DNA methylation rates scale with maximum lifespan across mammals

DNA methylation rates have previously been found to broadly correlate with maximum lifespan in mammals, yet no precise relationship has been observed. We developed a statistically robust framework to compare methylation rates at conserved age-related sites across mammals. We found that methylation rates negatively scale with maximum lifespan in both blood and skin. The emergence of explicit scaling suggests that methylation rates are, or are linked to, an evolutionary constraint on maximum lifespan acting across diverse mammalian lineages

Multiplexing for Oxidative Bisulfite Sequencing (oxBS-seq).

DNA modifications, especially methylation, are known to play a crucial part in many regulatory processes in the cell. Recently, 5-hydroxymethylcytosine (5hmC) was discovered, a DNA modification derived as an intermediate of 5-methylcytosine (5mC) oxidation. Efforts to gain insights into function of this DNA modification are underway and several methods were recently described to assess 5hmC levels using sequencing approaches. Here we integrate adaptation based multiplexing and high-efficiency library prep into the oxidative Bisulfite Sequencing (oxBS-seq) workflow reducing the starting amount and cost per sample to identify 5hmC levels genome-wide

Experimental design for single-cell RNA sequencing

Single-cell RNA sequencing (scRNA-seq) has opened new avenues for the characterization of heterogeneity in a large variety of cellular systems. As this is a relatively new technique, the field is fast evolving. Here, we discuss general considerations in experimental design and the two most popular approaches, plate-based Smart-Seq2 and microdroplet-based scRNA-seq at the example of 10x Chromium. We discuss advantages and disadvantages of both methods and point out major factors to consider in designing successful experiments

Purifying stem cell-derived red blood cells:a high-throughput label-free downstream processing strategy based on microfluidic spiral inertial separation and membrane filtration

Cell-based therapeutics, such as in vitro manufactured red blood cells (mRBC), are different to traditional biopharmaceutical products (the final product being the cells themselves as opposed to biological molecules such as proteins) and that presents a challenge of developing new robust and economically feasible manufacturing processes, especially for sample purification. Current purification technologies have limited throughput, rely on expensive fluorescent or magnetic immuno-labelling with a significant (up to 70%) cell loss and quality impairment. To address this challenge, previously characterised mechanical properties of umbilical cord blood CD34+ cells undergoing in vitro erythropoiesis were used to develop a mRBC purification strategy. The approach consists of two main stages: (1) a microfluidic separation using inertial focusing for deformability-based sorting of enucleated cells (mRBC) from nuclei and nucleated cells resulting in 70% purity and (2) membrane filtration to enhance the purity to 99%. Herein, we propose a new route for high-throughput (processing millions of cells /min and mls of medium /min) purification process for mRBC, leading to high mRBC purity while maintaining cells integrity and no alterations in their global gene expression profile. Further adaption of this separation approach offers a potential route for processing of a wide range of cellular products

Global reorganization of the nuclear landscape in senescent cells.

Cellular senescence has been implicated in tumor suppression, development, and aging and is accompanied by large-scale chromatin rearrangements, forming senescence-associated heterochromatic foci (SAHF). However, how the chromatin is reorganized during SAHF formation is poorly understood. Furthermore, heterochromatin formation in senescence appears to contrast with loss of heterochromatin in Hutchinson-Gilford progeria. We mapped architectural changes in genome organization in cellular senescence using Hi-C. Unexpectedly, we find a dramatic sequence- and lamin-dependent loss of local interactions in heterochromatin. This change in local connectivity resolves the paradox of opposing chromatin changes in senescence and progeria. In addition, we observe a senescence-specific spatial clustering of heterochromatic regions, suggesting a unique second step required for SAHF formation. Comparison of embryonic stem cells (ESCs), somatic cells, and senescent cells shows a unidirectional loss in local chromatin connectivity, suggesting that senescence is an endpoint of the continuous nuclear remodelling process during differentiation

Multi-layered spatial transcriptomics identify secretory factors promoting human hematopoietic stem cell development

Hematopoietic stem cells (HSCs) first emerge in the embryonic aorta-gonad-mesonephros (AGM) region. Studies of model organisms defined intersecting signaling pathways that converge to promote HSC emergence predominantly in the ventral domain of the dorsal aorta. Much less is known about mechanisms driving HSC development in humans. Here, to identify secreted signals underlying human HSC development, we combined spatial transcriptomics analysis of dorsoventral polarized signaling in the aorta with gene expression profiling of sorted cell populations and single cells. Our analysis revealed a subset of aortic endothelial cells with a downregulated arterial signature and a predicted lineage relationship with the emerging HSC/progenitor population. Analysis of the ventrally polarized molecular landscape identified endothelin 1 as an important secreted regulator of human HSC development. The obtained gene expression datasets will inform future studies on mechanisms of HSC development in vivo and on generation of clinically relevant HSCs in vitro

Clonal haematopoiesis of indeterminate potential: intersections between inflammation, vascular disease and heart failure

Ageing is a major risk factor for the development of cardiovascular disease (CVD) and cancer. Whilst the cumulative effect of exposure to conventional cardiovascular risk factors is important, recent evidence highlights clonal haematopoiesis of indeterminant potential (CHIP) as a further key risk factor. CHIP reflects the accumulation of somatic, potentially pro-leukaemic gene mutations within haematopoietic stem cells over time. The most common mutations associated with CHIP and CVD occur in genes that also play central roles in the regulation of inflammation. While CHIP carriers have a low risk of haematological malignant transformation (<1% per year), their relative risk of mortality is increased by 40% and this reflects an excess of cardiovascular events. Evidence linking CHIP, inflammation and atherosclerotic disease has recently become better defined. However, there is a paucity of information about the role of CHIP in the development and progression of heart failure, particularly heart failure with preserved ejection fraction (HFpEF). While systemic inflammation plays a role in the pathophysiology of both heart failure with reduced and preserved ejection fraction (EF), it may be of greater relevance in the pathophysiology of HFpEF, which is also strongly associated with ageing. This review describes CHIP and its pathogenetic links with ageing, inflammation and CVD, while providing insight into its putative role in HFpEF