Competing dynamic gene regulatory networks involved in fibroblast reprogramming to haematopoietic progenitor cells [ATAC-Seq]

Direct reprogramming of somatic cells offers a potentially safer therapeutic approach to generate patient-specific haematopoietic cells. However, this strategy is limited by stochasticity of reprogramming. Investigating the gene-regulatory networks involved during reprogramming would help generate functionally relevant cells in adequate numbers. To address this, we developed an inducible system to reprogram fibroblasts to HSPCs by ectopically expressing the two transcription factors SCL and LMO2. Transcriptome and epigenome analysis at different stages of reprogramming revealed uniform silencing of fibroblast genes and upregulation of the haemogenic endothelial program. Integrated analysis suggested that TFs FLI1, GATA1/2, KLF14 are direct targets of SCL/LMO2, which subsequently induce the haematopoietic programme. Single cell RNA-Seq revealed conflicting and competing fate decisions at intermediate stages of reprogramming. Inhibiting signalling pathways associated with competing neuronal fate significantly enhanced haematopoietic reprogramming efficiency. In conclusion, this study identifies early/intermediate reprogramming events and associated pathways that were targeted to improve reprogramming efficiency. To investigate the mechanisms of reprogramming mouse embryonic fibroblasts (MEFs) to blood progenitors, we have performed ATAC-Seq analysis on reprogramming cells (MEFs treated with doxycycline) harvested on days 2 and 4 of reprogramming. MEFs harvested at day 2 (without doxycycline) were used as controls. MEFs from three different embryos were used as biological replicates.