20 research outputs found
Generation of four induced pluripotent stem cell lines (FHUi003-A, FHUi003-B, FHUi004-A and FHUi004-B) from two affected individuals of a familial neurohypophyseal diabetes insipidus family
Four disease-specific induced pluripotent stem cell (iPSC) lines were respectively derived from peripheral blood mononuclear cells of two affected individuals in a family affected by familial neurohypophyseal diabetes insipidus carrying the c.314G>C mutation. The expression of pluripotency markers (NANOG, OCT4, and SOX2), maintenance of a normal karyotype, absence of episomal vectors used for iPSC generation, and presence of the original pathogenic mutation were confirmed for each iPSC line. The ability to differentiate into three germ layers was confirmed by a teratoma formation assay. These iPSC lines can help in disease recapitulation in vitro using organoids and elucidation of disease mechanisms
A prospective, randomized, open-label trial of early versus late povidone-iodine gargling in patients with COVID-19
Abstract Povidone-iodine (PVP–I) is a broad-spectrum antiseptic reagent that has been used for over 50 years. The purpose of this study is to look into the effect of gargling with PVP–I gargling on virus clearance and saliva infectivity in COVID-19. A prospective, randomized, open-label trial of intervention with PVP–I was conducted at three quarantine facilities in Osaka, Japan, enrolling adolescents and adults with asymptomatic-to-mild COVID-19. Patients were randomly allocated to the early and late intervention group at a 1:1 ratio. The early group gargled with PVP–I from days 2 to day 6; the late group gargled with water first, then with PVP–I from day 5 after sampling till day 6. The primary and secondary endpoints were viral clearance for SARS-CoV-2 using RT-qPCR at days 5 and 6, respectively, and the investigational endpoint was saliva infectivity clearance on day5. We enrolled 430 participants, with 215 assigned to each group, and 139 in the early group and 140 in the late had a positive saliva RT-qPCR test on day 2. On day 5, the early group had a significantly higher RT-qPCR negative rate than that of the late group (p = 0.015), and the early had a significantly higher clearance rate of infectivity (p = 0.025). During the PVP–I intervention, one participant reported oropharyngeal discomfort. Gargling with PVP–I may hasten virus clearance and reduce viral transmission via salivary droplets and aerosols in patients with asymptomatic-to-mild COVID-19. (Clinical trial registration numbers: jRCT1051200078 and dateof registration: 24/11/2020)
Tightly regulated and homogeneous transgene expression in human adipose-derived mesenchymal stem cells by lentivirus with tet-off system.
Genetic modification of human adipose tissue-derived multilineage progenitor cells (hADMPCs) is highly valuable for their exploitation in therapeutic applications. Here, we have developed a novel single tet-off lentiviral vector platform. This vector combines (1) a modified tetracycline (tet)-response element composite promoter, (2) a multi-cistronic strategy to express an improved version of the tet-controlled transactivator and the blasticidin resistance gene under the control of a ubiquitous promoter, and (3) acceptor sites for easy recombination cloning of the gene of interest. In the present study, we used the cytomegalovirus (CMV) or the elongation factor 1 α (EF-1α) promoter as the ubiquitous promoter, and EGFP was introduced as the gene of interest. hADMPCs transduced with a lentiviral vector carrying either the CMV promoter or the EF-1α promoter were effectively selected by blasticidin without affecting their stem cell properties, and EGFP expression was strictly regulated by doxycycline (Dox) treatment in these cells. However, the single tet-off lentiviral vector carrying the EF-1α promoter provided more homogenous expression of EGFP in hADMPCs. Intriguingly, differentiated cells from these Dox-responsive cell lines constitutively expressed EGFP only in the absence of Dox. This single tet-off lentiviral vector thus provides an important tool for applied research on hADMPCs
Generation of three induced pluripotent stem cell (iPSC) lines from a multiple endocrine neoplasia type 1 (MEN1) patient and three iPSC lines from an unaffected relative of the patient
Characterization of in vivo tumorigenicity tests using severe immunodeficient NOD/Shi-scid IL2Rγnull mice for detection of tumorigenic cellular impurities in human cell-processed therapeutic products
The contamination of human cell-processed therapeutic products (hCTPs) with tumorigenic cells is one of the major concerns in the manufacturing and quality control of hCTPs. However, no quantitative method for detecting the tumorigenic cellular impurities is currently standardized. NOD/Shi-scid IL2Rγnull (NOG) mice have shown high xeno-engraftment potential compared with other well-known immunodeficient strains, e.g. nude mice. Hypothesizing that tumorigenicity test using NOG mice could be a sensitive and quantitative method to detect a small amount of tumorigenic cells in hCTPs, we examined tumor formation after subcutaneous transplantation of HeLa cells, as a model of tumorigenic cells, in NOG mice and nude mice. Sixteen weeks after inoculation, the 50% tumor-producing dose (TPD50) values of HeLa cells were stable at 1.3 × 104 and 4.0 × 105 cells in NOG and nude mice, respectively, indicating a 30-fold higher sensitivity of NOG mice compared to that of nude mice. Transplanting HeLa cells embedded with Matrigel in NOG mice further decreased the TPD50 value to 7.9 × 10 cells, leading to a 5000-fold higher sensitivity, compared with that of nude mice. Additionally, when HeLa cells were mixed with 106 or 107 human mesenchymal stem cells as well as Matrigel, the TPD50 values in NOG mice were comparable to those of HeLa cells alone with Matrigel. These results suggest that the in vivo tumorigenicity test using NOG mice with Matrigel is a highly sensitive and quantitative method to detect a trace amount of tumorigenic cellular impurities in human somatic cells, which can be useful in the quality assessment of hCTPs
Schematic drawings of the single lentiviral vectors for tet-off system used in this work.
<p>(A) Gateway-compatible destination vectors containing <i>att</i>R recombination sites flanking a <i>ccd</i>B gene and a chloramphenicol-resistance gene, which allows an easy and rapid shuttling of gene of interest flanked by attL sites into the destination vectors using the Gateway LR recombination reaction. They also have an improved version of tetracycline-controlled transactivator (tTA) linked to the blasticidin resistant (Bsd) gene by the Thosea asigna virus 2A (2A) peptide sequence, whose expression is regulated by the CMV or EF-1α promoter. In the present study, we constructed an entry vector encoding EGFP flanked by attL, resulting in a destination clone, pTRE-EGFP-CMV-tTA-2A-Bsd or pTRE-EGFP-EF-tTA-2A-Bsd (B). In the absence of doxycycline (Dox), tTA-2A binds to the TRE-Tight promoter and activates EGFP transcription. For more details, see the Results section. CMV pro, CMV promoter; LTR, long terminal repeats; ψ, packaging signal; RRE, rev response elements; cPPT, central polypurine tract; TRE, tet-responsive element; Cm<sup>R</sup>, chloramphenicol resistance; tTA, tetracycline-controlled transactivator; Bsd, blasticidin resistance; WPRE, woodchuck hepatitis virus posttranscriptional control element; SIN, self-inactivating. (C) (D) hADMPCs were transduced with pTRE-EGFP-CMV-tTA-2A-Bsd or pTRE-EGFP-EF-tTA-2A-Bsd at m.o.i. of 250. Four days after transduction, the cells were divided into 2 populations; with 1 µg/mL of Dox (Dox (+)) and without Dox (Dox (-)). (C) Fluorescent and phase contrast images. Scale bar, 200 µm. (D) Log fluorescence histograms of EGFP by flow cytometry analysis. (E) The whole cell lysates from hADMPCs transduced with pTRE-EGFP-CMV-tTA-2A-Bsd or pTRE-EGFP-EF-tTA-2A-Bsd were subjected to western blotting to monitor the cleavage efficiency of tTA-2A-Bsd proteins. A primary antibody against TetR was used to detect either tTA-2A-Bsd (non-cleaved form) or tTA-2A (cleaved form). Asterisk indicates a nonspecific band.</p
Blasticidin selection of hADMPCs transduced with single tet-off lentiviral vector platform.
<p>hADMPCs were transduced with pTRE-EGFP-CMV-tTA-2A-Bsd (CMV) or pTRE-EGFP-EF-tTA-2A-Bsd (EF) at m.o.i. of 250. The cells were treated with 4 µg/mL blasticidin and 1 µg/mL Dox for 2 weeks. Then, the cells were cultured in the absence (Dox (-)) or presence (Dox (+)) of 1 µg/mL Dox for 4 days, and analyzed under a microscope (A) and flow cytometer (B). The cells were treated with 100 nM TSA (TSA), 5 µM 5-aza-dC (aza-dC), or both for 48 h before analyzed by flow cytometer. (C) A representative fluorescence histogram of EGFP. (D) The median fluorescence intensities of the EGFP-expressing populations. Error bars represent the standard error of 3 independent analyses. **, P<0.01 (Student's t test). Scale bar, 200 µm.</p
Differentiation potential of Dox-responsive hADMPCs.
<p>Dox-responsive hADMPCs were differentiated into adipocytes (A, E), osteocytes (B, F), chondrocytes (C, G), and neuronal cells (D, H). (A–D) Phase contrast (ph) and fluorescent (GFP) images. Dox-responsive hADMPCs were differentiated in the absence of Dox (Dox(−)) or in the presence of 1 µg/mL Dox (Dox(+)) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066274#s2" target="_blank">material and methods</a> section. (E–I) Confirmation of differentiated cells by oil red O staining for adipocytes (E), alizarin red staining for osteocytes (F), immunohistochemical staining against collagen II for chondrocytes (G), and immunohistochemical staining against β3-tubulin for neuronal cells (H). The percentages of differentiated cells to each cell type were calculated by the computerized image analysis (I). Cells that were not induced to differentiate (non-induced) were used as a negative control. CMV; hADMPCs transduced with pTRE-EGFP-CMV-tTA-2A-Bsd, EF; hADMPCs transduced with pTRE-EGFP-EF-tTA-2A-Bsd. Scale bar, 50 µm.</p