Portilho, N. A. | Search bioRxiv

bioRxiv2021

HSC-independent definitive hematopoietic cells persist into adult life

The stem cell theory that all blood cells are derived from hematopoietic stem cell (HSC) is a central dogma in hematology. However, various types of blood cells are already produced from hemogenic endothelial cells (HECs) before the first HSCs appear at embryonic day (E)11 in the mouse embryo. This early blood cell production from HECs, called HSC-independent hematopoiesis, includes primitive and definitive erythromyeloid progenitors that transiently support fetal blood homeostasis until HSC-derived hematopoiesis is established. Lymphoid potential has traditionally been detected in the extra-embryonic yolk sac (YS) and/or embryos before HSC emergence, but the actual presence of lymphoid progenitors at this stage remains unknown. In addition, whether HSCs in the fetal liver are the main source of innate-like B-1a cells has been controversial. Here, using complementary lineage tracing mouse models, we show that HSC-independent multipotent progenitors (MPPs) and HSC-independent adoptive B-lymphoid progenitors persist into adult life. Furthermore, HSCs minimally contribute to the peritoneal B-1a cell pool; most B-1a cells are originated directly from ECs in the YS and embryo and HSC-independent for life. Our discovery of extensive HSC-independent MPP and B-lymphoid progenitors in adults attests to the complex blood developmental dynamics through embryo to adult that underpin the immune system and challenges the paradigm of HSC theory in hematology.

developmental biology↗

bioRxiv2021

The DNMT1 inhibitor GSK-3484862 mediates global demethylation in murine embryonic stem cells

BackgroundDNA methylation plays an important role in regulating gene expression in mammals. The covalent DNMT1 inhibitors 5-azacytidine and decitabine are widely used in research to reduce DNA methylation levels, but they impart severe cytotoxicity which limits their demethylation capability and confounds interpretation of experiments. Recently, a non-covalent inhibitor of DNMT1 called GSK-3484862 was developed by GlaxoSmithKline. We sought to determine whether GSK-3484862 can induce demethylation more effectively than 5-azanucleosides. Murine embryonic stem cells (mESC) are an ideal cell type in which to conduct such experiments, as they have a high degree of DNA methylation but tolerate dramatic methylation loss. ResultsWe determined the cytotoxicity and optimal concentration of GSK-3484862 by treating wild-type (WT) or Dnmt1/3a/3b triple knockout (TKO) mESC with different concentrations of the compound, which was obtained from two commercial sources. Concentrations of 10 {micro}M or below were readily tolerated for 14 days of culture. Known DNA methylation targets such as germline genes and GLN-family transposons were upregulated within two days of the start of GSK-3484862 treatment. By contrast, 5-azacytidine and decitabine induced weaker upregulation of methylated genes and extensive cell death. Whole genome bisulfite sequencing (WGBS) showed that treatment with GSK-3484862 induced dramatic DNA methylation loss, with global CpG methylation levels falling from near 70% in WT mESC to less than 18% after 6 days of treatment with GSK-3484862, similar to the methylation level observed in Dnmt1 deficient mESCs. ConclusionsGSK-3484862 mediates striking demethylation in mESCs with minimal non-specific toxicity.

molecular biology↗