Osteoarthritis is a common disease character with progressive destruction of cartilage. MicroRNA (miR)-140-3p was validated as a biomarker for osteoarthritis. However, the mechanism by which ...miRNA-140-3p regulates osteoarthritis remains unclear. Thus, this study aims to evaluate the potential function of miRNA-140-3p during the pathogenesis of osteoarthritis. MiRNA-140-3p expression in tissue and CHON-001 chondrocyte cells was determined with quantitative real time (qRT)-PCR. In vitro osteoarthritis model was established by treatment of the chondrocyte cells CHON-001 with interleukin (IL)-1β. Cell proliferation and apoptosis were measured with cell counting kit-8 (CCK8) and Annexin V/propidium iodide (PI) apoptosis assay, respectively. Protein expressions were evaluated using Western blot. The target gene of miR-140-3p was predicted using Targetscan and miRDB. MiR-140-3p was downregulated in knee tissue from patients with osteoarthritis. IL-1β inhibited the proliferation of CHON-001 cells via inducing apoptosis. In addition, IL-1β significantly inhibited the expressions of collagen II and aggrecan and increased the level of matrix metalloproteinase (MMP)13. However, the effects of IL-1β could be ameliorated by the addition of miR-140-3p mimics. Moreover, luciferase reporter assay demonstrated CXCR4 as a target gene of miR-140-3p. IL-1β-induced upregulation of CXCR4 could be blocked by miR-140-3p mimics. Our study indicated that miR-140-3p could suppress the progression of osteoarthritis by directly targeting CXCR4. Therefore, miR-140-3p might serve as a potential therapeutic target for the treatment of osteoarthritis.
Human induced pluripotent stem cells (iPSCs) and genome editing provide a precise way to generate gene-corrected cells for disease modeling and cell therapies. Human iPSCs generated from sickle cell ...disease (SCD) patients have a homozygous missense point mutation in the HBB gene encoding adult β-globin proteins, and are used as a model system to improve strategies of human gene therapy. We demonstrate that the CRISPR/Cas9 system designer nuclease is much more efficient in stimulating gene targeting of the endogenous HBB locus near the SCD point mutation in human iPSCs than zinc finger nucleases and TALENs. Using a specific guide RNA and Cas9, we readily corrected one allele of the SCD HBB gene in human iPSCs by homologous recombination with a donor DNA template containing the wild-type HBB DNA and a selection cassette that was subsequently removed to avoid possible interference of HBB transcription and translation. We chose targeted iPSC clones that have one corrected and one disrupted SCD allele for erythroid differentiation assays, using an improved xeno-free and feeder-free culture condition we recently established. Erythrocytes from either the corrected or its parental (uncorrected) iPSC line were generated with similar efficiencies. Currently ∼6%-10% of these differentiated erythrocytes indeed lacked nuclei, characteristic of further matured erythrocytes called reticulocytes. We also detected the 16-kDa β-globin protein expressed from the corrected HBB allele in the erythrocytes differentiated from genome-edited iPSCs. Our results represent a significant step toward the clinical applications of genome editing using patient-derived iPSCs to generate disease-free cells for cell and gene therapies. Stem Cells 2015;33:1470-1479.
Home security system is needed for occupants' convenience and safety. In this paper, we present the design and implementation of a low cost, low power consumption, and GSM/GPRS (global system for ...mobile communication /general packet radio service) based wireless home security system. The system is a wireless home network which contains a GSM/GPRS gateway and three kinds of wireless security sensor nodes that are door security nodes, infrared security nodes and fire alarm nodes. The nodes are easy installing. The system can response rapidly to alarm incidents and has a friendly user interface including a LCD (liquid crystal display) and a capacitive sensor keyboard. The wireless communication protocol between the gateway and the nodes is also suitable for other home appliances. Furthermore, some methods are taken to ensure the security of system information.
The discovery that mature cells can be reprogrammed to become pluripotent and the development of engineered endonucleases for enhancing genome editing are two of the most exciting and impactful ...technology advances in modern medicine and science. Human pluripotent stem cells have the potential to establish new model systems for studying human developmental biology and disease mechanisms. Gene correction in patient-specific iPSCs can also provide a novel source for autologous cell therapy. Although historically challenging, precise genome editing in human iPSCs is becoming more feasible with the development of new genome-editing tools, including ZFNs, TALENs, and CRISPR. iPSCs derived from patients of a variety of diseases have been edited to correct disease-associated mutations and to generate isogenic cell lines. After directed differentiation, many of the corrected iPSCs showed restored functionality and demonstrated their potential in cell replacement therapy. Genome-wide analyses of gene-corrected iPSCs have collectively demonstrated a high fidelity of the engineered endonucleases. Remaining challenges in clinical translation of these technologies include maintaining genome integrity of the iPSC clones and the differentiated cells. Given the rapid advances in genome-editing technologies, gene correction is no longer the bottleneck in developing iPSC-based gene and cell therapies; generating functional and transplantable cell types from iPSCs remains the biggest challenge needing to be addressed by the research field.
T lymphocytes are the most abundant mononuclear blood cells and can serve as a source for generating induced pluripotent stem cells (iPSCs) for disease modeling or drug development. Here, we report ...the derivation of two iPSC lines from CD4+ helper T cells and CD8+ cytolytic T cells, respectively. The reprogramming was performed using Sendai virus encoding Klf-4, c-Myc, Oct-4 and Sox-2. Both iPSC lines displayed typical embryonic stem cell-like morphology and normal karyotype. Pluripotency was confirmed using immunocytochemistry methods and teratoma formation assay.
To identify accessible and permissive human cell types for efficient derivation of induced pluripotent stem cells （iPSCs）, we investigated epigenetic and gene expression signatures of multiple ...postnatal cell types such as fibroblasts and blood cells. Our analysis suggested that newborn cord blood （CB） and adult peripheral blood （PB） mononuclear cells （MNCs） display unique signatures that are closer to iPSCs and human embryonic stem cells （ESCs） than agematched fibroblasts to iPSCs/ESCs, thus making blood MNCs an attractive cell choice for the generation of integration-free iPSCs. Using an improved EBNA1/OriP plasmid expressing 5 reprogramming factors, we demonstrated highly efficient reprogramming of briefly cultured blood MNCs. Within 14 days of one-time transfection by one plasmid, up to 1000 iPSC-like colonies per 2 million transfected CB MNCs were generated. The efficiency of deriving iPSCs from adult PB MNCs was approximately 50-fold lower, but could be enhanced by inclusion of a second EBNA1/ OriP plasmid for transient expression of additional genes such as SV40 T antigen. The duration of obtaining bona fide iPSC colonies from adult PB MNCs was reduced to half （-14 days） as compared to adult fibroblastic cells （28- 30 days）. More than 9 human iPSC lines derived from PB or CB blood cells are extensively characterized, including those from PB MNCs of an adult patient with sickle cell disease. They lack V（D）J DNA rearrangements and vector DNA after expansion for 10-12 passages. This facile method of generating integration-free human iPSCs from blood MNCs will accelerate their use in both research and future clinical applications.
DNA base editors have enabled genome editing without generating DNA double strand breaks. The applications of this technology have been reported in a variety of animal and plant systems, however, ...their editing specificity in human stem cells has not been studied by unbiased genome-wide analysis. Here we investigate the fidelity of cytidine deaminase-mediated base editing in human induced pluripotent stem cells (iPSCs) by whole genome sequencing after sustained or transient base editor expression. While base-edited iPSC clones without significant off-target modifications are identified, this study also reveals the potential of APOBEC-based base editors in inducing unintended point mutations outside of likely in silico-predicted CRISPR-Cas9 off-targets. The majority of the off-target mutations are C:G->T:A transitions or C:G->G:C transversions enriched for the APOBEC mutagenesis signature. These results demonstrate that cytosine base editor-mediated editing may result in unintended genetic modifications with distinct patterns from that of the conventional CRISPR-Cas nucleases.
Several human postnatal somatic cell types have been successfully reprogrammed to induced pluripotent stem cells (iPSCs). Blood mononuclear cells (MNCs) offer several advantages compared with other ...cell types. They are easily isolated from umbilical cord blood (CB) or adult peripheral blood (PB), and can be used fresh or after freezing. A short culture allows for more efficient reprogramming, with iPSC colonies forming from blood MNCs in 14 d, compared with 28 d for age-matched fibroblastic cells. The advantages of briefly cultured blood MNCs may be due to favorable epigenetic profiles and gene expression patterns. Blood cells from adults, especially nonlymphoid cells that are replenished frequently from intermittently activated blood stem cells, are short-lived in vivo and may contain less somatic mutations than skin fibroblasts, which are more exposed to environmental mutagens over time. We describe here a detailed, validated protocol for effective generation of integration-free human iPSCs from blood MNCs by plasmid vectors.
PDF
