Mutant KLF1 in Adult Anemic Nan Mice Leads to Profound Transcriptome Changes and Disordered Erythropoiesis.

Anemic Nan mice carry a mutation (E339D) in the second zinc finger of erythroid transcription factor KLF1. Nan-KLF1 fails to bind a subset of normal KLF1 targets and ectopically binds a large set of genes not normally engaged by KLF1, resulting in a corrupted fetal liver transcriptome. Here, we performed RNAseq using flow cytometric-sorted spleen erythroid precursors from adult Nan and WT littermates rendered anemic by phlebotomy to identify global transcriptome changes specific to the Nan Klf1 mutation as opposed to anemia generally. Mutant Nan-KLF1 leads to extensive and progressive transcriptome corruption in adult spleen erythroid precursors such that stress erythropoiesis is severely compromised. Terminal erythroid differentiation is defective in the bone marrow as well. Principle component analysis reveals two major patterns of differential gene expression predicting that defects in basic cellular processes including translation, cell cycle, and DNA repair could contribute to disordered erythropoiesis and anemia in Nan. Significant erythroid precursor stage specific changes were identified in some of these processes in Nan. Remarkably, however, despite expression changes in large numbers of associated genes, most basic cellular processes were intact in Nan indicating that developing red cells display significant physiological resiliency and establish new homeostatic set points in vivo

Neomorphic effects of the neonatal anemia (Nan-Eklf) mutation contribute to deficits throughout development

Transcription factor control of cell-specific downstream targets can be significantly altered when the controlling factor is mutated. We show that the semi-dominant neonatal anemia (Nan) mutation in the EKLF/KLF1 transcription factor leads to ectopic expression of proteins that are not normally expressed in the red blood cell, leading to systemic effects that exacerbate the intrinsic anemia in the adult and alter correct development in the early embryo. Even when expressed as a heterozygote, the Nan-EKLF protein accomplishes this by direct binding and aberrant activation of genes encoding secreted factors that exert a negative effect on erythropoiesis and iron use. Our data form the basis for a novel mechanism of physiological deficiency that is relevant to human dyserythropoietic anemia and likely other disease states

Fibroblast growth factor-1 (FGF-1) promotes adipogenesis by downregulation of carboxypeptidase A4 (CPA4) – a negative regulator of adipogenesis implicated in the modulation of local and systemic insulin sensitivity

Fibroblast growth factor-1 (FGF-1) promotes differentiation of human preadipocytes into mature adipocytes via modulation of a BMP and Activin Membrane-Bound Inhibitor (BAMBI)/Peroxisome proliferator-activated receptor (PPAR?)-dependent network. Here, we combined transcriptomic and functional investigations to identify novel downstream effectors aligned with complementary analyses of gene expression in human adipose tissue to explore relationships with insulin sensitivity. RNA-Seq and qRT-PCR analysis revealed significant down-regulation of carboxypeptidase A4 (CPA4) following FGF-1 treatment or induction of differentiation of human preadipocytes in a BAMBI/PPAR?-independent manner. siRNA-mediated knockdown of CPA4 resulted in enhanced differentiation of human preadipocytes. Furthermore, expression of CPA4 in subcutaneous adipose tissue correlated negatively with indices of local and systemic (liver and muscle) insulin sensitivity. These results identify CPA4 as a negative regulator of adipogenesis that is down-regulated by FGF-1 and a putative deleterious modulator of local and systemic insulin sensitivity. Further investigations are required to define the molecular mechanism(s) involved and potential therapeutic opportunities

Non-Water-Suppressed 1H MR Spectroscopy with Orientational Prior Knowledge Shows Potential for Separating Intra- and Extramyocellular Lipid Signals in Human Myocardium

Conditions such as type II diabetes are linked with elevated lipid levels in the heart, and significantly increased risk of heart failure; however, metabolic processes underlying the development of cardiac disease in type II diabetes are not fully understood. Here we present a non-invasive method for in vivo investigation of cardiac lipid metabolism: namely, IVS-McPRESS. This technique uses metabolite-cycled, non-water suppressed 1H cardiac magnetic resonance spectroscopy with prospective and retrospective motion correction. High-quality IVS-McPRESS data acquired from healthy volunteers allowed us to investigate the frequency shift of extramyocellular lipid signals, which depends on the myocardial fibre orientation. Assuming consistent voxel positioning relative to myofibres, the myofibre angle with the magnetic field was derived from the voxel orientation. For separation and individual analysis of intra- and extramyocellular lipid signals, the angle myocardial fibres in the spectroscopy voxel take with the magnetic field should be within ±24.5°. Metabolite and lipid concentrations were analysed with respect to BMI. Significant correlations between BMI and unsaturated fatty acids in intramyocellular lipids, and methylene groups in extramyocellular lipids were found. The proposed IVS-McPRESS technique enables non-invasive investigation of cardiac lipid metabolism and may thus be a useful tool to study healthy and pathological conditions

Human Epidermal Neural Crest Stem Cells (hEPI-NCSC)—Characterization and Directed Differentiation into Osteocytes and Melanocytes

Here we describe the isolation, characterisation and ex-vivo expansion of human epidermal neural crest stem cells (hEPI-NCSC) and we provide protocols for their directed differentiation into osteocytes and melanocytes. hEPI-NCSC are neural crest-derived multipotent stem cells that persist into adulthood in the bulge of hair follicles. Multipotency and self-renewal were determined by in vitro clonal analyses. hEPI-NCSC generate all major neural crest derivatives, including bone/cartilage cells, neurons, Schwann cells, myofibroblasts and melanocytes. Furthermore, hEPI-NCSC express additional neural crest stem cell markers and global stem cell genes. To variable degrees and in a donor-dependent manner, hEPI-NCSC express the six essential pluripotency genes C-MYC, KLF4, SOX2, LIN28, OCT-4/POU5F1 and NANOG. hEPI-NCSC can be expanded ex vivo into millions of stem cells that remain mulitpotent and continue to express stem cell genes. The novelty of hEPI-NCSC lies in the combination of their highly desirable traits. hEPI-NCSC are embryonic remnants in a postnatal location, the bulge of hair follicles. Therefore they are readily accessible in the hairy skin by minimal invasive procedure. hEPI-NCSC are multipotent somatic stem cells that can be isolated reproducibly and with high yield. By taking advantage of their migratory ability, hEPI-NCSC can be isolated as a highly pure population of stem cells. hEPI-NCSC can undergo robust ex vivo expansion and directed differentiation. As somatic stem cells, hEPI-NCSC are conducive to autologous transplantation, which avoids graft rejection. Together, these traits make hEPI-NCSC novel and attractive candidates for future cell-based therapies and regenerative medicine