Dynamic DNA methylation reveals novel cis-regulatory elements in mouse hematopoiesis.

Differentiation of hematopoietic stem and progenitor cells to terminally differentiated immune cells is accompanied by large-scale remodeling of the DNA methylation landscape. Although significant insights into the molecular mechanisms of hematopoietic tissue regeneration were derived from mouse models, profiling of DNA methylation has been hampered by high cost or low resolution using available methods. The recent development of the Infinium Mouse Methylation BeadChip (MMBC) array facilitates methylation profiling of the mouse genome at a single CpG resolution at affordable cost. We extended the RnBeads package to provide a computational framework for the analysis of MMBC data. This framework was applied to a newly generated reference map of mouse hematopoiesis encompassing nine different cell types. Analysis of dynamically regulated CpG sites showed progressive and unidirectional DNA methylation changes from hematopoietic stem and progenitor cells to differentiated hematopoietic cells and allowed the identification of lineage- and cell type-specific DNA methylation programs. Comparison with previously published catalogs of cis-regulatory elements (CREs) revealed 12,856 novel putative CREs that were dynamically regulated by DNA methylation (mdCREs). These mdCREs were predominantly associated with patterns of cell type-specific DNA hypomethylation and could be identified as epigenetic control regions regulating the expression of key hematopoietic genes during differentiation. In summary, we established an analysis pipeline for MMBC data sets and provide a DNA methylation atlas of mouse hematopoiesis. This resource allowed us to identify novel putative CREs involved in hematopoiesis and will serve as a platform to study epigenetic regulation of normal and malignant hematopoiesis.

Classical and Non-classical Fibrosis Phenotypes Are Revealed by Lung and Cardiac Like Microvascular Tissues On-Chip.

Fibrosis, a hallmark of many cardiac and pulmonary diseases, is characterized by excess deposition of extracellular matrix proteins and increased tissue stiffness. This serious pathologic condition is thought to stem majorly from local stromal cell activation. Most studies have focused on the role of fibroblasts; however, the endothelium has been implicated in fibrosis through direct and indirect contributions. Here, we present a 3D vascular model to investigate vessel-stroma crosstalk in normal conditions and following induced fibrosis. Human-induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) are co-cultured with (and without) primary human cardiac and lung fibroblasts (LFs) in a microfluidic device to generate perfusable microvasculature in cardiac- and pulmonary-like microenvironments. Endothelial barrier function, vascular morphology, and matrix properties (stiffness and diffusivity) are differentially impacted by the presence of stromal cells. These vessels (with and without stromal cells) express inflammatory cytokines, which could induce a wound-healing state. Further treatment with transforming growth factor-ß (TGF-ß) induced varied fibrotic phenotypes on-chip, with LFs resulting in increased stiffness, lower MMP activity, and increased smooth muscle actin expression. Taken together, our work demonstrates the strong impact of stromal-endothelial interactions on vessel formation and extravascular matrix regulation. The role of TGF-ß is shown to affect co-cultured microvessels differentially and has a severe negative impact on the endothelium without stromal cell support. Our human 3D in vitro model has the potential to examine anti-fibrotic therapies on patient-specific hiPSCs in the future.