Ayşegül Doğan

Differentiation of human stem cells is promoted by amphiphilic pluronic block copolymers

Stem cell usage provides novel avenues of tissue regeneration and therapeutics across disciplines. Apart from ethical considerations, the selection and amplification of donor stem cells remain a challenge. Various biopolymers with a wide range of properties have been used extensively to deliver biomolecules such as drugs, growth factors and nucleic acids, as well as to provide biomimetic surface for cellular adhesion. Using human tooth germ stem cells with high proliferation and transformation capacity, we have investigated a range of biopolymers to assess their potential for tissue engineering. Tolerability, toxicity, and their ability to direct differentiation were evaluated. The majority of pluronics, consisting of both hydrophilic and hydrophobic poly(ethylene oxide) chains, either exerted cytotoxicity or had no significant effect on human tooth germ stem cells; whereas F68 increased the multi-potency of stem cells, and efficiently transformed them into osteogenic, chondrogenic, and adipogenic tissues. The data suggest that differentiation and maturation of stem cells can be promoted by selecting the appropriate mechanical and chemical properties of polymers. It has been shown for the first time that F68, with its unique molecular characteristics, has a great potential to increase the differentiation of cells, which may lead to the development of new tissue engineering strategies in regenerative medicine.

Boron enhances odontogenic and osteogenic differentiation of human tooth germ stem cells (hTGSCs) in vitro.

Stem cell technology has been a great hope for the treatment of many common problems such as Parkinsons disease, Alzheimers disease, diabetes, cancer, and tissue regeneration. Therefore, the main challenge in hard tissue engineering is to make a successful combination of stem cells and efficient inductors in the concept of stem cell differentiation into odontogenic and osteogenic cell types. Although some boron derivatives have been reported to promote bone and teeth growth in vivo, the molecular mechanism of bone formation has not been elucidated yet. Different concentrations of sodium pentaborate pentahydrate (NaB) were prepared for the analysis of cell toxicity and differentiation evaluations. The odontogenic, osteogenic differentiation and biomineralization of human tooth germ stem cells (hTGSCs) were evaluated by analyzing the mRNA expression levels, odontogenic and osteogenic protein expressions, alkaline phosphatase (ALP) activity, mineralization, and calcium deposits. The NaB-treated group displayed the highest ALP activity and expression of osteo- and odontogenic-related genes and proteins compared to the other groups and baseline. In the current study, increased in vitro odontogenic and osteogenic differentiation capacity of hTGSCs by NaB application has been shown for the first time. The study offers considerable promise for the development of new scaffold systems combined with NaB in both functional bone and tooth tissue engineering.

Boron containing poly-(lactide-co-glycolide) (PLGA) scaffolds for bone tissue engineering

Scaffold-based bone defect reconstructions still face many challenges due to their inadequate osteoinductive and osteoconductive properties. Various biocompatible and biodegradable scaffolds, combined with proper cell type and biochemical signal molecules, have attracted significant interest in hard tissue engineering approaches. In the present study, we have evaluated the effects of boron incorporation into poly-(lactide-co-glycolide-acid) (PLGA) scaffolds, with or without rat adipose-derived stem cells (rADSCs), on bone healing in vitro and in vivo. The results revealed that boron containing scaffolds increased in vitro proliferation, attachment and calcium mineralization of rADSCs. In addition, boron containing scaffold application resulted in increased bone regeneration by enhancing osteocalcin, VEGF and collagen type I protein levels in a femur defect model. Bone mineralization density (BMD) and computed tomography (CT) analysis proved that boron incorporated scaffold administration increased the healing rate of bone defects. Transplanting stem cells into boron containing scaffolds was found to further improve bone-related outcomes compared to control groups. Additional studies are highly warranted for the investigation of the mechanical properties of these scaffolds in order to address their potential use in clinics. The study proposes that boron serves as a promising innovative approach in manufacturing scaffold systems for functional bone tissue engineering.

Genetic Modifications of Saccharomyces cerevisiae for Ethanol Production from Starch Fermentation: A Review

There is a huge demand for developing new technologies for alternative energy sources due to the elevated costs of petroleum and its by-products, depletion of nonrenewable fuel sources, and to eliminate the disadvantages of geopolitical location and environmental pollution caused by high levels of carbon dioxide release. Science is striving to meet this demand and as molecular biology techniques have progressed, genetic engineering tools have been presented as promising future solutions in the form of optimizing the fermentation process to increase the ethanol yield from different carbon sources such as starch. As Saccharomyces cerevisiae is not naturally able to ferment starch, it can be genetically manipulated and modulated to improve the fuel production from starchy materials and the amount of cost that is required to produce ethanol would be decreased with these manipulations. General modifications in S. cerevisiae include specific gene expressions to gain new properties or improve existing pathways. This review aims to elicit the current status of ethanol production thorough alternative techniques from starch using current genetic engineering applications and to give further directions for high-throughput fermentations using genetically modified S. cerevisiae strains.

Boron increases the cell viability of mesenchymal stem cells after long-term cryopreservation.

The field of stem-cell biology has emerged as a key technology for the treatment of various disorders and tissue regeneration applications. However, a major problem remains in clinical practice, which is the question of whether stem cells preserve their self-renewal and differentiation potential in the culture conditions or not. In the current study, effects of boron on the cryopreservation of human tooth germ stem cells (hTGSCs) were evaluated for the first time. The impacts of various boron concentrations (sodium pentaborate pentahydrate (NaB)) were tested on characterized hTGSCs viability for different time intervals (24, 48, and 72 h). 20 μg/ml NaB with lower Me(2)SO concentration was found to display positive effects on hTGSCs during repeated freezing and defrosting cycles, and long-term cryopreservation. After thawing, cells were analyzed for their surface antigens and differentiation capacity. hTGSCs were successfully cryopreserved without any change in their mesenchymal stem cell characteristics as they were treated with boron containing freezing medium. In addition, fatty acid composition was examined to demonstrate membrane fatty acid profiles after freeze-thawing. Besides, NaB treatment extended osteogenic and chondrogenic differentiation of hTGSCs remarkably after long-term cryopreservation with respect to control groups. The study clearly suggests that NaB has a protective role on the survival of hTGSCs in short- and long-term cryopreservation. Due to the possible storage of hTGSCs at early ages, development of a functional and reliable cryopreservation media can be designed as a future solution to the dental stem cell banking.

Differentiation and Neuro-Protective Properties of Immortalized Human Tooth Germ Stem Cells

Stem cells are considered to be promising therapeutic options in many neuro-degenerative diseases and injuries to the central nervous system, including brain ischemia and spinal cord trauma. Apart from the gold standard embryonic and mesenchymal origin, human tooth germ stem cells (hTGSCs) have also been shown to enjoy the characteristics of mesenchymal stem cells (MSCs) and the ability to differentiate into adipo-, chondro-, osteo- and neuro-genic cells, suggesting that they might serve as potential alternatives in the cellular therapy of various maladies. Immortalization of stem cells may be useful to avoid senescence of stem cells and to increase their proliferation potential without altering their natural characteristics. This study evaluated the expression of stem cell markers, surface antigens, differentiation capacity, and karyotype of hTGSCs that have been immortalized by human telomerase reverse transcriptase (hTERT) or simian vacuolating virus 40 (SV40) large T antigen. These undying cells were also evaluated for their neuro-protective potential using an in vitro SH-SY5Y neuro-blastoma model treated with hydrogen-peroxide or doxo-rubicin. Although hTGSC-SV40 showed abnormal karyotypes, our results suggest that hTGSC-hTERT preserve their MSC characteristics, differentiation capacity and normal karyotype, and they also possess high proliferation rate and neuro-protective effects even at great passage numbers. These peculiars indicate that hTGSC-hTERT could be used as a viable model for studying adipo-, osteo-, odonto- and neuro-genesis, as well as neuro-protection of MSCs, which may serve as a springboard for potentially utilizing dental waste material in cellular therapy.

Boron and Poloxamer (F68 and F127) Containing Hydrogel Formulation for Burn Wound Healing

Burn injuries, the most common and destructive forms of wounds, are generally accompanied with life-threatening infections, inflammation, reduced angiogenesis, inadequate extracellular matrix production, and lack of growth factor stimulation. In the current study, a new antimicrobial carbopol-based hydrogel formulated with boron and pluronic block copolymers was evaluated for its healing activity using in vitro cell culture techniques and an experimental burn model. Cell viability, gene expression, and wound healing assays showed that gel formulation increased wound healing potential. In vitro tube-like structure formation and histopathological examinations revealed that gel not only increased wound closure by fibroblastic cell activity, but also induced vascularization process. Moreover, gel formulation exerted remarkable antimicrobial effects against bacteria, yeast, and fungi. Migration, angiogenesis, and contraction-related protein expressions including collagen, α-smooth muscle actin, transforming growth factor-β1, vimentin, and vascular endothelial growth factor were considerably enhanced in gel-treated groups. Macrophage-specific antigen showed an oscillating expression at the burn wounds, indicating the role of initial macrophage migration to the wound site and reduced inflammation phase. This is the first study indicating that boron containing hydrogel is able to heal burn wounds effectively. The formulation promoted burn wound healing via complex mechanisms including stimulation of cell migration, growth factor expression, inflammatory response, and vascularization.

In vitro differentiation of human tooth germ stem cells into endothelial‐ and epithelial‐like cells

Current clinical techniques in dental practice include stem cell and tissue engineering applications. Dental stem cells are promising primary cell source for mainly tooth tissue engineering. Interaction of mesenchymal stem cell with epithelial and endothelial cells is strictly required for an intact tooth morphogenesis. Therefore, it is important to investigate whether human tooth germ stem cells (hTGSCs) derived from wisdom tooth are suitable for endothelial and epithelial cell transformation in dental tissue regeneration approaches. Differentiation into endothelial and epithelial cell lineages were mimicked under defined conditions, confirmed by real time PCR, western blotting and immunocytochemical analysis by qualitative and quantitative methods. HUVECs and HaCaT cells were used as positive controls for the endothelial and epithelial differentiation assays, respectively. Immunocytochemical and western blotting analysis revealed that terminally differentiated cells expressed cell‐lineage markers including CD31, VEGFR2, VE‐Cadherin, vWF (endothelial cell markers), and cytokeratin (CK)‐17, CK‐19, EpCaM, vimentin (epithelial cell markers) in significant levels with respect to undifferentiated control cells. Moreover, high expression levels of VEGFR1, VEGFR2, VEGF, CK‐18, and CK‐19 genes were detected in differentiated endothelial and epithelial‐like cells. Endothelial‐like cells derived from hTGSCs were cultured on Matrigel, tube‐like structure formations were followed as an indication for functional endothelial differentiation. hTGSCs successfully differentiate into various cell types with a broad range of functional abilities using an in vitro approach. These findings suggest that hTGSCs may serve a potential stem cell source for tissue engineering and cell therapy of epithelial and endothelial tissue.

Improvements of Tolerance to Stress Conditions by Genetic Engineering in Saccharomyces Cerevisiae during Ethanol Production

Saccharomyces cerevisiae, industrial yeast isolate, has been of great interest in recent years for fuel ethanol production. The ethanol yield and productivity depend on many inhibitory factors during the fermentation process such as temperature, ethanol, compounds released as the result of pretreatment procedures, and osmotic stress. An ideal strain should be able to grow under different stress conditions occurred at different fermentation steps. Development of tolerant yeast strains can be achieved by reprogramming pathways supporting the ethanol metabolism by regulating the energy balance and detoxicification processes. Complex gene interactions should be solved for an in-depth comprehension of the yeast stress tolerance mechanism. Genetic engineering as a powerful biotechnological tool is required to design new strategies for increasing the ethanol fermentation performance. Upregulation of stress tolerance genes by recombinant DNA technology can be a useful approach to overcome inhibitory situations. This review presents the application of several genetic engineering strategies to increase ethanol yield under different stress conditions including inhibitor tolerance, ethanol tolerance, thermotolerance, and osmotolerance.

Effect of F68 on Cryopreservation of Mesenchymal Stem Cells Derived from Human Tooth Germ

The use of stem-cell-based therapies in regenerative medicine and in the treatment of disorders such as Parkinson, Alzheimers disease, diabetes, spinal cord injuries, and cancer has been shown to be promising. Among all stem cells, mesenchymal stem cells (MSCs) were reported to have anti-apoptotic, immunomodulatory, and angiogenic effects which are attributed to the restorative capacity of these cells. Human tooth germ stem cells (HTGSCs) having mesenchymal stem cell characteristics have been proven to exert high proliferation and differentiation capacity. Unlike bone-marrow-derived MSCs, HTGSCs can be easily isolated, expanded, and cryopreserved, which makes them an alternative stem cell source. Regardless of their sources, the stem cells are exposed to physical and chemical stresses during cryopreservation, hindering their therapeutic capacity. Amelioration of the side effects of cryopreservation on MSCs seems to be a priority in order to maximize the therapeutic efficacy of these cells. In this study, we tested the effect of Pluronic 188 (F68) on HTGSCs during long-term cryopreservation and repeated freezing and defrosting cycles. Our data revealed that F68 has a protective role on survival and differentiation of HTGSCs in long-term cryopreservation.