan Carlos Zuniga-Pflucker (Department of Immunology, University of Toronto) for supporting the OP9-DL1 cell line, and Dr. Kent E Vrana (Department of Pharmacology, Penn State University College of Medicine) for helping the design of this study. This project is funded, under grants with the Grant Number K18CA151798 from the National Cancer Institute, the Barsumian Trust and the Melanoma Research Foundation (J. Song).

<ol>
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No conflicts of interest declared.

For ACT-based therapies, the in vitro generation of large numbers of highly reactive Ag-specific T cells for in vivo re-infusion is an optimal approach. Although our in vitro method gives rise of functional T cells from iPS cells, large numbers of iPS cell-derived cells die in four weeks, especially in the fourth week. We conclude that the survival signals from Notch signaling mediated by the DL1 as well as IL-7 and FLt3L are not sufficient to maintain the survival of iPS cell-derived prog...

<table><tbody><tr><td>C57BL/6J mice</td><td>Jackson Laboratory</td><td>000664</td><td></td></tr><tr><td>B6.129S7-&lt;em&gt;Rag1&lt;sup&gt;tm1Mom&lt;/sup&gt;/J&lt;/em&gt;</td><td>Jackson Laboratory</td><td>002216</td><td></td></tr><tr><td>Anti-CD3 (2C11) antibody</td><td>BD Biosciences</td><td>553058</td><td></td></tr><tr><td>Anti-CD28 (37.51) antibody</td><td>BD Biosciences</td><td>553295</td><td></td></tr><tr><td>Anti-CD3 (17A2) antibody </td><td>BioLegend</td><td>100202</td><td></td></tr><tr><td>Anti-CD4 (GK1.5) antibody</td><td>BioLegend</td><td>100417</td><td></td></tr><tr><td>Anti-CD8 (53-6.7) antibody</td><td>BioLegend</td><td>100714</td><td></td></tr><tr><td>Anti-CD25 (3C7) antibody</td><td>BioLegend</td><td>101912</td><td></td></tr><tr><td>Anti-CD44 (1M7) antibody</td><td>BioLegend</td><td>103012</td><td></td></tr><tr><td>Anti-CD117 (2B8) antibody</td><td>BioLegend</td><td>105812</td><td></td></tr><tr><td>Anti-TCR-&amp;#946; (H57597) antibody</td><td>BioLegend</td><td>109220</td><td></td></tr><tr><td>Anti-IL-2 (JES6-5H4) antibody</td><td>BioLegend</td><td>503810</td><td></td></tr><tr><td>Anti-IFN-&amp;#947; (XMG1.2) antibody</td><td>BioLegend</td><td>505822</td><td></td></tr><tr><td>DMEM</td><td>Invitrogen</td><td>ABCD1234</td><td></td></tr><tr><td>&amp;alpha;-MEM</td><td>Invitrogen</td><td>A10490-01</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH3007.01</td><td></td></tr><tr><td>Brefeldin A</td><td>Sigma-Aldrich</td><td>B7651</td><td></td></tr><tr><td>Polybrene</td><td>Sigma-Aldrich</td><td>107689</td><td></td></tr><tr><td>GeneJammer</td><td>Integrated Sciences</td><td>204130</td><td></td></tr><tr><td>RNA kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>DNA kit</td><td>Qiagen</td><td>69504</td><td></td></tr><tr><td>CD8 Isolation Kit</td><td>Miltenyi Biotec</td><td>130-095-236</td><td></td></tr><tr><td>ACK lysis buffer</td><td>Lonza Inc.</td><td>10-548E</td><td></td></tr><tr><td>mFlt-3L</td><td>PeproTech Inc</td><td>250-31L</td><td></td></tr><tr><td>mIL-7</td><td>PeproTech Inc</td><td>217-17</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G9391</td><td></td></tr><tr><td>FITC-anti-OVA antibody</td><td>Rockland Immunochemicals</td><td>200-4233</td><td></td></tr><tr><td>Permeabilization buffer</td><td>Biolegend</td><td>421002</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>F8775</td><td></td></tr><tr><td>0.4 &amp;mu;m filter</td><td>EMD Millipore</td><td></td><td></td></tr><tr><td>Moflo Cell Sorter</td><td>Dako</td><td></td><td></td></tr><tr><td>Calibur Flow Cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>LSR II Flow Cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Mouse restrainer</td><td>Braintree Scientific, Inc.</td><td></td><td></td></tr></tbody></table>

directed differentiation of induced pluripotent stem cells towards t lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Watch this Scientific Journal Video about Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes at JoVE.com

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

18.5K Views.  Pennsylvania State University College of Medicine. Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Video: Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

Developmental Biology

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 ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Cancer Research

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of liver cancer. This is a major concern, especially in developing countries. Failure of the immune system to mount an effective response against HBV leads to chronic infection. Although HBV vaccine is present and novel antiviral medicines are being created, eradication of virus-reservoir cells remains a major health topic. Described here is a method for the generation of viral antigen (Ag) -specific CD8+ cytotoxic T lymphocytes (CTLs) derived from induced pluripotent stem cells (iPSCs) (i.e., iPSC-CTLs), which have the ability to suppress HBV replication. HBV replication is efficiently induced in mice through hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver. Then, HBV surface Ag-specific mouse iPSC-CTLs are adoptively transferred, which greatly suppresses HBV replication in the liver and blood as well as prevents HBV surface Ag expression in hepatocytes. This method demonstrates HBV replication in mice after hydrodynamic injection and that stem cell-derived viral Ag-specific CTLs can suppress HBV replication. This protocol provides a useful method for HBV immunotherapy.

Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of ...

Generating Single Positive CD8 T Cells from Induced Pluripotent Stem Cells

Source: Good, M. L., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59997">Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells.</a> J. Vis. Exp. (2019).
This video demonstrates an assay to generate tumor antigen-specific induced pluripotent stem cell (iPSC)-derived CD8&#945;&#946;+ T Cells. Tumor antigen-specific iPSCs are grown on a stromal cell monolayer and differentiated into hematopoietic progenitor cells (HPCs). The HPCs are differentiated into CD4+ CD8+ double-positive T cells using a differentiation medium. These cells are then stimulated to generate single-positive T cells expressing CD8.

1. Culturing Human iPSCs (hiPSCs) on Mouse Embryonic Fibroblasts (MEF)
NOTE: Alternative methods for culturing hiPSCs can also be used, including but not ...

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Cellular Immunotherapy

Differentiation of Human-Induced Pluripotent Stem Cells into Macrophages

To begin the differentiation process on day zero, add 2.25 milliliters of stage 1 media into two wells of an ultra-low attachment 6-well plate. Then, for 180% confluent well of iPSCs in a 6-well plate, replace the maintenance media with 1.5 milliliters of stage 1 media. Cut colonies using a new cell passaging tool. Using a pipette, transfer the cut colonies into the two wells of the ultra-low attachment 6-well plate.
On day 2, adjust the cytokine concentrations by adding 0.5 milliliters of hESC-SFM media to the 6-well well plate containing the day two embryoid bodies.
On day 4, coat four wells of a 6-well tissue culture plate with 0.1% gelatin. After incubating for at least 10 minutes, remove the gelatin, and add 2.5 milliliters of stage 2 media.
Collect formed embryoid bodies from the two wells of a 6-well plate, and place them in a 50-milliliter centrifuge tube. Allow them to settle at the bottom of the tube by gravity. Then, carefully aspirate the media from the tube, and resuspend the embryoid bodies in 2 milliliters of stage 2 media.
Transfer 10 to 15 embryoid bodies to a gelatin-coated well, containing 2.5 milliliters of stage 2 media, and incubate at 37 degrees Celsius with 5% carbon dioxide. For two to three weeks, change the media every three to four days.
Plating of the EBs is crucial. Having fewer than 10 or more than 15 EBs, or having them unevenly distributed, can lead to low yields of macrophages.
After two to three weeks, the embryoid bodies start releasing non-adherent hematopoietic cells into suspension. Using a 40-micrometer strainer, collect these cells into a 50-milliliter tube, and then, replenish the media. Centrifuge the suspension of hematopoietic cells at 200 x g for three minutes.
Resuspend the hematopoietic cells in stage 3 media. Then, plate the cells at a density of 0.2 x 106 per milliliter. Change the stage 3 media every five days.
After nine to 11 days the macrophages will be fully differentiated and fully functional.

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Summary

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