Papers by Stelios Andreadis
Proceedings of the Second Joint 24th Annual Conference and the Annual Fall Meeting of the Biomedical Engineering Society] [Engineering in Medicine and Biology
We developed 2D and 3D models of wound regeneration to assess the efficacy of fibrin and keratino... more We developed 2D and 3D models of wound regeneration to assess the efficacy of fibrin and keratinocyte growth factor (KGF or FGF-7) to accelerate skin reepithelialization. A modified scratch wounding assay in culture was used to study the fibrillar. structure of fibrin and its effect on keratinocyte migration. A skin equivalent model of wound healing was utilized to examine the same conditions in 3D. Fibrin accelerated the healing of wounds in 2D and 3D when used at physiologic concentrations (2 - 4 mg/ml). Interestingly, higher fibrinogen concentrations had a deleterious effect in 2D but did not alter the rate of healing in the 3D model. We also evaluated the effects of heparin and fibrillar structure of fibrin gels on the kinetics of KGF release. Fibrin gels were then used to deliver KGF to wounds created in skin equivalents grafted to athymic mice. We found that fibrin-mediated delivery of KGF accelerated reepithelialization and increased vascularization of wounds in vivo. Our results suggest that the structure of fibrin may differentially affect growth factor delivery and keratinocyte migration in 2D vs. 3D models.
Experimental Cell Research, 2017
Adherens junctions (AJs) are a key structural component for tissue organization and function. Und... more Adherens junctions (AJs) are a key structural component for tissue organization and function. Under fluid shear stress, AJs exhibit dynamic assembly/disassembly, but how shear stress couples to AJs is unclear. In MDCK cells we measured simultaneously the forces in cytoskeletal α-actinin and the density and length of AJs using a genetically coded optical force sensor, actinin-sstFRET, and fluorescently labeled E-cadherin (E-cad). We found that shear stress of 0.74 dyn/cm 2 for 3 hours significantly enhanced E-cad expression at cell-cell contacts and this phenomenon has two phases. The initial formation of segregated AJ plaques coincided with a decrease in cytoskeletal tension, but an increase in tension was necessary for expansion of the plaques and the formation of continuous AJs in the later phase. The changes in cytoskeletal tension and reorganization appear to be an upstream process in response to flow since it occurred in both wild type and dominant negative E-cad cells. Disruption of F-actin with a Rho-ROCK 2 inhibitor eliminated AJ growth under flow. These results delineate the shear stress transduction paths in cultured cells, which helps to understand pathology of a range of diseases that involve dysfunction of E-cadherin.
Tissue Engineering Part A, 2017
Mesenchymal stem cells (MSCs) have been extensively used in the field of tissue engineering as a ... more Mesenchymal stem cells (MSCs) have been extensively used in the field of tissue engineering as a source of smooth muscle cells (SMCs). However, recent studies showed deficits in the contractile function of SMCs derived from senescent MSCs and there are no available strategies to restore the contractile function that is impaired due to cellular or organismal senescence. In this study, we developed a tetracycline-regulatable system and employed micropost tissue arrays to evaluate the effects of the embryonic transcription factor, NANOG, on the contractility of senescent MSCs. Using this system, we show that expression of NANOG fortified the actin cytoskeleton and restored contractile function that was impaired in senescent MSCs. NANOG increased the expression of smooth muscle a-actin (ACTA2) as well as the contractile force generated by cells in three-dimensional microtissues. Interestingly, NANOG worked together with transforming growth factor-beta1 to further enhance the contractility of senescent microtissues. The effect of NANOG on contractile function was sustained for about 10 days after termination of its expression. Our results show that NANOG could reverse the effects of stem cell senescence and restore the myogenic differentiation potential of senescent MSCs. These findings may enable development of novel strategies to restore the function of senescent cardiovascular and other SMC-containing tissues.
Emerging Trends in Cell and Gene Therapy, 2013
The adult body harbors powerful reservoirs of stem cells that enable tissue regeneration under ho... more The adult body harbors powerful reservoirs of stem cells that enable tissue regeneration under homeostatic conditions or in response to disease or injury. The hair follicle is a readily accessible mini organ within the skin and contains stem cells from diverse developmental origins that are shown to have surprisingly broad differentiation potential. In this chapter, we discuss the biology of the hair follicle with particular emphasis on the various stem cell populations residing within the tissue. We summarize the existing knowledge on putative hair follicle stem cell markers, the differentiation potential, and technologies to isolate and expand distinct stem cell populations. We also discuss the potential of hair follicle stem cells for drug and gene delivery, tissue engineering, and regenerative medicine. We propose that the abundance of stem cells with broad differentiation potential and the ease of accessibility make the hair follicle an ideal source of stem cells for gene and cell therapies.
Trends in Biotechnology, 2006
Tissue Engineering Part B: Reviews, 2012
This review focuses on the stem cell sources with the potential to be used in vascular tissue eng... more This review focuses on the stem cell sources with the potential to be used in vascular tissue engineering and to promote vascular regeneration. The first clinical studies using tissue-engineered vascular grafts are already under way, supporting the potential of this technology in the treatment of cardiovascular and other diseases. Despite progress in engineering biomaterials with the appropriate mechanical properties and biological cues as well as bioreactors for generating the correct tissue microenvironment, the source of cells that make up the vascular tissues remains a major challenge for tissue engineers and physicians. Mature cells from the tissue of origin may be difficult to obtain and suffer from limited proliferative capacity, which may further decline as a function of donor age. On the other hand, multipotent and pluripotent stem cells have great potential to provide large numbers of autologous cells with a great differentiation capacity. Here, we discuss the adult multipotent as well as embryonic and induced pluripotent stem cells, their differentiation potential toward vascular lineages, and their use in engineering functional and implantable vascular tissues. We also discuss the associated challenges that need to be addressed in order to facilitate the transition of this technology from the bench to the bedside. Vascular Tissue Engineering: Unmet Clinical Need C ardiovascular disease, and coronary artery disease (CAD) in particular, is the leading cause of mortality in the United States, necessitating *500,000 coronary artery bypass graft (CABG) surgeries annually. 1 Surgically harvested autologous grafts, such as the left internal mammary and radial arteries or the greater saphenous vein, from patients are considered the gold standard for CABG procedures. 2-5 Other autologous arterial/venous grafts, cryopreserved cadaveric grafts, umbilical vein grafts, and arterial allografts have also been tried but with limited success because of associated complications. 6-11 Although autologous vessels from patients remain the grafts of choice, in many cases, previous harvest, morbidity at the donor site, or disease progression limit the availability of native grafts. 12,13 Clinical studies suggest that only a limited number of patients undergoing CABG surgeries have suitable arterial grafts and up to 30% of patients requiring venous grafts for peripheral vascular diseases lack transplantable veins. 14,15 While synthetic vascular prostheses such as expanded polytetrafluoroethylene (ePTFE) and Dacron are available alternatives for high-flow, low-resistance, large peripheral vessel pathologies, their clinical outcome for small-diameter (< 6 mm) vessel replacement has been grim. 16-20 Prosthetic graft failure has been attributed to intimal hyperplasia, thrombogenicity, compliance mismatch, and diameter mismatch between the graft and native artery. 21-24 Despite decades of effort, the successful fabrication of an ideal vascular graft still remains a challenge. Ideally, a vascular graft should be strong, biocompatible, nontoxic, nonimmunogenic, antithrombotic, compliant, vasoactive, and amenable to postimplantation remodeling by the host tissue. To this end, tissue-engineered vessels (TEVs) that can withstand the challenging arterial hemodynamic microenvironment and are amenable to physiological remodeling represent an attractive alternative. Vascular Tissue Engineering Approaches Three major approaches have been proposed for the tissue engineering of vascular grafts: (1) decellularized matrices; (2) cell-sheet engineering; and (3) biodegradable scaffolds from natural or synthetic polymers. Decellularized blood vessels as well as small intestinal submucosa (SIS) have been used to fabricate vascular grafts. The main advantage of using decellularized tissue is that the native three-dimensional (3D) architecture of matrix molecules-mainly type 1 collagen and elastin-is preserved 25 and might be helpful in guiding tissue repair and remodeling postimplantation. Decellularized blood vessels
Tissue Engineering, 2004
Advances in understanding the complex process of wound healing and development of novel growth fa... more Advances in understanding the complex process of wound healing and development of novel growth factor and gene therapies would benefit from models that mimic closely the physiology of human wounds. To this end, we developed a hybrid wound-healing model based on human tissue-engineered skin transplanted onto athymic mice. Grafted tissues were infiltrated with mouse mesenchymal cells as native and foreign dermal regions fused together. Immunohistochemical staining for human involucrin revealed that the transplanted epithelium maintained its human origin, whereas the dermis was infiltrated by numerous mouse fibroblasts and blood vessels. Grafted tissues were wounded with a 4-mm punch to create full-thickness excisional wounds. At 1 and 2 weeks, the tissues were excised and assessed for reepithelialization, differentiation, and neovascularization. Interestingly, the average rate of keratinocyte migration (120 microm/day) was similar to migration rates observed in human subjects and significantly lower than migration in mouse epidermis. Immunohistochemical staining for keratin 10, laminin, and involucrin revealed a normal pattern of differentiation in the neoepidermis. Neovascularization was significantly elevated in the granulation tissue at 1 week and subsided to the level of unwounded tissue at 2 weeks postwounding. Our data suggest that skin equivalents grafted to a mouse model may serve as a realistic model of human wound regeneration. Because skin equivalents can be prepared with patient cells and genetically modified to stimulate or suppress gene expression, this model may be ideal for addressing mechanistic questions and evaluating the efficacy of biomaterials and gene therapeutics for promoting wound healing.
The FASEB Journal, 2013
Functional smooth muscle engineering requires isolation and expansion of smooth muscle cells (SMC... more Functional smooth muscle engineering requires isolation and expansion of smooth muscle cells (SMCs), and this process is particularly challenging for visceral smooth muscle tissue where progenitor cells have not been clearly identified. Herein we showed for the first time that efficient SMCs can be obtained from human amniotic fluid stem cells (hAFSCs). Clonal lines were generated from c-kit ؉ hAFSCs. Differentiation toward SM lineage (SMhAFSCs) was obtained using a medium conditioned by PDGF-BB and TGF-1. Molecular assays revealed higher level of ␣ smooth muscle actin (␣-SMA), desmin, calponin, and smoothelin in SMhAFSCs when compared to hAFSCs. Ultrastructural analysis demonstrated that SMhAFSCs also presented in the cytoplasm increased intermediate filaments, dense bodies, and glycogen deposits like SMCs. SMhAFSC metabolism evaluated via mass spectrometry showed higher glucose oxidation and an enhanced response to mitogenic stimuli in comparison to hAFSCs. Patch clamp of transduced hAFSCs with lentiviral vectors encoding ZsGreen under the control of the ␣-SMA promoter was performed demonstrating that SMhAFSCs retained a smooth muscle cell-like electrophysiological fingerprint. Eventually SMhAFSCs contractility was evident both at single cell level and on a collagen gel. In conclusion, we showed here that hAFSCs under selective culture conditions are able to give rise to functional SMCs.
Journal of Investigative Dermatology, 2003
Journal of Investigative Dermatology, 2003
Journal of Cell Science, 2013
We demonstrate that JNK responds to substrate stiffness and regulates AJ formation in epithelial ... more We demonstrate that JNK responds to substrate stiffness and regulates AJ formation in epithelial cells in 2D cultures and in 3D tissues in vitro and in vivo. Rigid substrates led to JNK activation and AJ disassembly, while soft matrices suppressed JNK activity leading to AJ formation. Expression of constitutively active JNK (MKK7-JNK1) induced AJ dissolution even on soft substrates, while JNK knockdown (shJNK) induced AJ formation even on hard substrates. In human epidermis, basal cells expressed phosphorylated (p)-JNK but lacked AJ, while suprabasal keratinocytes contained strong AJ but lacked p-JNK. AJ formation was significantly impaired even in the upper suprabasal layers of bioengineered epidermis when prepared with stiffer scaffold or MKK7-JNK1 expressing keratinocytes. In contrast, shJNK1 or shJNK2 epidermis exhibited strong AJ even in the basal layer. The results with bioengineered epidermis were in full agreement with the epidermis of jnk1−/− or jnk2−/− mice. In conclusion,...
Experimental Cell Research, 2009
EGF is known to affect adherens junctions and disrupt cell-cell adhesion in a variety of carcinom... more EGF is known to affect adherens junctions and disrupt cell-cell adhesion in a variety of carcinomas but the underlying mechanisms are not completely understood. Using human tumor epithelial cells overexpressing EGFR we demonstrated that EGF-induced cell scattering was mediated by protein kinase C-delta (PKC-delta). PKC-delta knockdown by siRNA significantly inhibited EGF-induced internalization of E-cadherin into the cytoplasm and blocked cell scattering. EGF phosphorylated PKC-delta at Y311 and ectopic expression of the mutant Y311F prevented PKC-delta binding to E-cadherin and EGF-induced cell scattering. Moreover, depletion of Src using siRNA decreased EGF-induced phosphorylation of PKC-delta at Y311 and blocked scattering. Finally, EGF reduced expression of the tight junction protein, occludin, and this effect was also mediated by PKC-delta through Src. In summary, PKC-delta mediated the effects of EGF on adherens and tight junctions thereby playing an important role in cell-cell adhesion with possible wider implications in tumor metastasis or epithelial-to-mesenchymal transition.
Current Opinion in Biotechnology, 2013
Due to rise in cardiovascular disease throughout the world, there is increasing demand for small ... more Due to rise in cardiovascular disease throughout the world, there is increasing demand for small diameter blood vessels as replacement grafts. The present review focuses on the animal models that have been used to test small-diameter TEVs with emphasis on the attributes of each model. Small animal models are used to test short-term patency and address mechanistic hypotheses; and large, pre-clinical animal models are employed to test long-term patency, remodeling and function in an environment mimicking human physiology. We also discuss recent clinical trials that employed laboratory fabricated TEVs and showed very promising results. Ultimately, animal models provide a testing platform for optimizing vascular grafts before clinical use in patients without suitable autologous vessels.
Biotechnology Progress, 2014
There have been many clinical trials recently using ex vivo-expanded human mesenchymal stem cells... more There have been many clinical trials recently using ex vivo-expanded human mesenchymal stem cells (MSCs) to treat several indications such as graft-versus-host disease, acute myocardial infarction, Crohn's disease, and multiple sclerosis. However, the conventional 2-dimensional (2D) culture of MSCs is laborious and limited in scale potential. The large dosage requirement for many of the indications further exacerbates this manufacturing challenge. In contrast, spheroid MSC culture does not require a cell attachment surface and is amenable to large-scale suspension cell culture techniques, such as stirred-tank bioreactors. In this present study, we developed and optimized serum free media for culturing MSC spheroids. We used Design of Experiment (DoE)based strategies to systematically evaluate media mixtures and a panel of different components. The optimization yielded two prototype media that could allow MSCs to form aggregates and proliferate in both static cultures and dynamic cultures. The expanded MSCs expressed the expected surface markers for mesenchymal cells (CD73, CD90 and CD105). In addition, the expanded cells demonstrated multipotency and differentiated to the osteocyte, chondrocyte and adipocyte lineages, which showed similar or enhanced differentiation levels compared with serumcontaining adherent cultures.
Biotechnology and Bioengineering, 2005
Although recombinant retroviruses are widely used in gene therapy and as gene transfer vehicles f... more Although recombinant retroviruses are widely used in gene therapy and as gene transfer vehicles for basic biological studies, their titers are very low as compared to other recombinant viral systems, e.g., adenovirus. We investigated the rate-limiting steps in production of LacZ-encoding ecotropic (CRE BAG 2) and amphotropic (É-CRIP) retrovirus. We found that ecotropic retrovirus producer cells produced a large number of inactive viral particles because they were severely limited by the amount of mRNA that was packaged into viral capsids. Introduction of the gene for green fluorescence protein (GFP) increased retroviral titers 40-fold, without affecting the viral matrix protein, p30, or the activity of reverse transcriptase. Surprisingly, while transfer of GFP gene increased retrovirus production, b-gal activity and X-gal titer decreased significantly. Quantitative real-time polymerase chain reaction (PCR) showed that although producer cells synthesized similar amounts of both mRNAs, retroviral supernatants contained significantly lower amount of LacZ mRNA, possibly due to competition between LacZ and GFP mRNAs for encapsidation into virions. In contrast to ecotropic producers, introduction of GFP gene copies into amphotropic producers resulted in a moderate twofold increase in retrovirus production. However, delivery of genes encoding for the viral proteins gp70 and p30 increased virus production by fivefold, suggesting that amphotropic producers may also be limited by synthesis of structural viral proteins. Our data show that in addition to the amount of viral genome or proteins, assembly of viral components into active viral particles may limit production of high titer retroviral preparations.
American Journal of Obstetrics and Gynecology, 2010
This study was undertaken to isolate and characterize multipotent mesenchymal stem cells from ter... more This study was undertaken to isolate and characterize multipotent mesenchymal stem cells from term human placenta (placenta-derived mesenchymal stem cells, PD-MSCs). STUDY DESIGN: Sequential enzymatic digestion was used to isolate PD-MSCs in which trypsin removes the trophoblast layer, followed by collagenase treatment of remaining placental tissue. Karyotype, phenotype, growth kinetics, and differentiability of PD-MSC isolates from collagenase digests were analyzed. RESULTS: PD-MSC isolation was successful in 14 of 17 cases. Karyotyping of PD-MSC isolates from deliveries with a male fetus revealed that these cells are of maternal origin. Flow cytometry and immunocytochemistry confirmed the mesenchymal stem cell phenotype. Proliferation rates of PD-MSCs remained constantly high up to passage 20. These cells could be differentiated toward mesodermal lineage in vitro up to passage 20. Nonconfluent culture was critical to maintain the MSC stemness during long-term culture. CONCLUSION: Term placenta constitutes a rich, very reliable source of maternal mesenchymal stem cells that remain differentiable, even at high passage numbers.
Tissue engineering. Part A, Jul 7, 2016
Bone is a highly vascularized tissue and efficient bone regeneration requires neovascularization,... more Bone is a highly vascularized tissue and efficient bone regeneration requires neovascularization, especially for critical size bone defects. We developed a novel hybrid biomaterial comprising of calcium sulfate (nCS) nanoparticles and fibrin hydrogel to deliver mesenchymal stem cells (MSCs) and angiogenic factors VEGF and FGF9 in order to promote neovascularization and bone formation. MSC and growth factor(s) loaded scaffolds were implanted subcutaneously into mice to examine their angiogenic and osteogenic potential. Micro CT, alkaline phosphatase activity assay and histological analysis were used to evaluate bone formation, while immunohistochemistry was employed to assess neovessel formation. The presence of fibrin preserved the nCS scaffold structure and promoted de novo bone formation. In addition, the presence of BMP2-expressing MSC in nCS and fibrin hydrogels improved bone regeneration significantly. While FGF9 alone had no significant effect, the combination FGF9 and VEGF co...
PloS one, 2017
Hyposalivation reduces the patient quality of life, as saliva is important for maintaining oral h... more Hyposalivation reduces the patient quality of life, as saliva is important for maintaining oral health. Current treatments for hyposalivation are limited to medications such as the muscarinic receptor agonists, pilocarpine and cevimeline. However, these therapies only provide temporary relief. Therefore, alternative therapies are essential to restore salivary gland function. An option is to use bioengineered scaffolds to promote functional salivary gland regeneration. Previous studies demonstrated that the laminin-111 protein is critical for intact salivary gland cell cluster formation and organization. However, laminin-111 protein as a whole is not suitable for clinical applications as some protein domains may contribute to unwanted side effects such as degradation, tumorigenesis and immune responses. Conversely, the use of synthetic laminin-111 peptides makes it possible to minimize the immune reactivity or pathogen transfer. In addition, it is relatively simple and inexpensive as...
TECHNOLOGY
Recent advances in vascular tissue engineering have led to the development of cell-free grafts th... more Recent advances in vascular tissue engineering have led to the development of cell-free grafts that are available off-the-shelf for on demand surgery. Challenges associated with cell-based technologies including cell sourcing, cell expansion and long-term bioreactor culture motivated the development of completely cell-free vascular grafts. These are based on decellularized arteries, decellularized cultured cell-based tissue engineered grafts or biomaterials functionalized with biological signals that promote in situ tissue regeneration. Clinical trials undertaken to demonstrate the applicability of these grafts are also discussed. This comprehensive review summarizes recent developments in vascular graft technologies, with potential applications in coronary artery bypass procedures, lower extremity bypass, vascular injury and trauma, congenital heart diseases and dialysis access shunts, to name a few.
Stem cells (Dayton, Ohio), May 31, 2017
During development, neural crest cells are induced by signaling events at the neural plate border... more During development, neural crest cells are induced by signaling events at the neural plate border of all vertebrate embryos. Initially arising within the central nervous system, neural crest cells subsequently undergo an epithelial to mesenchymal transition to migrate into the periphery, where they differentiate into diverse cell types. Here we provide evidence that postnatal human epidermal keratinocytes, in response to FGF2 and IGF1 signals, can be reprogrammed toward a neural crest fate. Genome-wide transcriptome analyses show that keratinocyte-derived neural crest cells are similar to those derived from human embryonic stem cells. Moreover, they give rise in vitro and in vivo to neural crest derivatives such as peripheral neurons, melanocytes, Schwann cells and mesenchymal cells (osteocytes, chondrocytes, adipocytes and smooth muscle). By demonstrating that human KRT14+ keratinocytes can form neural crest cells, even from clones of single cells, our results have important implic...
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Papers by Stelios Andreadis