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RNA sequencing analyses of fused heterokaryons between mouse embryonic stem cells and human B lymphocytes collected at early timepoints after fusion

ABSTRACT: Tcf3 is an effector of Wnt canonical pathway and deletion of Tcf3 in mouse embryonic stem cells (mESCs) strongly enhances reprogramming efficiency after fusion. We fused murine Tcf3-/- mESCs with human B-lymphocytes using the Polyethylene glycol (PEG) reagent and isolated heterokaryons at different time points to analyze early reprogramming events starting from 4 hours after fusion. We performed a genome-wide expression profile of the heterokaryons by RNA-sequencing at a high resolution.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE114238 | GEO | 2020/11/12

REPOSITORIES: GEO

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Project description:Purpose: Current methods to repair ablation-type peripheral nerve injuries (PNIs) using peripheral nerve allografts (PNAs) often result in poor functional recovery due to immunological rejection as well as slow and inaccurate outgrowth of regenerating axon sprouts. In contrast, ablation-type PNIs repaired by PNAs using a multistep protocol, in which one step uses the membrane fusogen polyethylene glycol (PEG), permanently restore sciatic-mediated behaviors within weeks. Axons and cells within the PNA remain viable, even though outbred host and donor tissues are neither immunosuppressed nor tissue matched. PEG-fused PNAs exhibit significantly reduced T cell and macrophage infiltration, apoptosis, and expression of major histocompatibility complex I/II. In this study, we analyzed the coding transcriptome of PEG-fused PNAs to determine possible mechanisms underlying immunosuppression. Methods: Ablation-type sciatic PNIs in adult Sprague Dawley rats were repaired using PNAs and a PEG-fusion protocol combined with neurorrhaphy. Electrophysiological and behavioral recovery tests confirmed successful PEG-fusion of PNAs. RNA sequencing analyzed differential expression profiles of protein-coding genes between PEG-fused PNAs and NC PNAs (not treated with PEG) at 14d PO, along with Unoperated nerves. Sequencing results were validated by Quantitative Reverse Transcription PCR (RT-qPCR). Results: PEG-fused PNAs display significant downregulation of a great many gene transcripts associated with innate and adaptive allorejection responses. Many Schwann cell-associated transcripts are upregulated, and many cellular processes such as extracellular matrix remodeling, cell and tissue development are particularly enriched. Transcripts encoding several potentially immunosuppressive proteins (e.g. Thrombospondins 1 and 2) are upregulated in PEG-fused PNAs. Conclusions: This study is the first to characterize the coding transcriptome of PEG-fused PNAs, and identifies possible links between alterations of the extracellular matrix and suppression of the allorejection response. The results establish a molecular-basis to understand mechanisms underlying PEG-mediated immunosuppression.

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RNA sequencing analyses of fused heterokaryons between mouse embryonic stem cells and human B lymphocytes collected at early timepoints after fusion

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