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0 Q&A 1483 Views Nov 20, 2023

Cancer cells evade the immune system by downregulating antigen presentation. Although immune checkpoint inhibitors (ICI) and adoptive T-cell therapies revolutionized cancer treatment, their efficacy relies on the intrinsic immunogenicity of tumor cells and antigen presentation by dendritic cells. Here, we describe a protocol to directly reprogram murine and human cancer cells into tumor-antigen-presenting cells (tumor-APCs), using the type 1 conventional dendritic cell (cDC1) transcription factors PU.1, IRF8, and BATF3 delivered by a lentiviral vector. Tumor-APCs acquire a cDC1 cell-like phenotype, transcriptional and epigenetic programs, and function within nine days (Zimmermannova et al., 2023). Tumor-APCs express the hematopoietic marker CD45 and acquire the antigen presentation complexes MHC class I and II as well as co-stimulatory molecules required for antigen presentation to T cells, but do not express high levels of negative immune checkpoint regulators. Enriched tumor-APCs present antigens to Naïve CD8+ and CD4+ T cells, are targeted by activated cytotoxic T lymphocytes, and elicit anti-tumor responses in vivo. The tumor-APC reprogramming protocol described here provides a simple and robust method to revert tumor evasion mechanisms by increasing antigen presentation in cancer cells. This platform has the potential to prime antigen-specific T-cell expansion, which can be leveraged for developing new cancer vaccines, neoantigen discovery, and expansion of tumor-infiltrating lymphocytes.


Key features

• This protocol describes the generation of antigen-presenting cells from cancer cells by direct reprogramming using lineage-instructive transcription factors of conventional dendritic cells type I.

• Verification of reprogramming efficiency by flow cytometry and functional assessment of tumor-APCs by antigen presentation assays.

0 Q&A 3913 Views Oct 20, 2020
Induced pluripotent stem cells (iPSCs) are genetically reprogrammed somatic cells that exhibit features identical to those of embryonic stem cells (ESCs). Multiple approaches are available to derive iPSCs, among which the Sendai virus is the most effective at reprogramming different cell types. Here we describe a rapid, efficient, safe, and reliable approach to reprogram human fibroblasts into iPSCs that are compatible with future iPSCs uses such as genome editing and differentiation to a transplantable cell type.
0 Q&A 3705 Views Oct 5, 2019
The elucidation of the molecular mechanisms of long-term synaptic plasticity has been hindered by both the compensation that can occur after chronic loss of the core plasticity molecules and by ex vivo conditions that may not reproduce in vivo plasticity. Here we describe a novel method to rapidly suppress gene expression by antisense oligodeoxynucleotides (ODNs) applied to rodent brain slices in an “Oslo-type” interface chamber. The method has three advantageous features: 1) rapid blockade of new synthesis of the targeted proteins that avoids genetic compensation, 2) efficient oxygenation of the brain slice, which is critical for reproducing in vivo conditions of long-term synaptic plasticity, and 3) a recirculation system that uses only small volumes of bath solution (< 5 ml), reducing the amount of reagents required for long-term experiments lasting many hours. The method employs a custom-made recirculation system involving piezoelectric micropumps and was first used for the acute translational blockade of protein kinase Mζ (PKMζ) synthesis during long-term potentiation (LTP) by Tsokas et al., 2016. In that study, applying antisense-ODN rapidly prevents the synthesis of PKMζ and blocks late-LTP without inducing the compensation by other protein kinase C (PKC) isoforms that occurs in PKCζ/PKMζ knockout mice. In addition, we show that in a low-oxygenation submersion-type chamber, applications of the atypical PKC inhibitor, zeta inhibitory peptide (ZIP) can result in unstable baseline synaptic transmission, but in the high-oxygenation, "Oslo-type" interface electrophysiology chamber, the drug reverses late-LTP without affecting baseline synaptic transmission. This comparison reveals that the interface chamber, but not the submersion chamber, reproduces the effects of ZIP in vivo. Therefore, the protocol combines the ability to acutely block new synthesis of specific proteins for the study of long-term synaptic plasticity, while maintaining properties of synaptic transmission that reproduce in vivo conditions relevant for long-term memory.
1 Q&A 10129 Views Jan 20, 2018
Primary mature hepatocytes (MHs) or their progenitor cells are candidate cell sources for cell transplantation therapy in severe liver diseases. However, stable culture of these cells or generation of equivalent cells from pluripotent stem cells has been limited. Using a cocktail of small molecules that we previously found useful in stable culture of multiple types of stem/progenitor cells, we recently established a novel method to generate bipotent liver progenitor cells, named chemically induced liver progenitors (CLiPs), from adult rat MHs. Here, we describe a detailed protocol for the induction of rat CLiPs. We first describe the method to isolate primary rat MHs and then describe how to induce CLiPs from these MHs. In addition, we describe a method to evaluate the bipotentiality of generated CLiPs to differentiate into hepatocytes and biliary epithelial cells. We also describe how to establish stable CLiPs through long-term culture with detailed example data. Primary CLiPs can be generated within 2 weeks, and stable CLiPs, which undergo 10 passages, can be established within 2.5-4 months with batch-to-batch variability.



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