Highly reliable GIGA-sized synthetic human therapeutic antibody library construction.

Background: Monoclonal antibodies (mAbs) and their derivatives are the fastest expanding category of pharmaceuticals. Efficient screening and generation of appropriate therapeutic human antibodies are important and urgent issues in the field of medicine. The successful in vitro biopanning method for antibody screening largely depends on the highly diverse, reliable and humanized CDR library. To rapidly obtain potent human antibodies, we designed and constructed a highly diverse synthetic human single-chain variable fragment (scFv) antibody library greater than a giga in size by phage display. Herein, the novel TIM-3-neutralizing antibodies with immunomodulatory functions derived from this library serve as an example to demonstrate the library's potential for biomedical applications. Methods: The library was designed with high stability scaffolds and six complementarity determining regions (CDRs) tailored to mimic human composition. The engineered antibody sequences were optimized for codon usage and subjected to synthesis. The six CDRs with variable length CDR-H3s were individually subjected to ß-lactamase selection and then recombined for library construction. Five therapeutic target antigens were used for human antibody generation via phage library biopanning. TIM-3 antibody activity was verified by immunoactivity assays. Results: We have designed and constructed a highly diverse synthetic human scFv library named DSyn-1 (DCB Synthetic-1) containing 2.5 × 1010 phage clones. Three selected TIM-3-recognizing antibodies DCBT3-4, DCBT3-19, and DCBT3-22 showed significant inhibition activity by TIM-3 reporter assays at nanomolar ranges and binding affinities in sub-nanomolar ranges. Furthermore, clone DCBT3-22 was exceptionally superior with good physicochemical property and a purity of more than 98% without aggregation. Conclusion: The promising results illustrate not only the potential of the DSyn-1 library for biomedical research applications, but also the therapeutic potential of the three novel fully human TIM-3-neutralizing antibodies.

Ocular surface microbiota: Ophthalmic infectious disease and probiotics.

Recently, increasing studies have emphasized the importance of commensal bacteria in humans, including microbiota in the oral cavity, gut, vagina, or skin. Ocular surface microbiota (OSM) is gaining great importance as new methodologies for bacteria DNA sequencing have been published. This review outlines the current understanding and investigation of OSM and introduces the new concept of the gut-eye axis. Moreover, we have collected current studies that focus on the relationship between ophthalmic infectious disease and alterations in the OSM or human gut microbiota. Finally, we discuss the current application of probiotics in ophthalmic infectious disease, its limitations to date, and futural directions.

More than Antibiotics: Latest Therapeutics in the Treatment and Prevention of Ocular Surface Infections.

Ocular surface infections have been common issues for ophthalmologists for decades. Traditional strategies for infection include antibiotics, antiviral agents, and steroids. However, multiple drug-resistant bacteria have become more common with the prevalence of antibiotic use. Furthermore, an ideal treatment for an infectious disease should not only emphasize eliminating the microorganism but also maintaining clear and satisfying visual acuity. Immunogenetic inflammation, tissue fibrosis, and corneal scarring pose serious threats to vision, and they are not attenuated or prevented by traditional antimicrobial therapeutics. Herein, we collected information about current management techniques including stem-cell therapy, probiotics, and gene therapy as well as preventive strategies related to Toll-like receptors. Finally, we will introduce the latest research findings in ocular drug-delivery systems, which may enhance the bioavailability and efficiency of ocular therapeutics. The clinical application of improved delivery systems and novel therapeutics may support people suffering from ocular surface infections.

Regulatory RNAs, microRNA, long-non coding RNA and circular RNA roles in colorectal cancer stem cells.

The properties of cancer stem cells (CSCs), such as self-renewal, drug resistance, and metastasis, have been indicated to be responsible for the poor prognosis of patients with colon cancers. The epigenetic regulatory network plays a crucial role in CSC properties. Regulatory non-coding RNA (ncRNA), including microRNAs, long noncoding RNAs, and circular RNAs, have an important influence on cell physiopathology. They modulate cells by regulating gene expression in different ways. This review discusses the basic characteristics and the physiological functions of colorectal cancer (CRC) stem cells. Elucidation of these ncRNAs will help us understand the pathological mechanism of CRC progression, and they could become a new target for cancer treatment.

Current Development, Obstacle and Futural Direction of Induced Pluripotent Stem Cell and Mesenchymal Stem Cell Treatment in Degenerative Retinal Disease.

Degenerative retinal disease is one of the major causes of vision loss around the world. The past several decades have witnessed emerging development of stem cell treatment for retinal disease. Nevertheless, sourcing stem cells remains controversial due to ethical concerns and their rarity. Furthermore, induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) are both isolated from patients' mature tissues; thus, issues such as avoiding moral controversy and adverse events related to immunosuppression and obtaining a large number of cells have opened a new era in regenerative medicine. This review focuses on the current application and development, clinical trials, and latest research of stem cell therapy, as well as its limitations and future directions.

Chitosan 3D cell culture system promotes naïve-like features of human induced pluripotent stem cells: A novel tool to sustain pluripotency and facilitate differentiation.

A simplified and cost-effective culture system for maintaining the pluripotency of human induced pluripotent stem cells (hiPSCs) is crucial for stem cell applications. Although recombinant protein-based feeder-free hiPSC culture systems have been developed, their manufacturing processes are expensive and complicated, which hinders hiPSC technology progress. Chitosan, a versatile biocompatible polysaccharide, has been reported as a biomaterial for three-dimensional (3D) cell culture system that promotes the physiological activities of mesenchymal stem cells and cancer cells. In the current study, we demonstrated that chitosan membranes sustained proliferation and pluripotency of hiPSCs in long-term culture (up to 365 days). Moreover, using vitronectin as the comparison group, the pluripotency of hiPSCs grown on the membranes was altered into a naïve-like state, which, for pluripotent stem cells, is an earlier developmental stage with higher stemness. On the chitosan membranes, hiPSCs self-assembled into 3D spheroids with an average diameter of ~100 µm. These hiPSC spheroids could be directly differentiated into lineage-specific cells from the three germ layers with 3D structures. Collectively, chitosan membranes not only promoted the naïve pluripotent features of hiPSCs but also provided a novel 3D differentiation platform. This convenient biomaterial-based culture system may enable the effective expansion and accessibility of hiPSCs for regenerative medicine, disease modeling, and drug screening.

SIRT1-Mediated Expression of CD24 and Epigenetic Suppression of Novel Tumor Suppressor miR-1185-1 Increases Colorectal Cancer Stemness.

NAD-dependent deacetylase sirtuin-1 (SIRT1) is a class III histone deacetylase that positively regulates cancer-related pathways such as proliferation and stress resistance. SIRT1 has been shown to promote progression of colorectal cancer and is associated with cancer stemness, yet the precise mechanism between colorectal cancer stemness and SIRT1 remains to be further clarified. Here we report that SIRT1 signaling regulates colorectal cancer stemness by enhancing expression of CD24, a colorectal cancer stemness promoter. A novel miRNA, miR-1185-1, suppressed the expression of CD24 by targeting its 3'UTR (untranslated region) and could be inhibited by SIRT1 via histone deacetylation. Targeting SIRT1 by RNAi led to elevated H3 lysine 9 acetylation on the promoter region of miR-1185-1, which increased expression of miR-1185-1 and further repressed CD24 translation and colorectal cancer stemness. In a mouse xenograft model, overexpression of miR-1185-1 in colorectal cancer cells substantially reduced tumor growth. In addition, expression of miR-1185-1 was downregulated in human colorectal cancer tissues, whereas expression of CD24 was increased. In conclusion, this study not only demonstrates the essential roles of a SIRT1-miR-1185-1-CD24 axis in both colorectal cancer stemness properties and tumorigenesis but provides a potential therapeutic target for colorectal cancer treatment. SIGNIFICANCE: A novel tumor suppressor miR-1185-1 is involved in molecular regulation of CD24- and SIRT1-related cancer stemness networks, marking it a potential therapeutic target in colorectal cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/23/5257/F1.large.jpg.

The use of micro-needle arrays to deliver cells for cellular therapies.

Cell therapy is used to treat various diseases and to repair injuries. Cell delivery is a crucial process that delivers cells to target sites. Cells must be precisely delivered to a target site and the cells that are delivered must be localized to the target site to repair damaged tissue. For stem cell therapy, the most convenient method of cell delivery involves directly injecting cells into damaged tissue. Other strategies use carriers to transplant stem cells into damaged tissue. These are termed, stem cell delivery systems (SCDSs). Micro-needle arrays are minimally invasive transdermal delivery systems. The devices can pass through the stratum corneum barrier and deliver macromolecules into the skin. They can also access the microcirculation system in the skin. This study fabricates PMMA micro-needle using a two-stage micro-molding method. Cells are seeded on the micro-needle arrays and then transferred into the target tissue. Collagen hydrogel is used as a model biomimetic tissue. Cells are efficiently delivered to regions of interest, collagen hydrogel, by using this system. The delivery rate is about 83.2%. This demonstrates that micro-needle arrays allow very efficient delivery of cells.

NanoMuscle: controllable contraction and extension of mechanically interlocked DNA origami.

Artificial molecular machines synthesized in supramolecular chemistry have attracted great interest over the past decades. DNA origami presents an alternative approach to construct nano-machines by directly designing its thermodynamically stable state by DNA sequences. Here, we construct a molecular device, named NanoMuscle, with mechanically interlocked DNA origami. NanoMuscle's configuration - either extended or contracted - can be controlled by adding specific DNA strands. We monitored NanoMuscle's multistep synthesis with gel electrophoresis, and verified that monomers of the NanoMuscle are interlocked at correct orientation with transmission electron microscopy (TEM). We then validated that NanoMuscle can switch between extended and contracted configuration. By converting binding energy from DNA hybridization and Brownian motion to mechanical movements, NanoMuscle may serve as a novel building block for future mesoscale machinery.

Patient-derived induced pluripotent stem cells for models of cancer and cancer stem cell research.

Induced pluripotent stem cells (iPSCs) are embryonic stem cell-like cells reprogrammed from somatic cells by four transcription factors, OCT4, SOX2, KLF4 and c-MYC. iPSCs derived from cancer cells (cancer-iPSCs) could be a novel strategy for studying cancer. During cancer cell reprogramming, the epigenetic status of the cancer cell may be altered, such that it acquires stemness and pluripotency. The cellular behavior of the reprogrammed cells exhibits dynamic changes during the different stages of reprogramming. The cells may acquire the properties of cancer stem cells (CSCs) during the process of reprogramming, and lose their carcinogenic properties during reprogramming into a cancer-iPSCs. Differentiation of cancer-iPSCs by teratoma formation or organoid culturing could mimic the process of tumorigenesis. Some of the molecular mechanisms associated with cancer progression could be elucidated using the cancer-iPSC model. Furthermore, cancer-iPSCs could be expanded in culture system or bioreactors, and serve as cell sources for research, and as personal disease models for therapy and drug screening. This article introduces cancer studies that used the cell reprogramming strategy.

Highly Efficient Intracellular Protein Delivery by Cationic Polyethyleneimine-Modified Gelatin Nanoparticles.

Intracellular protein delivery may provide a safe and non-genome integrated strategy for targeting abnormal or specific cells for applications in cell reprogramming therapy. Thus, highly efficient intracellular functional protein delivery would be beneficial for protein drug discovery. In this study, we generated a cationic polyethyleneimine (PEI)-modified gelatin nanoparticle and evaluated its intracellular protein delivery ability in vitro and in vivo. The experimental results showed that the PEI-modified gelatin nanoparticle had a zeta potential of approximately +60 mV and the particle size was approximately 135 nm. The particle was stable at different biological pH values and temperatures and high protein loading efficiency was observed. The fluorescent image results revealed that large numbers of particles were taken up into the mammalian cells and escaped from the endosomes into the cytoplasm. In a mouse C26 cell-xenograft cancer model, particles accumulated in cancer cells. In conclusion, the PEI-modified gelatin particle may provide a biodegradable and highly efficient protein delivery system for use in regenerative medicine and cancer therapy.

Chitosan promotes cancer progression and stem cell properties in association with Wnt signaling in colon and hepatocellular carcinoma cells.

Cancer stem cells (CSCs), a small population of cancer cells, have been considered to be the origin of cancer initiation, recurrence, and metastasis. Tumor microenvironment provides crucial signals for CSCs to maintain stem cell properties and promotes tumorigenesis. Therefore, establishment of an appropriate cell culture system to mimic the microenvironment for CSC studies is an important issue. In this study, we grew colon and hepatocellular carcinoma (HCC) cells on chitosan membranes and evaluated the tumor progression and the CSC properties. Experimental results showed that culturing cancer cells on chitosan increased cell motility, drug resistance, quiescent population, self-renewal capacity, and the expression levels of stemness and CSC marker genes, such as OCT4, NANOG, CD133, CD44, and EpCAM. Furthermore, we demonstrated that chitosan might activate canonical Wnt/ß-catenin-CD44 axis signaling in CD44positive colon cancer cells and noncanonical Wnt-STAT3 signaling in CD44negative HCC cells. In conclusion, chitosan as culture substrates activated the essential signaling of CSCs and promoted CSC properties. The chitosan culture system provides a convenient platform for the research of CSC biology and screening of anticancer drugs.

Y-box binding protein-1 promotes hepatocellular carcinoma-initiating cell progression and tumorigenesis via Wnt/ß-catenin pathway.

Y-box binding protein-1 (YB-1) is a pleiotropic molecule that binds DNA to regulate genes on a transcriptional level in the nucleus and binds RNA to modulate gene translation in the cytoplasm. In our previous studies, YB-1 was also characterized as a fetal hepatic protein that regulates the maturation of hepatocytes and is upregulated during liver regeneration. Moreover, YB-1 has been shown to be expressed in human hepatocellular carcinoma (HCC). However, the role of YB-1 in HCC remains unclear. Here, we aimed to characterize the role of YB-1 in HCC. Based on the results of loss-of-function in HCC and gain-of-function in mice liver using hydrodynamic gene delivery, YB-1 promoted hepatic cells proliferation in vitro and in vivo. YB-1 was also involved in HCC cell proliferation, migration, and drug-resistance. The results of extreme limiting dilution sphere forming analysis and cancer initiating cell marker analysis were also shown that YB-1 maintained HCC initiating cells population. YB-1 also induced the epithelial-mesenchymal transition and stemness-related gene expression. Knockdown of YB-1 suppressed the expression of Wnt ligands and ß-catenin, impaired Wnt/ß-catenin signaling pathway and reduced the numbers of HCC initiating cells. Moreover, YB-1 displayed nuclear localization particularly in the HCC initiating cells, the EpCAM+ cells or sphere cells. Our findings suggested that YB-1 was a key factor in HCC tumorigenesis and maintained the HCC initiating cell population.

CPM Is a Useful Cell Surface Marker to Isolate Expandable Bi-Potential Liver Progenitor Cells Derived from Human iPS Cells.

To develop a culture system for large-scale production of mature hepatocytes, liver progenitor cells (LPCs) with a high proliferation potential would be advantageous. We have found that carboxypeptidase M (CPM) is highly expressed in embryonic LPCs, hepatoblasts, while its expression is decreased along with hepatic maturation. Consistently, CPM expression was transiently induced during hepatic specification from human-induced pluripotent stem cells (hiPSCs). CPM(+) cells isolated from differentiated hiPSCs at the immature hepatocyte stage proliferated extensively in vitro and expressed a set of genes that were typical of hepatoblasts. Moreover, the CPM(+) cells exhibited a mature hepatocyte phenotype after induction of hepatic maturation and also underwent cholangiocytic differentiation in a three-dimensional culture system. These results indicated that hiPSC-derived CPM(+) cells share the characteristics of LPCs, with the potential to proliferate and differentiate bi-directionally. Thus, CPM is a useful marker for isolating hiPSC-derived LPCs, which allows development of a large-scale culture system for producing hepatocytes and cholangiocytes.