Identification of the niche and phenotype of the first human hematopoietic stem cells

SummaryIn various vertebrate species, the dorsal aorta (Ao) is the site of specification of adult hematopoietic stem cells (HSCs). It has been observed that the upregulation of essential hematopoietic transcription factors and the formation of specific intra-aortic hematopoietic cell clusters occur predominantly in the ventral domain of the Ao (AoV). In the mouse, the first HSCs emerge in the AoV. Here, we demonstrate that in the human embryo the first definitive HSCs also emerge asymmetrically and are localized to the AoV, which thus identifies a functional niche for developing human HSCs. Using magnetic cell separation and xenotransplantations, we show that the first human HSCs are CD34+VE-cadherin+CD45+C-KIT+THY-1+Endoglin+RUNX1+CD38−/loCD45RA−. This population harbors practically all committed hematopoietic progenitors and is underrepresented in the dorsal domain of the Ao (AoD) and urogenital ridges (UGRs). The present study provides a foundation for analysis of molecular mechanisms underpinning embryonic specification of human HSCs

Competence and competency in higher education, simple terms yet with complex meanings: Theoretical and practical issues for university teachers and assessors implementing Competency-Based Education (CBE)

There are different and conflicting definitions of competence, competency, and competency-based learning. Consequently, multiple interpretations and understandings are held by educators in respect of what these terms mean, when applied to their own teaching and assessment practices. Whilst UK and American universities are increasingly adopting competency-based learning, unless informed and considered discussion has taken place amongst staff about their individual understandings and interpretations, any development of new, competency-based assessment processes and procedures, is problematic. This paper provides an overview of the main issues involved in defining competency and assessing competence, along with recommendations for action. The purpose is to stimulate reflection and discussion, so that teaching staff can arrive at a common understanding and interpretation of the terminology of competency-based education, so that they may develop appropriate, authentic and fair assessment processes

Optimal ex vivo expansion of neutrophils from PBSC CD34+ cells by a combination of SCF, Flt3-L and G-CSF and its inhibition by further addition of TPO

<p>Abstract</p> <p>Background</p> <p>Autologous mobilised peripheral blood stem cell (PBSC) transplantation is now a standard approach in the treatment of haematological diseases to reconstitute haematopoiesis following myeloablative chemotherapy. However, there remains a period of severe neutropenia and thrombocytopenia before haematopoietic reconstitution is achieved. <it>Ex vivo </it>expanded PBSC have been employed as an adjunct to unmanipulated HSC transplantation, but have tended to be produced using complex cytokine mixtures aimed at multilineage (neutrophil and megakaryocyte) progenitor expansion. These have been reported to reduce or abrogate neutropenia but have little major effect on thrombocytopenia. Selective megakaryocyte expansion has been to date ineffective in reducing thrombocytopenia. This study was implemented to evaluate neutrophil specific rather than multilineage <it>ex vivo </it>expansion of PBSC for specifically focusing on reduction or abrogation of neutropenia.</p> <p>Methods</p> <p>CD34⁺cells (PBSC) were enriched from peripheral blood mononuclear cells following G-CSF-mobilisation and cultured with different permutations of cytokines to determine optimal cytokine combinations and doses for expansion and functional differentiation and maturation of neutrophils and their progenitors. Results were assessed by cell number, morphology, phenotype and function.</p> <p>Results</p> <p>A simple cytokine combination, SCF + Flt3-L + G-CSF, synergised to optimally expand and mature neutrophil progenitors assessed by cell number, phenotype, morphology and function (superoxide respiratory burst measured by chemiluminescence). G-CSF appears mandatory for functional maturation. Addition of other commonly employed cytokines, IL-3 and IL-6, had no demonstrable additive effect on numbers or function compared to this optimal combination. Addition of TPO, commonly included in multilineage progenitor expansion for development of megakaryocytes, reduced the maturation of neutrophil progenitors as assessed by number, morphology and function (respiratory burst activity).</p> <p>Conclusion</p> <p>Given that platelet transfusion support is available for autologous PBSC transplantation but granulocyte transfusion is generally lacking, and that multilineage expanded PBSC do not reduce thrombocytopenia, we suggest that instead of multilineage expansion selective neutrophil expansion based on this relatively simple cytokine combination might be prioritized for development for clinical use as an adjunct to unmanipulated PBSC transplantation to reduce or abrogate post-transplant neutropenia.</p

Absence of a relationship between immunophenotypic and colony enumeration analysis of endothelial progenitor cells in clinical haematopoietic cell sources

<p>Abstract</p> <p>Background</p> <p>The discovery of adult endothelial progenitor cells (EPC) offers potential for vascular regenerative therapies. The expression of CD34 and VEGFR2 by EPC indicates a close relationship with haematopoietic progenitor cells (HPC), and HPC-rich sources have been used to treat cardiac and limb ischaemias with apparent clinical benefit. However, the laboratory characterisation of the vasculogenic capability of potential or actual therapeutic cell autograft sources is uncertain since the description of EPC remains elusive. Various definitions of EPC based on phenotype and more recently on colony formation (CFU-EPC) have been proposed.</p> <p>Methods</p> <p>We determined EPC as defined by proposed phenotype definitions (flow cytometry) and by CFU-EPC in HPC-rich sources: bone marrow (BM); cord blood (CB); and G-CSF-mobilised peripheral blood (mPB), and in HPC-poor normal peripheral blood (nPB).</p> <p>Results</p> <p>As expected, the highest numbers of cells expressing the HPC markers CD34 or CD133 were found in mPB and least in nPB. The proportions of CD34⁺cells co-expressing CD133 is of the order mPB>CB>BM≈nPB. CD34⁺cells co-expressing VEGFR2 were also most frequent in mPB. In contrast, CFU-EPC were virtually absent in mPB and were most readily detected in nPB, the source lowest in HPC.</p> <p>Conclusion</p> <p>HPC sources differ in their content of putative EPC. Normal peripheral blood, poor in HPC and in HPC-related phenotypically defined EPC, is the richest source of CFU-EPC, suggesting no direct relationship between the proposed EPC immunophenotypes and CFU-EPC potential. It is not apparent whether either of these EPC measurements, or any, is an appropriate indicator of the therapeutic vasculogenic potential of autologous HSC sources.</p

Identifying nonalcoholic fatty liver disease patients with active fibrosis by measuring extracellular matrix remodeling rates in tissue and blood.

Excess collagen synthesis (fibrogenesis) in the liver plays a causal role in the progression of nonalcoholic fatty liver disease (NAFLD). Methods are needed to identify patients with more rapidly progressing disease and to demonstrate early response to treatment. We describe here a novel method to quantify hepatic fibrogenesis flux rates both directly in liver tissue and noninvasively in blood. Twenty-one patients with suspected NAFLD ingested heavy water (2 H2 O, 50-mL aliquots) two to three times daily for 3-5 weeks prior to a clinically indicated liver biopsy. Liver collagen fractional synthesis rate (FSR) and plasma lumican FSR were measured based on 2 H labeling using tandem mass spectrometry. Patients were classified by histology for fibrosis stage (F0-F4) and as having nonalcoholic fatty liver or nonalcoholic steatohepatitis (NASH). Magnetic resonance elastography measurements of liver stiffness were also performed. Hepatic collagen FSR in NAFLD increased with advancing disease stage (e.g., higher in NASH than nonalcoholic fatty liver, positive correlation with fibrosis score and liver stiffness) and correlated with hemoglobin A1C. In addition, plasma lumican FSR demonstrated a significant correlation with hepatic collagen FSR.ConclusionUsing a well-characterized cohort of patients with biopsy-proven NAFLD, this study demonstrates that hepatic scar in NASH is actively remodeled even in advanced fibrosis, a disease that is generally regarded as static and slowly progressive. Moreover, hepatic collagen FSR correlates with established risks for fibrotic disease progression in NASH, and plasma lumican FSR correlates with hepatic collagen FSR, suggesting applications as direct or surrogate markers, respectively, of hepatic fibrogenesis in humans. (Hepatology 2017;65:78-88)

Systematic assessment in an animal model of the angiogenic potential of different human cell sources for therapeutic revascularization

INTRODUCTION: Endothelial progenitor cells (EPC) capable of initiating or augmenting vascular growth were recently identified within the small population of CD34-expressing cells that circulate in human peripheral blood and which are considered hematopoietic progenitor cells (HPC). Soon thereafter human HPC began to be used in clinical trials as putative sources of EPC for therapeutic vascular regeneration, especially in myocardial and critical limb ischemias. However, unlike HPC where hematopoietic efficacy is related quantitatively to CD34(+ )cell numbers implanted, there has been no consensus on how to measure EPC or how to assess cellular graft potency for vascular regeneration. We employed an animal model of spontaneous neovascularization to simultaneously determine whether human cells incorporate into new vessels and to quantify the effect of different putative angiogenic cells on vascularization in terms of number of vessels generated. We systematically compared competence for therapeutic angiogenesis in different sources of human cells with putative angiogenic potential, to begin to provide some rationale for optimising cell procurement for this therapy. METHODS: Human cells employed were mononuclear cells from normal peripheral blood and HPC-rich cell sources (umbilical cord blood, mobilized peripheral blood, bone marrow), CD34(+ )enriched or depleted subsets of these, and outgrowth cell populations from these. An established sponge implant angiogenesis model was adapted to determine the effects of different human cells on vascularization of implants in immunodeficient mice. Angiogenesis was quantified by vessel density and species of origin by immunohistochemistry. RESULTS: CD34(+ )cells from mobilized peripheral blood or umbilical cord blood HPC were the only cells to promote new vessel growth, but did not incorporate into vessels. Only endothelial outgrowth cells (EOC) incorporated into vessels, but these did not promote vessel growth. CONCLUSIONS: These studies indicate that, since EPC are very rare, any benefit seen in clinical trials of HPC in therapeutic vascular regeneration is predominantly mediated by indirect proangiogenic effects rather than through direct incorporation of any rare EPC contained within these sources. It should be possible to produce autologous EOC for therapeutic use, and evaluate the effect of EPC distinct from, or in synergy with, the proangiogenic effects of HPC therapies

The Embedded Super Star Cluster of SBS0335-052

We analyze the infrared (6-100 micron) spectral energy distribution of the blue compact dwarf and metal-poor (Z=Z_solar/41) galaxy SBS0335-052. With the help of DUSTY (Ivezic et al. 1999), a program that solves the radiation transfer equations in a spherical environment, we evaluate that the infrared (IR) emission of SBS0335-052 is produced by an embedded super-star cluster (SSC) hidden under 10^5 M_solar of dust, causing 30 mag of visual extinction. This implies that one cannot detect any stellar emission from the 2x10^6 M_solar stellar cluster even at near-infrared (NIR) wavelengths. The derived grain size distribution departs markedly from the widely accepted size distribution inferred for dust in our galaxy (the so-called MRN distribution, Mathis et al. 1977), but resembles what is seen around AGNs, namely an absence of PAH and smaller grains, and grains that grow to larger sizes (around 1 micron). The fact that a significant amount of dust is present in such a low-metallicity galaxy, hiding from UV and optical view most of the star formation activity in the galaxy, and that the dust size distribution cannot be reproduced by a standard galactic law, should be borne in mind when interpreting the spectrum of primeval galaxies.Comment: 32 pages, 3 figures,accepted for publication in A

From Classical Four-Wave Mixing to Parametric Fluorescence in Silicon micro-ring resonators

Four-wave mixing can be stimulated or occur spontaneously. The first process is intrinsically much stronger, and well understood through classical nonlinear optics. The latter, also known as parametric fluorescence, can be explained only in the framework of a quantum theory of light. We experimentally demonstrate that, in a micro-ring resonator, there exists a simple relation between the efficiencies of these two processes, which is independent of the nonlinearity and size of the ring. In particular we show that the average power generated by parametric fluorescence can be immediately estimated from a classical FWM experiment. These results suggest that classical nonlinear characterization of a photonic integrated structure can provide accurate information on its nonlinear quantum properties.Comment: 4 pages, 3 figure

Cell necrosis, intrinsic apoptosis and senescence contribute to the progression of exencephaly to anencephaly in a mice model of congenital chranioschisis

Amniotic fluid; Neonatal mortality; ExencephalyLíquido amniótico; Mortalidad neonatal; ExencefaliaLíquid amniòtic; Mortalitat neonatal; ExencefàliaExencephaly/anencephaly is one of the leading causes of neonatal mortality and the most extreme open neural tube defect with no current treatments and limited mechanistic understanding. We hypothesized that exencephaly leads to a local neurodegenerative process in the brain exposed to the amniotic fluid as well as diffuse degeneration in other encephalic areas and the spinal cord. To evaluate the consequences of in utero neural tissue exposure, brain and spinal cord samples from E17 exencephalic murine fetuses (maternal intraperitoneal administration of valproic acid at E8) were analyzed and compared to controls and saline-injected shams (n = 11/group). Expression of apoptosis and senescence genes (p53, p21, p16, Rbl2, Casp3, Casp9) was determined by qRT-PCR and protein expression analyzed by western blot. Apoptosis was measured by TUNEL assay and PI/AV flow cytometry. Valproic acid at E8 induced exencephaly in 22% of fetuses. At E17 the fetuses exhibited the characteristic absence of cranial bones. The brain structures from exencephalic fetuses demonstrated a loss of layers in cortical regions and a complete loss of structural organization in the olfactory bulb, hippocampus, dental gyrus and septal cortex. E17 fetuses had reduced expression of NeuN, GFAP and Oligodendrocytes in the brain with primed microglia. Intrinsic apoptotic activation (p53, Caspase9 and 3) was upregulated and active Caspase3 localized to the layer of brain exposed to the amniotic fluid. Senescence via p21-Rbl2 was increased in the brain and in the spinal cord at the lamina I-II of the somatosensory dorsal horn. The current study characterizes CNS alterations in murine exencephaly and demonstrates that degeneration due to intrinsic apoptosis and senescence occurs in the directly exposed brain but also remotely in the spinal cord.This work was supported by Prof. Jose L. Peiro internal Cincinnati Children's Hospital funding