Absorption and Fluorescence Spectra of 10-Hydroxy-benzo [h] quinoline and 10-Methoxy-benzo [h] quinoline in various Solvents

Article信州大学工学部紀要 73: 39-48 (1993)departmental bulletin pape

Seeding technique for lowering temperature during synthesis of α-alumina

This paper reports a method for producing α-Al2O3 at low temperature using a seeding technique. A white product obtained by hydrolyzing aluminum isopropoxide in water at 80 °C was peptized using acetic acid at 80 °C, which transformed the white product to a transparent alumina sol. α-Al2O3 particles were added to the alumina sol as seed material; the sol containing α-Al2O3 particles was then transformed to an α-Al2O3-seeded alumina gel by drying the sol at room temperature. The non-seeded alumina gel remained boehmite after annealing at 300 °C and crystallized into γ-Al2O3 and α-Al2O3 at temperatures between 300 and 500 °C and between 900 and 1100 °C, respectively. The α-Al2O3 seeding promoted crystallization of the alumina gel into α-Al2O3. The promotion of crystallization was significant with an increase in α-Al2O3 particle content by weight in the final seeded alumina gel. With an α-Al2O3 particle content of 5%, the seeded alumina gel was partially crystallized into α-Al2O3 by annealing at a temperature as low as 900 °C

Adipose stromal cells contain phenotypically distinct adipogenic progenitors derived from neural crest.

Recent studies have shown that adipose-derived stromal/stem cells (ASCs) contain phenotypically and functionally heterogeneous subpopulations of cells, but their developmental origin and their relative differentiation potential remain elusive. In the present study, we aimed at investigating how and to what extent the neural crest contributes to ASCs using Cre-loxP-mediated fate mapping. ASCs harvested from subcutaneous fat depots of either adult P0-Cre/or Wnt1-Cre/Floxed-reporter mice contained a few neural crest-derived ASCs (NCDASCs). This subpopulation of cells was successfully expanded in vitro under standard culture conditions and their growth rate was comparable to non-neural crest derivatives. Although NCDASCs were positive for several mesenchymal stem cell markers as non-neural crest derivatives, they exhibited a unique bipolar or multipolar morphology with higher expression of markers for both neural crest progenitors (p75NTR, Nestin, and Sox2) and preadipocytes (CD24, CD34, S100, Pref-1, GATA2, and C/EBP-delta). NCDASCs were able to differentiate into adipocytes with high efficiency but their osteogenic and chondrogenic potential was markedly attenuated, indicating their commitment to adipogenesis. In vivo, a very small proportion of adipocytes were originated from the neural crest. In addition, p75NTR-positive neural crest-derived cells were identified along the vessels within the subcutaneous adipose tissue, but they were negative for mural and endothelial markers. These results demonstrate that ASCs contain neural crest-derived adipocyte-restricted progenitors whose phenotype is distinct from that of non-neural crest derivatives

RT-PCR primer sequences.

<p>RT-PCR primer sequences.</p

Characterization of neural crest-derived ASCs.

<p><b><i>A</i></b>. Quantification of surface marker expression in both GFP+ and GFP− ASCs of either P0-Cre/FR or Wnt1-Cre/FR mice with flow cytometric analysis. The majority of both GFP+ and GFP− cells expressed MSC markers including CD29, CD44, CD90, CD105, and Sca-1. <b><i>B</i></b>. The proportion of both CD24- and CD34-positive cells in the GFP+ population was also higher, and the proportion of double-positive cells in the GFP+ population was more than five-fold higher compared to those in GFP− cells. Representative flow cytometry profiles of CD24/CD34 double-positive cells from P0-Cre/FR mice are shown. <b><i>C</i></b>. The GFP+ population had a significantly higher proportion of p75NTR-positive cells. Data (<i>A–C</i>) are shown as the mean + SEM of 4 independent experiments for each condition. *<i>p</i><0.05 versus GFP− cells, <i>t</i> test. <b><i>D</i></b>. Immunofluorescent staining for p75NTR, S100, Nestin, GFAP, and fibronectin (<i>red</i>) of either P0-Cre/FR ASCs (<i>upper five panels</i>) or Wnt1-Cre/FR (<i>lower two panels</i>) shows that the majority of GFP+ cells (<i>green</i>) co-expressed p75NTR, S100, and Nestin (<i>arrow</i>) but were negative for GFAP and fibronectin. A few GFP-negative but p75NTR- S100-, or Nestin-positive cells were also observed (<i>arrowhead</i>). Note that the majority of GFP− cells were positive for fibronectin. Scale bar  = 50 µm. <i>E</i>. RT-qPCR analysis for pluripotent markers <i>Sox2, Nanog</i>, and <i>Oct3/4</i> on FACS-purified GFP− and GFP+ cells from P0-Cre/FR mice. Results are normalized based on <i>GAPDH</i> expression and shown as relative changes to GFP− cells. Data are shown as the mean + SEM of 3–4 independent experiments for each condition.</p

Osteogenic and chondrogenic potential of neural crest-derived ASCs.

<p><b><i>A</i></b>. Bright-field images of alizarin staining (<i>top</i>) and fluorescent images of osteopontin immunostaning (<i>red in bottom</i>) in FACS-purified GFP- and GFP+ cells after osteogenic induction. Nuclei were stained with DAPI (<i>blue</i>). Scale bar  = 50 µm. <b><i>B</i></b>. The osteopontin-positive area was measured and expressed as a percentage of the total area. Data are shown as mean +SEM, n = 3. **<i>p</i><0.01 versus GFP− cells, <i>t</i> test. <b><i>C</i></b>. Bright-field images of Alcian blue staining in FACS-purified GFP+ and GFP− cells after chondrogenic induction. Scale bar = 100 µm. <b><i>D</i></b>. RT-qPCR analysis for chondrogenic markers <i>Aggrecan, COL2a1</i>, and <i>Sox9</i> in the GFP− and the GFP+ cells after chondrogenic differentiation. Results are normalized based on <i>GAPDH</i> expression and shown as relative changes to GFP-cells. Data are shown as the mean + SEM of 3–4 independent experiments for each condition.</p

Contribution of neural crest-derived cells to adult ASCs.

<p><b><i>A</i></b>. Schematic diagram of the Cre-loxP-mediated fate mapping of NC-derived cells. Either P0-Cre or Wnt1-Cre transgenic mice are crossed with CAG-CAT-EGFP mice to generate NC-specific Cre/Foxed-Reporter (FR) mice in which NC-derived cells can be traced by the expression of the reporter protein GFP. <b><i>B</i></b>. Flow cytometry profiles of ASCs from adult Cre/FR mice. The x axis is GFP fluorescent intensity, and the y axis is the side scatter channel (SSC). ASCs from non-transgenic controls served as a gating control. The proportions of GFP+ cells per total cells were 0.98±0.24% and 1.48±0.35% in P0-Cre/FR and in Wnt1-Cre/FR mice, respectively. <b><i>C</i></b>. Phase contrast and fluorescent micrographs of second passage ASC cultures isolated from adult P0-Cre/FR (<i>left</i>) and Wnt1-Cre/FR mice (<i>right</i>). The majority of GFP+ cells possess small soma with long slender processes (<i>arrow in bottom</i>), whereas a few cells show a flat/polygonal shape similar to GFP- ASCs (<i>arrowhead</i>). Scale bar  = 50 µm. <b><i>D</i></b>. The proportions of GFP+ cells in ASC cultures from P0-Cre/FR mice of different ages (1 week-, 3 week-, and 16 week-old) at different passages (from passage 0 to 6). Data are shown as the mean + SEM of 6 independent cultures. <b><i>E</i></b>. Growth curves of GFP+ and GFP− cells sorted from second passage ASCs of P0-Cre/FR mice. Results are shown as relative changes to the cell number on day 0 (after sorting). There was no significant difference in the population doubling time between GFP+ and GFP− cells. Data are shown as the mean ± SEM of 3 independent experiments for each condition.</p

<i>In vivo</i> localization of neural crest-derived cells in the subcutaneous adipose tissue.

<p>Confocal micrographs of the reporter protein GFP (<i>green</i>) combined with immunofluorescence of various markers (<i>red</i>) in the adult subcutaneous tissue of either P0-Cre/FR (<i>A, B, D–I</i>) or Wnt1-Cre/FR (<i>C, J</i>) mice. <b><i>A–A″</i></b>. A small portion of perilipin-positive adipocytes expressed GFP in the cephalic region. <b><i>B</i></b>. Peripheral nerve tissue was GFP+. <b><i>C</i></b>. GFP+ cells with slender processes were identified in the stroma of both cephalic and trunk regions. <b><i>D–J</i></b>. These cells (<i>arrow</i>) were localized along the vessels or occasionally in the vicinity to GFP+ adipocytes (<i>arrowhead in E</i>). They were negative for an endothelial marker CD31 (<i>D</i>), mural markers PDGFRβ and αSMA (<i>E, F</i>), and a glial marker GFAP (<i>G</i>), but positive for p75NTR and S100 (<i>H–J</i>). Nuclei were stained with DAPI (<i>blue, D–F, H–J</i>). Scale bar = 20 µm (<i>A, B</i>), 10 µm (<i>C–J</i>).</p

Adipogenic potential of neural crest-derived ASCs.

<p><b><i>A</i></b>. Phase contrast and fluorescent images show that both GFP+ (<i>arrow</i>) and GFP− ASCs (<i>arrowhead</i>) from P0-Cre/FR mice contain lipid droplets after adipogenic differentiation. Scale bar  = 50 µm. <b><i>B</i></b>. FACS-purified GFP+ cells are positive for both perilipin immunostaining (<i>top left, top right, and bottom left</i>) and Oil red O staining (<i>bottom right</i>) after adipogenic differentiation. Nuclei were stained with DAPI (<i>blue</i>). Scale bar  = 50 µm. <b><i>C</i></b>. Representative images and quantification of adipogenic differentiation of FACS-purified GFP+ and GFP− cells. The percentage of perilipin-positive cells per total cells was more than 1.5-fold higher in the GFP+ cells than that of the GFP-cells. Data are shown as the mean + SEM of 4 independent experiments for each condition. *<i>p</i><0.05 versus GFP− cells, <i>t</i> test. <b><i>D</i></b>. RT-qPCR analysis for preadipocyte markers <i>Pref-1, C/EBPδ</i>, and <i>GATA2</i>, as well as for mature adipocyte markers <i>PPARγ</i> and <i>aP2</i> on purified GFP− and GFP+ cells before or after (<i>adipo</i>) adipogenic induction. Results are normalized based on <i>GAPDH</i> expression and shown as relative changes to undifferentiated GFP− cells. Data are shown as the mean + SEM of 3–4 independent experiments for each condition.</p