Physiological mouse brain Abeta levels are not related to the phosphorylation state of threonine-668 of Alzheimer's APP.

Amyloid-beta peptide species ending at positions 40 and 42 (Abeta40, Abeta42) are generated by the proteolytic processing of the Alzheimer's amyloid precursor protein (APP). Abeta peptides accumulate in the brain early in the course of Alzheimer's disease (AD), especially Abeta42. The cytoplasmic domain of APP regulates intracellular trafficking and metabolism of APP and its carboxyl-terminal fragments (CTFalpha, CTFbeta). The role of protein phosphorylation in general, and that of the phosphorylation state of APP at threonine-668 (Thr668) in particular, has been investigated in detail by several laboratories (including our own). Some investigators have recently proposed that the phosphorylation state of Thr668 plays a pivotal role in governing brain Abeta levels, prompting the current study

Fabricating retinal pigment epithelial cell sheets derived from human induced pluripotent stem cells in an automated closed culture system for regenerative medicine.

Regenerative medicine has received a lot of attention as a novel strategy for injuries and diseases that are difficult to cure using current techniques. Cell production, which is vital for regenerative medicine, has undergone remarkable progress via breakthroughs in developmental biology and tissue engineering; currently, cell production requires numerous experimental operators performing manual, small-scale cell cultures. Other major obstacles for cell production and regenerative medicine include the variable quality of products based on the experimental procedure, the skills of operators, the level of labor required for production, and costs. Technological developments are required to overcome this, including automation instead of manual culture. Age-related macular regeneration (AMD) is a refractory ocular disease that causes severe deterioration in central vision due to senescence in the retinal pigment epithelium (RPE). Recently, we performed an autologous transplantation of induced pluripotent stem (iPS) cell-derived RPE cell sheets and started clinical research on allografts from RPE cell suspensions differentiated from iPS cells. The use of regenerative therapies for AMD using iPS cell-derived RPE is expected to become more widespread. In the present study, human iPS cell-derived RPE cells were cultured to form RPE cell sheets using equipment with a closed culture module. The quality of the automated cultured RPE cell sheets was confirmed by comparing their morphological and biological properties with those of manually generated RPE cell sheets. As a result, machine-cultured RPE sheets displayed the same quality as manually cultured RPE sheets, showing that iPS cell-derived RPE cell sheets were successfully cultured by an automated process

A novel, flexible and automated manufacturing facility for cell-based health care products: Tissue Factory

Introduction: Current production facilities for Cell-Based Health care Products (CBHPs), also referred as Advanced-Therapy Medicinal Products or Regenerative Medicine Products, are still dependent on manual work performed by skilled workers. A more robust, safer and efficient manufacturing system will be necessary to meet the expected expansion of this industrial field in the future. Thus, the ‘flexible Modular Platform (fMP)’ was newly designed to be a true “factory” utilizing the state-of-the-art technology to replace conventional “laboratory-like” manufacturing methods. Then, we built the Tissue Factory as the first actual entity of the fMP. Methods: The Tissue Factory was designed based on the fMP in which several automated modules are combined to perform various culture processes. Each module has a biologically sealed chamber that can be decontaminated by hydrogen peroxide. The asepticity of the processing environment was tested according to a pharmaceutical sterility method. Then, three procedures, production of multi-layered skeletal myoblast sheets, expansion of human articular chondrocytes and passage culture of human induced pluripotent stem cells, were conducted by the system to confirm its ability to manufacture CHBPs. Results: Falling or adhered microorganisms were not detected either just after decontamination or during the cell culture processes. In cell culture tests, multi-layered skeletal myoblast sheets were successfully manufactured using the method optimized for automatic processing. In addition, human articular chondrocytes and human induced-pluripotent stem cells could be propagated through three passages by the system at a yield comparable to manual operations. Conclusions: The Tissue Factory, based on the fMP, successfully reproduced three tentative manufacturing processes of CBHPs without any microbial contamination. The platform will improve the manufacturability in terms of lower production cost, improved quality variance and reduced contamination risks. Moreover, its flexibility has the potential to adapt to the modern challenges in the business environment including employment issues, low operational rates, and relocation of facilities. The fMP is expected to become the standard design basis of future manufacturing facilities for CBHPs. Keywords: Regenerative medicine, Automation, Cell processing facility, Manufacturing, Decontaminatio

The EZH2–PRC2–H3K27me3 axis governs the endometrial cell cycle and differentiation for blastocyst invasion

Abstract Infertility occurs in 15% of couples worldwide. Recurrent implantation failure (RIF) is one of the major problems in in vitro fertilization and embryo transfer (IVF–ET) programs, and how to manage patients with RIF to achieve successful pregnancy outcomes remains unresolved. Here, a uterine polycomb repressive complex 2 (PRC2)-regulated gene network was found to control embryo implantation. Our RNA-seq analyses of the human peri-implantation endometrium obtained from patients with RIF and fertile controls revealed that PRC2 components, including its core enzyme enhancer of zeste homolog 2 (EZH2)-catalyzing H3K27 trimethylation (H3K27me3) and their target genes are dysregulated in the RIF group. Although fertility of uterine epithelium-specific knockout mice of Ezh2 (eKO mice) was normal, Ezh2-deleted mice in the uterine epithelium and stroma (uKO mice) exhibited severe subfertility, suggesting that stromal Ezh2 plays a key role in female fertility. The RNA-seq and ChIP-seq analyses revealed that H3K27me3-related dynamic gene silencing is canceled, and the gene expression of cell-cycle regulators is dysregulated in Ezh2-deleted uteri, causing severe epithelial and stromal differentiation defects and failed embryo invasion. Thus, our findings indicate that the EZH2–PRC2–H3K27me3 axis is critical to preparing the endometrium for the blastocyst invasion into the stroma in mice and humans

Reliability and Validity of Radiographic Measurement of the Humerus-Elbow-Wrist Angle in Healthy Children

Background:. We conducted a retrospective cohort study to evaluate the normal value, range, reliability, and validity of measurement of the humerus-elbow-wrist angle, an index of valgus-varus angulation of the elbow, in healthy children. This measurement has been used to assess postoperative radiographic results. Methods:. Radiographs of the elbow in 62 healthy children ranging from 2 to 11 years of age were reviewed by 6 examiners at 2 sessions. The mean value and the reliability of measurement of the humerus-elbow-wrist angle, the carrying angle, and the Baumann angle were assessed. Intraobserver and interobserver reliability were calculated with use of intraclass correlation coefficients (ICCs). To determine concurrent validity, the association between the humerus-elbow-wrist angle and carrying angle measurements was examined with use of Pearson correlation coefficients. Results:. The mean humerus-elbow-wrist angle value was 12.0° (range, 1° to 24°), and the mean carrying angle was 14.6° (range, 4° to 28°). The ICCs for intraobserver measurements of the humerus-elbow-wrist angle were almost perfect for 4 examiners and were substantial for 2 examiners, with a mean value of 0.85 (range, 0.73 to 0.94). The ICCs for interobserver reliability with regard to the first and second measurements of the humerus-elbow-wrist angle were both substantial (0.76 and 0.78). A significant association between the humerus-elbow-wrist angle and the carrying angle was observed, with the Pearson correlation coefficients ranging from 0.74 to 0.90 (p < 0.001). Conclusions:. Measurement of the humerus-elbow-wrist angle demonstrated good reliability and validity. The humerus-elbow-wrist angle is a reliable radiographic measure of coronal alignment of the humerus and forearm