Whole-Genome Sequencing Can Identify Clinically Relevant Variants from a Single Sub-Punch of a Dried Blood Spot Specimen.

The collection of dried blood spots (DBS) facilitates newborn screening for a variety of rare, but very serious conditions in healthcare systems around the world. Sub-punches of varying sizes (1.5-6 mm) can be taken from DBS specimens to use as inputs for a range of biochemical assays. Advances in DNA sequencing workflows allow whole-genome sequencing (WGS) libraries to be generated directly from inputs such as peripheral blood, saliva, and DBS. We compared WGS metrics obtained from libraries generated directly from DBS to those generated from DNA extracted from peripheral blood, the standard input for this type of assay. We explored the flexibility of DBS as an input for WGS by altering the punch number and size as inputs to the assay. We showed that WGS libraries can be successfully generated from a variety of DBS inputs, including a single 3 mm or 6 mm diameter punch, with equivalent data quality observed across a number of key metrics of importance in the detection of gene variants. We observed no difference in the performance of DBS and peripheral-blood-extracted DNA in the detection of likely pathogenic gene variants in samples taken from individuals with cystic fibrosis or phenylketonuria. WGS can be performed directly from DBS and is a powerful method for the rapid discovery of clinically relevant, disease-causing gene variants.

Different niches for stem cells carrying the same oncogenic driver affect pathogenesis and therapy response in myeloproliferative neoplasms.

Aging facilitates the expansion of hematopoietic stem cells (HSCs) carrying clonal hematopoiesis-related somatic mutations and the development of myeloid malignancies, such as myeloproliferative neoplasms (MPNs). While cooperating mutations can cause transformation, it is unclear whether distinct bone marrow (BM) HSC-niches can influence the growth and therapy response of HSCs carrying the same oncogenic driver. Here we found different BM niches for HSCs in MPN subtypes. JAK-STAT signaling differentially regulates CDC42-dependent HSC polarity, niche interaction and mutant cell expansion. Asymmetric HSC distribution causes differential BM niche remodeling: sinusoidal dilation in polycythemia vera and endosteal niche expansion in essential thrombocythemia. MPN development accelerates in a prematurely aged BM microenvironment, suggesting that the specialized niche can modulate mutant cell expansion. Finally, dissimilar HSC-niche interactions underpin variable clinical response to JAK inhibitor. Therefore, HSC-niche interactions influence the expansion rate and therapy response of cells carrying the same clonal hematopoiesis oncogenic driver.

A cholinergic neuroskeletal interface promotes bone formation during postnatal growth and exercise.

The autonomic nervous system is a master regulator of homeostatic processes and stress responses. Sympathetic noradrenergic nerve fibers decrease bone mass, but the role of cholinergic signaling in bone has remained largely unknown. Here, we describe that early postnatally, a subset of sympathetic nerve fibers undergoes an interleukin-6 (IL-6)-induced cholinergic switch upon contacting the bone. A neurotrophic dependency mediated through GDNF-family receptor-α2 (GFRα2) and its ligand, neurturin (NRTN), is established between sympathetic cholinergic fibers and bone-embedded osteocytes, which require cholinergic innervation for their survival and connectivity. Bone-lining osteoprogenitors amplify and propagate cholinergic signals in the bone marrow (BM). Moderate exercise augments trabecular bone partly through an IL-6-dependent expansion of sympathetic cholinergic nerve fibers. Consequently, loss of cholinergic skeletal innervation reduces osteocyte survival and function, causing osteopenia and impaired skeletal adaptation to moderate exercise. These results uncover a cholinergic neuro-osteocyte interface that regulates skeletogenesis and skeletal turnover through bone-anabolic effects.

Cholinergic signals preserve haematopoietic stem cell quiescence during regenerative haematopoiesis.

The sympathetic nervous system has been evolutionary selected to respond to stress and activates haematopoietic stem cells via noradrenergic signals. However, the pathways preserving haematopoietic stem cell quiescence and maintenance under proliferative stress remain largely unknown. Here we found that cholinergic signals preserve haematopoietic stem cell quiescence in bone-associated (endosteal) bone marrow niches. Bone marrow cholinergic neural signals increase during stress haematopoiesis and are amplified through cholinergic osteoprogenitors. Lack of cholinergic innervation impairs balanced responses to chemotherapy or irradiation and reduces haematopoietic stem cell quiescence and self-renewal. Cholinergic signals activate α7 nicotinic receptor in bone marrow mesenchymal stromal cells leading to increased CXCL12 expression and haematopoietic stem cell quiescence. Consequently, nicotine exposure increases endosteal haematopoietic stem cell quiescence in vivo and impairs hematopoietic regeneration after haematopoietic stem cell transplantation in mice. In humans, smoking history is associated with delayed normalisation of platelet counts after allogeneic haematopoietic stem cell transplantation. These results suggest that cholinergic signals preserve stem cell quiescence under proliferative stress.

Bone Marrow Mesenchymal Stem Cells Support Acute Myeloid Leukemia Bioenergetics and Enhance Antioxidant Defense and Escape from Chemotherapy.

Like normal hematopoietic stem cells, leukemic stem cells depend on their bone marrow (BM) microenvironment for survival, but the underlying mechanisms remain largely unknown. We have studied the contribution of nestin+ BM mesenchymal stem cells (BMSCs) to MLL-AF9-driven acute myeloid leukemia (AML) development and chemoresistance in vivo. Unlike bulk stroma, nestin+ BMSC numbers are not reduced in AML, but their function changes to support AML cells, at the expense of non-mutated hematopoietic stem cells (HSCs). Nestin+ cell depletion delays leukemogenesis in primary AML mice and selectively decreases AML, but not normal, cells in chimeric mice. Nestin+ BMSCs support survival and chemotherapy relapse of AML through increased oxidative phosphorylation, tricarboxylic acid (TCA) cycle activity, and glutathione (GSH)-mediated antioxidant defense. Therefore, AML cells co-opt energy sources and antioxidant defense mechanisms from BMSCs to survive chemotherapy.

Neuronal regulation of bone marrow stem cell niches.

The bone marrow (BM) is the primary site of postnatal hematopoiesis and hematopoietic stem cell (HSC) maintenance. The BM HSC niche is an essential microenvironment which evolves and responds to the physiological demands of HSCs. It is responsible for orchestrating the fate of HSCs and tightly regulates the processes that occur in the BM, including self-renewal, quiescence, engraftment, and lineage differentiation. However, the BM HSC niche is disturbed following hematological stress such as hematological malignancies, ionizing radiation, and chemotherapy, causing the cellular composition to alter and remodeling to occur. Consequently, hematopoietic recovery has been the focus of many recent studies and elucidating these mechanisms has great biological and clinical relevance, namely to exploit these mechanisms as a therapeutic treatment for hematopoietic malignancies and improve regeneration following BM injury. The sympathetic nervous system innervates the BM niche and regulates the migration of HSCs in and out of the BM under steady state. However, recent studies have investigated how sympathetic innervation and signaling are dysregulated under stress and the subsequent effect they have on hematopoiesis. Here, we provide an overview of distinct BM niches and how they contribute to HSC regulatory processes with a particular focus on neuronal regulation of HSCs under steady state and stress hematopoiesis.

Characterisation of Cultured Mesothelial Cells Derived from the Murine Adult Omentum.

The human omentum has been long regarded as a healing patch, used by surgeons for its ability to immunomodulate, repair and vascularise injured tissues. A major component of the omentum are mesothelial cells, which display some of the characteristics of mesenchymal stem/stromal cells. For instance, lineage tracing studies have shown that mesothelial cells give rise to adipocytes and vascular smooth muscle cells, and human and rat mesothelial cells have been shown to differentiate into osteoblast- and adipocyte-like cells in vitro, indicating that they have considerable plasticity. However, so far, long-term cultures of mesothelial cells have not been successfully established due to early senescence. Here, we demonstrate that mesothelial cells isolated from the mouse omentum could be cultured for more than 30 passages. While epithelial markers were downregulated over passages in the mesothelial cells, their mesenchymal profile remained unchanged. Early passage mesothelial cells displayed clonogenicitiy, expressed several stem cell markers, and up to passage 5 and 13, respectively, could differentiate along the adipogenic and osteogenic lineages, demonstrating stem/progenitor characteristics and differentiation potential.