Embryonic Hematopoietic Progenitor Cells Reside in Muscle before Bone Marrow Hematopoiesis

<div><p>In mice, hematopoietic cells home to bone marrow from fetal liver prenatally. To elucidate mechanisms underlying homing, we performed immunohistochemistry with the hematopoietic cell marker c-Kit, and observed c-Kit(+) cells localized inside muscle surrounding bone after 14.5 days post coitum. Flow cytometric analysis showed that CD45(+) c-Kit(+) hematopoietic cells were more abundant in muscle than in bone marrow between 14.5 and 17.5 days post coitum, peaking at 16.5 days post coitum. CD45(+) c-Kit(+) cells in muscle at 16.5 days post coitum exhibited higher expression of <i>Gata2</i>, among several hematopoietic genes, than did fetal liver or bone marrow cells. Colony formation assays revealed that muscle hematopoietic cells possess hematopoietic progenitor activity. Furthermore, <i>exo utero</i> transplantation revealed that fetal liver hematopoietic progenitor cells home to muscle and then to BM. Our findings demonstrate that hematopoietic progenitor cell homing occurs earlier than previously reported and that hematopoietic progenitor cells reside in muscle tissue before bone marrow hematopoiesis occurs during mouse embryogenesis.</p></div

Summary of <i>exo utero</i> surgical transplantation data.

<p>Summary of <i>exo utero</i> surgical transplantation data.</p

Colony forming capacity of muscle CD45(+) c-Kit(+) cells.

<p>Muscle CD45(+) c-Kit(+) cells exhibit hematopoietic activity. (A) One thousand sorted muscle CD45(+) c-Kit(+) cells at 16.5 dpc were cultured in semisolid medium containing stem cell factor (SCF), interleukin (IL)-3, IL-6 and erythropoietin (Epo). On day 14, the number of CFU-G (colony-forming units of granulocytes), CFU-M (of macrophages), CFU-GM (of granulocytes and macrophages), CFU-Mk (of megakaryocytes), and CFU-GEMM (of granulocytes, erythrocytes, monocytes and macrophages) and the total colony number were counted. Bars represent means and SD of three culture dishes. (B) Fetal muscle CD45(+) c-Kit(+) cells obtained at 16.5 dpc were co-cultured with OP9 or OP9 Delta1 lines to assess lymphoid potential. Shown are surface expression of CD19 and B220 (B lymphoid markers) on cells cultured with OP9 cells for 16 days (left panel) and surface expression of CD4 and CD8 (T lymphoid markers) on cells cultured with OP9 Delta1 cell line for 16 days (right panel). (C) Experimental design of the colony formation assay after an organ culture step. Muscle tissue at 16.5 dpc was cultured on filter paper for 72 hours and a single cell suspension was prepared. One thousand cells were cultured and evaluated as in (A). (D) Shown are the number of cells of each colony type and total number of colonies on day 14. *<i>p</i><0.01.</p

Immunohistochemistry analysis of c-Kit(+) cells in bone marrow and muscle tissue surrounding femurs.

<p>Cryosections of femurs and surrounding muscle tissues from C57BL/6 mouse embryos at 14.5 to 19.5 dpc co-stained with c-Kit as an HPC marker and CD31 as an endothelial cell marker and observed by confocal microscopy. (A) Representative images showing hematoxylin-eosin (left) and immunohistochemical (right) staining of femur and surrounding muscle tissue at 16.5 dpc. Shown is c-Kit (green), CD31 (red) and TOTO-3 iodide (blue) staining. Scale bar represents 500 μm (left) and 50 μm (right). Green c-Kit(+) cells are observed in muscle tissue and indicated by arrows in right panel. (B) Confocal images of femur and surrounding muscle tissues at 14.5 dpc, 15.5 dpc and 16.5 dpc. Shown is c-Kit (green), CD31 (red) and TOTO-3 iodide (blue) staining. Scale bar represents 10 μm for all panels. (C) Localization of CD45(+) c-Kit(+) cells in serial sections (10 μm) of muscle tissue co-stained with antibodies against CD45 (red), c-Kit (green), CD31 (magenta) and DAPI (blue) at 14.5 dpc (left) and 16.5 dpc (right). Scale bar represents 10 μm for all panels. Arrows indicate CD45(+) c-Kit(+) cells, most located in muscle tissue rather than inside blood vessels. (D) The number of CD45(+) c-Kit(+) cells inside or outside of blood vessels in two sets of femur-surrounding muscle tissues was determined by microscopic observation of serial sections. Bars represent mean values and SD of cell numbers from three individual embryos. *<i>p</i><0.01.</p

Flow cytometric analysis of hematopoietic cells in fetal muscle tissue.

<p>Single cell suspensions of fetal muscle tissue surrounding both left and right femurs at stages 14.5 to 19.5 dpc, as analyzed by flow cytometry. (A) Surface phenotypes of fetal muscle cells. Among CD45(+) cells (upper panels), surface expression of Sca-1 and c-Kit was analyzed (lower panels). (B) The number of CD45(+) c-Kit(+) Sca-1(–) cells per two femurs and per two sets of femur-surrounding muscle tissue at 14.5 to 19.5 dpc. Bars represent mean values and SD of three individual experiments. (C) The number of CD45(+) c-Kit(+) Sca-1(+) cells per two femurs and per two sets of femur-surrounding muscle tissue at 14.5 to 19.5 dpc. Bars represent mean values and SD of three individual experiments. (D) Morphology of CD45(+) c-Kit(+) Sca-1(–) cells (left) and CD45(+) c-Kit(+) Sca-1(+) cells (right) from 16.5 dpc muscle. Cells are stained with May-Grünwald Giemsa solution. Scale bar represents 10 μm for all panels. *<i>p</i><0.01.</p

Expression of hematopoietic transcription factors and proliferation-related genes in muscle hematopoietic cells.

<p>RNA was extracted from sorted 14.5 dpc and 16.5 dpc FL CD45(+) c-Kit(+) Sca-1(–) and CD45(+) c-Kit(+) Sca-1(+) cells, 16.5 dpc muscle CD45(+) c-Kit(+) Sca-1(–) F4/80(–) and CD45(+) c-Kit(+) Sca-1(+) F4/80(–) cells, 19.5 dpc fetal BM CD45(+) c-Kit(+) Sca-1(–) and CD45(+) c-Kit(+) Sca-1(+) cells, and 3-month-old adult BM Lin(–) CD34(–) c-Kit(+) Sca-1(–) and CD45(+) c-Kit(+) Sca-1(+) cells, and expression of indicated factors assessed by real-time PCR. (A) Relative expression of hematopoietic transcription factors in the CD45(+) c-Kit(+) Sca-1(–) cell population in indicated samples. Each bar represents mean value and SD of three replicates. (B) Similar analysis in the CD45(+) c-Kit(+) Sca-1(+) cell population in indicated samples. Each bar represents mean value and SD of three technical replicates. (C) Relative expression of the proliferation-related genes <i>Myc</i> and <i>Ccnd1</i> in samples noted in (A). (D) Relative expression of proliferation-related genes in samples noted in (B). (E) Confocal images of Ki-67 in 16.5 dpc FL CD45(+) c-Kit(+) Sca-1(+) cells, 16.5 dpc muscle CD45(+) c-Kit(+) Sca-1(+) F4/80(–) cells, and 3-month-old adult BM Lin(–) CD34(–) c-Kit(+) Sca-1(+) cells. Shown is Ki-67 (green) and TOTO-3 iodide (blue) staining. Scale bar represents 50 μm for all panels. (F) The proportion of Ki-67(+) cells per total TOTO-3(+) cells in 16.5 dpc FL CD45(+) c-Kit(+) Sca-1(+) cells, 16.5 dpc muscle CD45(+) c-Kit(+) Sca-1(+) F4/80(–) cells, and 3-month-old adult BM Lin(–) CD34(–) c-Kit(+) Sca-1(+) cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138621#pone.0138621.s006" target="_blank">S5 Fig</a> shows unstained Ki-67 control. *<i>p</i><0.01.</p

Fetal CD45(+) c-Kit(+) cells migrate from liver to muscle and then to BM.

<p>Fetal CD45(+) c-Kit(+) cell migration was assessed by <i>exo utero</i> surgical transplantation. (A) Experimental design showing the transplantation protocol. EGFP(+) CD45(+) c-Kit(+) cells were sorted from FL of EGFP Tg mouse embryos at 14.5 dpc and from muscle tissues surrounding femurs at 16.5 dpc and transplanted into corresponding tissues of recipient C57BL/6 mouse embryos at the same developmental stage. After 24 hours, the presence of EGFP(+) cells in muscle tissue and BM was analyzed by flow cytometry and/or immunohistochemistry. (B) Flow cytometric profile exhibiting gate setting used to sort EGFP(+) CD45(+) c-Kit(+) cells from FL of EGFP Tg embryos at 14.5 dpc (upper). At 24 hours after transplantation, muscle tissue was dissociated into single cells and analyzed by flow cytometry. Representative flow cytometric profile shows EGFP(+) CD45(+) c-Kit(+) cells present in muscle tissue of 15.5 dpc recipients (middle). Muscle tissue was sectioned and immunostained with CD31 (red) and TOTO-3 iodide (blue). Representative confocal image showing EGFP(+) donor cells (arrow; green) in muscle (lower). Scale bar represents 10 μm for all panels. (C) Flow cytometric profile exhibiting gate setting used to sort EGFP(+) CD45(+) c-Kit(+) cells from muscle tissue of EGFP Tg embryos at 16.5 dpc (upper). At 24 hours after transplantation, femurs were sectioned and immunostained with CD31 (red) and TOTO-3 iodide (blue). Scale bar represents 100 μm. EGFP(+) donor cells (green) are present in BM at 17.5 dpc. Boxed area of merge panel is shown at higher magnification.</p

Sistematización de la experiencia de un ambiente de aprendizaje enriquecido por TIC durante la práctica clínica en fisioterapia cardiopulmonar en un hospital de nivel II de la ciudad de Cali

Esta investigación se centra en la caracterización de la experiencia de 4 estudiantes de fisioterapia de IX semestre de la Institución Universitaria Escuela Nacional del Deporte (IUEND) durante la implementación de un ambiente de aprendizaje enriquecido con Tecnologías de la Información y la Comunicación (TIC) en la práctica clínico – asistencial en Salud Cardiopulmonar; la cual se fundamenta en el hacer y pone a prueba las bases conceptuales del ciclo de fundamentación; todo esto con el fin de identificar las experiencias significativas que facilitan el aprendizaje y desarrollo de competencias clínicas, además analizar si este tipo de estrategias de enseñanza -aprendizaje permite al estudiante y al docente asesor superar inconvenientes propios de la práctica clínica como: optimizar tiempos de atención a pacientes, estudio independiente y trabajo colaborativo, retomar e integrar gran cantidad de conceptos y procedimientos aprendidos en IV semestre con las nuevas experiencias y la realidad del paciente; y a la vez cumplir con funciones administrativas propios del rol del fisioterapeuta asistencial (estadística, indicadores, desarrollo de guías, etc.) que dificultan el proceso de aprendizaje; concluyendo que los ambientes mediados por TIC pueden lograr superar estas dificultades y favorecer finalmente el aprendizaje significativo (juicio clínico), en el que se fundamenta el ciclo de práctica profesional

Collision detection on transmission lines with optical interferometer

V diplomski nalogi skušamo ugotoviti, v kolikšni meri je možno zaznavati in klasificirati trke na jeklenicah daljnovodov z optičnim interferometrom. Na začetku predstavimo osnovne pojme interferometrije in opišemo uporabljen optični interferometer. V jedru diplomske naloge natančneje opišemo eksperimentalni protokol in obdelavo signalov. Nadaljujemo z implementacijo algoritmov za segmentacijo in klasifikacijo zajetih signalov ter predstavimo dobljene rezultate. Segmentacijo izvedemo v domeni števila prehodov signala skozi ničlo, za klasifikacijo pa uporabimo večplastno nevronsko mrežo z algoritmom vzvratnega učenja. Rezultati študije nakazujejo, da sta implementirani segmentacija in klasifikacija uspešni v 77 % izvedenih trkov različnih predmetov.We analyse feasibility of collision detection on transmission lines with optical interferometer. We first provide a brief introduction into interferometry, along with a description of the optical interferometer used for measurements in this study. Afterwards, we describe the conducted experimental protocol and signal processing methodology. The focus is on implementation of algorithms for signal segmentation and collision classification. We used zero-crossing algorithm to transform signals into segmentation domain. Classification of collisions is done with a multilayer neural network trained by the backpropagation algorithm. The results demonstrate an average success rate of 77% for segmentation and classification of collision with five different objects

DRA001027

List of DRA (http://trace.ddbj.nig.ac.jp/dra/index_e.html) accession numbers of the FANTOM5 samples, sequences and genomic coordinations. Files are in tab-delimited format, which includes * Library ID * FFID * BioSamples accession number * DRA experiment accession number * DRA run accession numbers * DRA analysis accession number for genomic coordination (BAM file) * DRA analysis accession number for CTSS (BED file) * Experiment method (CAGE/RNA-Seq/sRNA-Seq