Journal: Cytotherapy

Article Title: Ex vivo -expanded highly pure ABCB5 + mesenchymal stromal cells as Good Manufacturing Practice-compliant autologous advanced therapy medicinal product for clinical use: process validation and first in-human data

doi: 10.1016/j.jcyt.2020.08.012

Figure Lengend Snippet: Effect of topical application of ABCB5 + MSCs on healing of experimental full-thickness skin wounds in NSG mice. (A–C) Wound sizes (means of both wounds of each animal) at 9 days (A), 11 days (B) and 13 days (C) after vehicle (control) or MSC application. (D) Total number of fully closed wounds at 13 days after vehicle (control) or MSC application, shown as percentage value for each group. (E–G) Immunohistochemical evaluation of the wounds for human CD31 (E), unspecific CD31 (antibody-detected mouse and human CD31) (F) and cytokeratin 14 (G). Expression was semi-quantitatively quantified as no (−), slight (1+), moderate (2+), strong (3+) and intense (4+) positivity. Shown are group means ± SD. Control: vehicle only (n = 12 wounds, six animals). Low dose: 1.875 × 10 5 ABCB5 + MSCs per wound (n = 18 wounds, nine animals; one animal was prematurely euthanized). Low to mid dose: 3.75 × 10 5 ABCB5 + MSCs per wound (n = 19 wounds, 10 animals; one wound was pathologically not evaluable). Mid to high: 7.5 × 10 5 ABCB5 + MSCs per wound (n = 20 wounds, 10 animals). High dose: 1.5 × 10 6 ABCB5 + MSCs per wound (n = 20 wounds, 10 animals). The high-dose group was excluded from wound size analysis (A–C) because of significantly smaller baseline wound size values (not shown). Non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.01 versus control. d., dose; IHC, immunohistochemistry; SD, standard deviation. (Color version of figure is available online).

Article Snippet: Paraffin-embedded sections were immunohistochemically stained using rabbit anti-human/mouse CD31 at 1:50 (ab28364; Abcam), rabbit anti-human CD31 at 1:50 (IHC-00055; Bethyl, Montgomery, TX, USA) and rabbit anti-human/mouse cytokeratin 14 at 1:1000 (PRB-155P; Covance/BioLegend), respectively, following validated multistep labeling protocols.

Techniques: Control, Immunohistochemical staining, Expressing, Immunohistochemistry, Standard Deviation

Journal: Cellular and molecular bioengineering

Article Title: Mouse Pluripotent Stem Cell Differentiation Under Physiological Oxygen Reduces Residual Teratomas.

doi: 10.1007/s12195-021-00687-8

Figure Lengend Snippet: FIGURE 2. Expression of pluripotency markers decrease substantially with time during differentiation and extended culture under reduced oxygen. Cells were differentiated for 10 days followed by up to 80 days of extended culture (90 days total). (a) Flow cytometric quantification of the fraction and number of Oct4-GFP1 for differentiating mESCs and miPSCs at 142, 36, and 7 mmHg pO2gas (n = 3). (b) Representative en face bright field and Oct4-GFP images of cell aggregates after extended culture at 142 or 7 mmHg pO2gas. (c) Relative expression of Oct4 and Nanog mRNA with time in mESCs, miPSCs, and HIF-1a2/2 mESCs at 142, 36, and 7 mmHg pO2gas (n = 3) measured with qPCR.

Article Snippet: Cells were incubated overnight at 4 C with primary antibodies against Oct4 (sc-9081; Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:100, Nanog (IHC-00205; Bethyl Laboratories, Montogomery, TX, USA) diluted 1:100, SSEA-1 (MC-480; DSHB) diluted 1:10, Foxa2 (sc-6554; Santa Cruz Biotechnology) diluted 1:200, Nestin diluted 1:50, cardiac troponin T (cTnT; MS-295-P1; NeoMarkers, Fremont, CA, USA) diluted 1:50, or Nkx2.5 (sc-8697; Santa Cruz Biotechnology) in 3% DS, 0.1% Triton X-100 solution, washed once with PBS, and incubated with secondary donkey AlexaFluor 488 (green) or 594 (red) antibodies (Invitrogen) against the appropriate species diluted 1:250 in 3% DS, 0.1% Triton X-100 solution for 2 h in the dark.

Techniques: Expressing

Journal: Molecular Biology of the Cell

Article Title: Kdm3a lysine demethylase is an Hsp90 client required for cytoskeletal rearrangements during spermatogenesis

doi: 10.1091/mbc.E13-08-0471

Figure Lengend Snippet: Two Kdm3a mouse models present arrested spermatogenesis with globozoospermia. (A) Diagram illustrates Cre-mediated deletion of exons 22–24 containing JmjC catalytic domain of Kdm3a in Kdm3a ΔJC mice ( Tateishi et al. , 2009 ). (a), (b), and (c) indicate the position of primers used to genotype mutant mice. Color code: green oval, C6-type zinc finger (ZF); blue, LXXLL nuclear receptor binding; red, JmjC domain; black, exons surrounding and affected by deletion. pAb, polyclonal antibody. (B) Genomic PCR identifying ΔJC deletion mutants. (C) Immunoblot of total MEF extracts with Kdm3a antibody (N-terminal) shows absence of full-length Kdm3a protein (FL, red arrow) in Kdm3a ΔJC/ΔJC . A shorter Kdm3a protein product result of Cre-induced deletion is indicated (ΔJC, black arrow). A nonspecific band is indicated (*). (D) DAPI staining of Kdm3a ΔJC/ΔJC epididymis reveals few mature sperm with rounded nuclei. (E) DAPI staining of testis cross-sections shows some abnormal tubules in Kdm3a ΔJC/+ (boxed region). Kdm3a ΔJC/ΔJC show arrested spermatogenesis. (F) Higher magnification of indicated regions from (E). es, elongated spermatids; arn, accumulation of round nuclei. Numbers represent the proportion of abnormal to normal tubules in each slide. (G) Diagram illustrates the position of the gene-trap insertion in Kdm3a GT/GT . Arrows indicate primers used for RT-PCR shown in (H). (H) Absence of Kdm3a transcript in Kdm3a GT/GT MEFs. Loading control used was 18S. (I) Immunoblot of total MEF extracts with a Kdm3a antibody directed to the N-terminus shows absence of full-length protein (FL, red arrow) in Kdm3a GT/GT . β-gal antibody detects the gene-trap product Kdm3a GT/GT only. (J) Kdm3a GT/GT testes are smaller than wild-type (WT) and heterozygous littermates. (K) Hematoxylin and eosin stain show disorganized tubules in testis sections of homozygous. (L) Immunohistochemistry of testis sections with a commercial antibody to Kdm3b (green) stained the acrosome of elongating spermatids. Note abnormal acrosome shape in Kdm3a GT/GT sections only. (M) DAPI panel shows absence of mature sperm in homozygous-GT epididymis. (N) Bright-field images show round-headed spermatozoon in Kdm3a GT/GT mice.

Article Snippet: The antibodies used along this study are to KDM3A (12835; Proteintech; and NB100-77282; Novus Biologicals, Littleton, CO); KDM3B (IHC 00189; Bethyl Laboratories, Montgomery, TX); Cct4 (ARP34271; Aviva); anti–mono- and dimethylated lysines (ab23366 and ab76118; Abcam, Cambridge, MA); anti-GFP (sc-8334), monoclonal to GAPDH (5019A-2; Imgenex, San Diego, CA), β-gal (A-11132; Molecular Probes), GST (C83271; LSBio); HP1a (clone 15.1952; Upstate); γ-tubulin (GTU-88; Sigma-Aldrich, St. Louis, MO), β-actin (ab8229; Abcam), Hsp90ab1 (MAB32861; R&D Systems), Hsp90aa1 (10713715; Pierce, Rockford, IL), Actbl2 (ab134977; Abcam).

Techniques: Mutagenesis, Binding Assay, Western Blot, Staining, Reverse Transcription Polymerase Chain Reaction, Control, H&E Stain, Immunohistochemistry