The process of reprogramming and recloning.

<p>(A) Phase contrast light microscopic observation of cells during reprogramming and recloning. Images captured on a Pronectin F-coated dish prior to colony picking on days four, nine, 13, 17 and 24 (upper panels). Note that human ES cell-like colonies emerged within a cobblestone like morphology. (B) Efficiency of generating reprogrammed cells on various coating materials and the number of colonies characterized. All the experiments used 1×10⁴ CD34⁺ CBCs, SeV TS vectors at 20 M.O.I. and ReproFF medium. Three independent experiments for Matrigel, Pronectin F and two independent experiments for fibronectin and laminin (Laminin-extracts), Pro<i>nectin L we</i>re performed. (C) Frequency of generating human ES cell-like colonies in various culture media. Five thousand CD34⁺ CBCs were infected with 20 M.O.I. of SeV carrying four factors and cultured in ReproFF, ReproFF2, mTeSR1, or E8 medium on Pronectin F-coated dishes to reprogram CD34+ CBCs. D: Endogenous gene expression of <i>Klf4, c-Myc, Oct3/4,</i> and <i>Sox2</i> in feeder like-cells (lane 1) and first pick up of a human ES cell like-colony (lane 2).</p

Expression of surface molecules on CD34⁺ cells and iPSCs.

<p>(A) Adhesion molecules integrin α5, β1, syndecan-2, and -4 on CD34⁺ CBCs (upper panels) and iPSC colonies (lower panels) detected by immunostaining with the relevant antibody. Alexa 594- and Alexa 488-conjugated secondary antibodies (red and green, respectively) were used to visualize the staining. Nuclei were stained with DAPI (lower photos). Means of the percentages of positive cells with standard deviation are appended in the right top of the photos. (B) Protocol for generation of iPSCs from CD34⁺ CBCs on Pronectin F-coated dishes with temperature sensitive SeV vectors. P: passage.</p

Generation of reprogrammed cell clone from a single cell via the naïve state.

<p>Human ES cell-like colonies (first prime state) were picked up at day 24 and cultured on Pronectin F-coated dishes. The colonies were subjected to heat treatment (38°C, three days) at passage three (P3). Light microscopic image and ALP staining at P3 are shown in upper and lower panels, respectively. Colonies emerged from single cells in Pronectin F-coated 96-well plates under naïve conditions at P4, dome-shaped colonies at P5 under naïve conditions, ES cell-like colonies (second primed) cultured under primed culture conditions at P6 or long-term passaged clone (PFX#9) at P45 are shown.</p

Exploration of Trends in Antimicrobial Use and Their Determinants Based on Dispensing Information Collected from Pharmacies throughout Japan: A First Report

The purpose of this study was to evaluate the defined daily doses (DDD)/1000 prescriptions/month (DPM) as a new indicator that can be used in pharmacies, and to describe antimicrobial use patterns in pharmacies nationwide in Japan. Dispensing volumes, number of prescriptions received, and facility information were obtained from 2638 pharmacies that participated in a survey. DPM was calculated based on the dispensing volume and number of prescriptions, which are routinely collected data that are simple to use. Use of third-generation cephalosporins, quinolones, and macrolides in pharmacies that received prescriptions primarily from hospitals or clinics decreased from January 2019 to January 2021. In particular, the antimicrobial use was higher in otorhinolaryngology departments than in other departments, despite a decrease in the antimicrobial use. In the linear multiple regression analysis, otorhinolaryngology department was independently associated with the third-generation cephalosporin, quinolone, and macrolide prescription in all periods. This study reveals for the first-time trends in antimicrobial use through a new indicator using the volume of drugs dispensed in pharmacies throughout Japan. Antimicrobial use differed by the medical department, suggesting the need to target interventions according to the department type

Tumorigenicity Studies of Induced Pluripotent Stem Cell (iPSC)-Derived Retinal Pigment Epithelium (RPE) for the Treatment of Age-Related Macular Degeneration

<div><p>Basic studies of human pluripotential stem cells have advanced rapidly and stem cell products are now seeing therapeutic applications. However, questions remain regarding the tumorigenic potential of such cells. Here, we report the tumorigenic potential of induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) for the treatment of wet-type, age-related macular degeneration (AMD). First, immunodeficient mouse strains (nude, SCID, NOD-SCID and NOG) were tested for HeLa cells’ tumor-forming capacity by transplanting various cell doses subcutaneously with or without Matrigel. The 50% Tumor Producing Dose (TPD₅₀ value) is the minimal dose of transplanted cells that generated tumors in 50% of animals. For HeLa cells, the TPD₅₀ was the lowest when cells were embedded in Matrigel and transplanted into NOG mice (TPD₅₀ = 10^1.1, n = 75). The TPD₅₀ for undifferentiated iPSCs transplanted subcutaneously to NOG mice in Matrigel was 10^2.12; (n = 30). Based on these experiments, 1×10⁶ iPSC-derived RPE were transplanted subcutaneously with Matrigel, and no tumor was found during 15 months of monitoring (n = 65). Next, to model clinical application, we assessed the tumor-forming potential of HeLa cells and iPSC 201B7 cells following subretinal transplantation of nude rats. The TPD₅₀ for iPSCs was 10^4.73 (n = 20) and for HeLa cells 10^1.32 (n = 37) respectively. Next, the tumorigenicity of iPSC-derived RPE was tested in the subretinal space of nude rats by transplanting 0.8–1.5×10⁴ iPSC-derived RPE in a collagen-lined (1 mm×1 mm) sheet. No tumor was found with iPSC-derived RPE sheets during 6–12 months of monitoring (n = 26). Considering the number of rodents used, the monitoring period, the sensitivity of detecting tumors via subcutaneous and subretinal administration routes and the incidence of tumor formation from the iPSC-derived RPE, we conclude that the tumorigenic potential of the iPSC-derived RPE was negligible.</p></div

Detection of human cells in host mouse tissue by <i>Alu</i> PCR.

<p>DNA from hiPSC-derived RPEs (positive control, Lane 1), NOG mouse subcutaneous tissue just beneath the transplants (2), mouse liver (3), mouse heart (4), mouse spleen (5), mouse kidney (6) and mouse lung (7) were used as PCR templates. M: 1 kb marker (A). <i>Alu</i> PCR detects ≥0.1% human cells included in mouse cells determined by visual assessment of PCR products generated from various ratios of human: mouse DNA template mixtures. Percentage of human DNA in DNA mixture is shown in a respective lane number (1–8) (B). M: 1 kb marker.</p

Tumorigenicity testing by subcutaneous transplantation of hiPSC-derived RPE into NOG mice.

<p>Log₁₀TPD₅₀ value for hiPSC 201B7 determined by subcutaneously transplanting cells in Matrigel into NOG was calculated by the Trimmed Spearman-Karber method (upper panel). Tumor formation from 1×10⁶ hiPSC-derived RPE cells prior to making RPE sheets (cell suspension) or after making RPE sheets (cell sheet) transplanted subcutaneously in various conditions into NOG mice. Animals were monitored for 13–85 weeks (lower panel).</p

Histological analyses of hiPSCs or HeLa cells transplanted into the subretinal space of nude rats.

<p>Eye balls were excised from a nude rat 7 weeks after subretinal transplantation of hiPSC. Non-transplanted right eye ball (NT) and left eye ball transplanted with 1×10⁴ hiPSCs (hiPSC) (A). HE staining of cross section of NT eye ball (B) and hiPSC-transplanted eye ball (C). HE staining of hiPSC-derived teratoma with three germ layers: cartilage-like tissue (mesoderm, D), intestinal epithelium-like tissue (endoderm, E) and neuron-like tissue (ectoderm, F) in hiPSC-transplanted eye ball. (G – O) Eye balls were excised from a nude rat 5 weeks after subretinal transplantation of HeLa cells. Non-transplanted right eye ball (NT) and left eye ball transplanted with 1×10⁵ HeLa cells (HeLa) (G). HE staining of cross section of HeLa cell-transplanted eye ball (H) and HeLa-derived tumor tissue (I). Anti-Ki-67-antibody (J), Hoechst 33258 (K) and HE staining (L) of serial sections of hiPSC-derived teratoma. Anti-Lamin A antibody (M), Hoechst 33258 (N) staining and microscopic image (O) of serial cross sections containing a boundary of hiPSC-derived teratoma and host rat tissue. Anti-Lamin A antibody specifically recognizes human cells in rat tissue.</p