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  • Direct Reprogramming of Mouse Embryonic Fibroblasts to Conventional Type 1 Dendritic Cells by Enforced Expression of Transcription Factors.

Direct Reprogramming of Mouse Embryonic Fibroblasts to Conventional Type 1 Dendritic Cells by Enforced Expression of Transcription Factors.

Bio-protocol (2021-03-05)
Fábio F Rosa, Cristiana F Pires, Olga Zimmermannova, Carlos-Filipe Pereira
ABSTRACT

Ectopic expression of transcription factor combinations has been recently demonstrated to reprogram differentiated somatic cells towards the dendritic cell (DC) lineage without reversion to a multipotent state. DCs have the ability to induce potent and long-lasting adaptive immune responses. In particular, conventional type 1 DCs (cDC1s) excel on antigen cross-presentation, a critical step for inducing CD8+ T cell cytotoxic responses. The rarity of naturally occurring cDC1s and lack of in vitro methodologies for the generation of pure cDC1 populations strongly hinders the study of cDC1 lineage specification and function. Here, we describe a protocol for the generation of induced DCs (iDCs) by lentiviral-mediated expression of the transcription factors PU.1, IRF8 and BATF3 in mouse embryonic fibroblasts. iDCs acquire DC morphology, cDC1 phenotype and transcriptional signatures within 9 days. iDCs generated with this protocol acquire functional ability to respond to inflammatory stimuli, engulf dead cells, process and cross-present antigens to CD8+ T cells. DC reprogramming provides a simple and tractable system to generate high numbers of cDC1-like cells for high content screening, opening new avenues to better understand cDC1 specification and function. In the future, faithful induction of cDC1 fate in fibroblasts may lead to the generation of patient-specific DCs for vaccination.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Gelatin from porcine skin, powder, gel strength ~300 g Bloom, Type A, BioReagent, suitable for electrophoresis, suitable for cell culture
Sigma-Aldrich
Doxycycline hyclate
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Hexadimethrine bromide, ≥94% (titration)
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Micro-dissecting scissors, size 4 1/4 in., straight, sharp point stainless steel
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DAPI, for nucleic acid staining
Sigma-Aldrich
Dimethyl sulfoxide, for molecular biology
Sigma-Aldrich
Polyethylenimine, branched, average Mw ~25,000 by LS, average Mn ~10,000 by GPC, branched