Synthetic Hydrogels with Stiffness Gradients for Durotaxis Study and Tissue Engineering Scaffolds

Migration of cells along the right direction is of paramount importance in a number of in vivo circumstances such as immune response, embryonic developments, morphogenesis, and healing of wounds and scars. While it has been known for a while that spatial gradients in chemical cues guide the direction of cell migration, the significance of the gradient in mechanical cues, such as stiffness of extracellular matrices (ECMs), in directed migration of cells has only recently emerged. With advances in synthetic chemistry, micro-fabrication techniques, and methods to characterize mechanical properties at a length scale even smaller than a single cell, synthetic ECMs with spatially controlled stiffness have been created with variations in design parameters. Since then, the synthetic ECMs have served as platforms to study the migratory behaviors of cells in the presence of the stiffness gradient of ECM and also as scaffolds for the regeneration of tissues. In this review, we highlight recent studies in cell migration directed by the stiffness gradient, called durotaxis, and discuss the mechanisms of durotaxis. We also summarize general methods and design principles to create synthetic ECMs with the stiffness gradients and, finally, conclude by discussing current limitations and future directions of synthetic ECMs for the study of durotaxis and the scaffold for tissue engineering.

Artificial antigen-presenting cells plus IL-15 and IL-21 efficiently induce melanoma-specific cytotoxic CD8+ CD28+ T lymphocyte responses

OBJECTIVE@#To develop a novel artificial antigen-presenting system for efficiently inducing melanoma-specific CD8(+) CD28(+) cytotoxic T lymphocyte (CTL) responses.@*METHODS@#Cell-sized Dynabeads® M-450 Epoxy beads coated with H-2K(b): Ig-TRP2180-188 and anti-CD28 antibody were used as artificial antigen-presenting cells (aAPCs) to induce melanoma-specific CD8(+)CD28(+) CTL responses with the help of IL-21 and IL-15. Dimer staining, proliferation, ELISPOT, and cytotoxicity experiments were conducted to evaluate the frequency and activity of induced CTLs.@*RESULTS@#Dimer staining demonstrated that the new artificial antigen-presenting system efficiently induced melanoma TRP2-specific CD8(+)CD28(+)CTLs. Proliferation and ELISPOT assays indicated that the induced CTLs rapidly proliferate and produce increased IFN- γ under the stimulation of H-2K(b): Ig-TRP2-aAPCs, IL-15, and IL-21. In addition, cytotoxicity experiments showed that induced CTLs have specific killing activity of target cells.@*CONCLUSIONS@#The new artificial antigen-presenting system including aAPCs plus IL-21 and IL-15 can induce a large number of antigen-specific CD8(+) CD28(+) CTLs against the melanoma. Our study provides evidence for a novel adoptive immunotherapy against tumors.

Protein budget: cost estimating criteria for synthetic biology / 生物工程学报

The aim of synthetic biology is to design artificial life systems. Such system is hoped to create a better production process with desired ability for bioproduction, biotransformation, adaption and environmental monitoring. However, to design a life system involves understanding the cellular regulation networks at multiple levels, in which the controls of protein level, subcelluar location, and activity are especially critical. Thus tuning protein expression has become essential tools in synthetic biology studies, such as part design, module assembly and compatibility optimization. Protein budget, just like budget for a factory, can be thought as the cost estimating criteria for an artificial cell factory. Protein budget control has provided a powerful optimization strategy for synthetic biology.

Essential genes, minimal genome and synthetic cell of bacteria: a review / 生物工程学报

Single-cell prokaryotes represent a simple and primitive cellular life form. The identification of the essential genes of bacteria and the minimal genome for the free-living cellular life could provide insights into the origin, evolution, and essence of life forms. The principles, methodology, and recent progresses in the identification of essential genes and minimal genome and the creation of synthetic cells are reviewed and particularly the strategies for creating the minimal genome and the potential applications are introduced.

Improvement of osteogenic potential of biphasic calcium phosphate bone substitute coated with synthetic cell binding peptide sequences

PURPOSE: The aim of this study was to evaluate the improvement of osteogenic potential of biphasic calcium phosphate (BCP) bone substitute coated with synthetic cell-binding peptide sequences in a standardized rabbit sinus model. METHODS: Standardized 6-mm diameter defects were created bilaterally on the maxillary sinus of ten male New Zealand white rabbits, receiving BCP bone substitute coated with synthetic cell binding peptide sequences on one side (experimental group) and BCP bone substitute without coating (control group) on the other side. Histologic and histomorphometric analysis of bone formation was carried out after a healing period of 4 or 8 weeks. RESULTS: Histological analysis revealed signs of new bone formation in both experimental groups (4- and 8-week healing groups) with a statistically significant increase in bone formation in the 4-week healing group compared to the control group. However, no statistically significant difference in bone formation was found between the 8-week healing group and the control group. CONCLUSIONS: This study found that BCP bone substitute coated with synthetic cell-binding peptide sequences enhanced osteoinductive potential in a standardized rabbit sinus model and its effectiveness was greater in the 4-week healing group than in the 8-week healing group.

Comparative study on the cellular activities of osteoblast-like cells and new bone formation of anorganic bone mineral coated with tetra-cell adhesion molecules and synthetic cell binding peptide

PURPOSE: We have previously reported that tetra-cell adhesion molecule (T-CAM) markedly enhanced the differentiation of osteoblast-like cells grown on anorganic bone mineral (ABM). T-CAM comprises recombinant peptides containing the Arg-Gly-Asp (RGD) sequence in the tenth type III domain, Pro-His-Ser-Arg-Asn (PHSRN) sequence in the ninth type III domain of fibronectin (FN), and the Glu-Pro-Asp-Ilu-Met (EPDIM) and Tyr-His (YH) sequence in the fourth fas-1 domain of betaig-h3. Therefore, the purpose of this study was to evaluate the cellular activity of osteoblast-like cells and the new bone formation on ABM coated with T-CAM, while comparing the results with those of synthetic cell binding peptide (PepGen P-15). METHODS: To analyze the cell viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed, andto analyze gene expression, northernblot was performed. Mineral nodule formations were evaluated using alizarin red stain. The new bone formations of each group were evaluated using histologic observation and histomorphometrc analysis. RESULTS: Expression of alkaline phosphatase mRNA was similar in all groups on days 10 and 20. The highest expression of osteopontin mRNA was observed in the group cultured with ABM/P-15, followed by those with ABM/T-CAM and ABM on days 20 and 30. Little difference was seen in the level of expression of collagen type I mRNA on the ABM, ABM/T-CAM, and ABM/P-15 cultured on day 20. There were similar growth and proliferation patterns for the ABM/T-CAM and ABM/P-15. The halo of red stain consistent with Ca2+ deposition was wider and denser around ABM/T-CAM and ABM/P-15 particles than around the ABM particles. The ABM/T-CAM group seemed to have bone forming bioactivity similar to that of ABM/P-15. A complete bony bridge was seen in two thirds of the defects in the ABM/T-CAM and ABM/P-15 groups. CONCLUSIONS: ABM/T-CAM, which seemed to have bone forming bioactivity similar to ABM/P-15, was considered to serve as effective tissue-engineered bone graft material.

Clinical application of human embryonic stem cells

Recent advances in stem cell biology, including the development of optimized cell type-specific culture systems, and the broader understandings of biochemical and molecular signals involved in cell self-renewal and differentiation have brought cell-based therapy closer to practical application. As of now, at least 250 adult stem cell therapies are being used or tested in clinical situations. Stem cells have two important properties that distinguish them from other types of cells; they can both proliferate without changing their phenotypes indefinitely, and they also can differentiate into one or more new kinds of cells depending on their culture conditions. Thus, stem cell therapy could be most effective for treating the diseases that are marked by the loss of cells. The typical examples are Parkinson's disease, Alzheimer's disease, diabetes, heart failure, blindness, spinal cord injury, and stroke. Additionally, stem cell derivatives can be used in drug discovery as well. In the last decade, various types of stem cells have been identified from preimplantation stage embryos, fetuses, placentas, and adult tissues. Moreover, it is now almost a common practice to produce induced pluripotent stem (iPS) cells from various adult somatic cells using only a few defined factors. Thus, it is feasible that patient-specific stem cells will be generated with less controversy in the near future. However, human embryonic stem (ES) cells firmly remain "the gold standard" because of their greatest potential to become any type of cell in the body. The vast knowledge obtained from human ES cell research in the past decade has made cell-based therapy more promising than ever. Even the recent establishment of iPS cell technology is the culmination of human ES cells research. In our laboratory, interesting human cardiovascular cells including endothelial precursor cells and beating myocardiac cells, artificial blood cells, and retinal pigment epithelial cells were successfully differentiated and their therapeutic potential was confirmed after cell transplantation into animal models. Thus, here, the current research status of human embryonic stem cell-based therapy will be introduced and the future directions of stem cell applications in clinical trials will be discussed.

How to make a minimal genome for synthetic minimal cell

As a key focus of synthetic biology, building a minimal artificial cell has given rise to many discussions. A synthetic minimal cell will provide an appropriate chassis to integrate functional synthetic parts, devices and systems with functions that cannot generally be found in nature. The design and construction of a functional minimal genome is a key step while building such a cell/chassis since all the cell functions can be traced back to the genome. Kinds of approaches, based on bioinformatics and molecular biology, have been developed and proceeded to derive essential genes and minimal gene sets for the synthetic minimal genome. Experiments about streamlining genomes of model bacteria revealed genome reduction led to unanticipated beneficial properties, such as high electroporation efficiency and accurate propagation of recombinant genes and plasmids that were unstable in other strains. Recent achievements in chemical synthesis technology for large DNA segments together with the rapid development of the whole-genome sequencing, have transferred synthesis of genes to assembly of the whole genomes based on oligonucleotides, and thus created strong preconditions for synthesis of artificial minimal genome. Here in this article, we review briefly the history and current state of research in this field and summarize the main methods for making a minimal genome. We also discuss the impacts of minimized genome on metabolism and regulation of artificial cell.