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PubMed2006

Photopatterned polymer brushes promoting cell adhesion gradients.

The ability to spatially control cellular adhesion in a continuous manner on a biocompatible substrate is an important factor in designing new biomaterials for use in wound healing and tissue engineering applications. In this work, a novel method of engineering cell-adhesive RGD-ligand density gradients to control specific cell adhesion across a substrate is presented. Polymer brushes exhibiting spatially defined gradients in chain density are created and subsequently functionalized with RGD to create ligand density gradients capable of inducing cell adhesion on an otherwise weakly adhesive substrate. Cell studies indicate that these ligand-functionalized surfaces are noncytotoxic, with cellular adhesion increasing with RGD-ligand density across the gradient brush surface.

3T3 Cells↗

PubMed2025

Engineering recombination machinery facilitates the construction of yeast cell factories.

Advances in genome editing have been promoted by programmable nucleases like CRISPR-Cas9, which triggers endogenous DNA repair mechanisms by inducing double-strand break (DSB). Cellular responses to DSBs are governed by competing repair pathways: error-prone non-homologous end joining (NHEJ) and high-fidelity homologous recombination (HR). This review systematically compares the molecular mechanisms and key regulators of NHEJ and HR, with a focus on recent breakthroughs in recombination engineering in non-conventional yeasts. These advances address challenges in precise genome editing, enabling robust metabolic engineering of yeast cell factories for sustainable bioproduction.

Metabolic Engineering↗

PubMed2006

Modeling suppression of cell death by Bcl-2 over-expression in myeloma NS0 6A1 cells.

A novel population-balance model was employed to evaluate the suppression of cell death in myeloma NS0 6A1 cells metabolically engineered to over-express the apoptotic suppressor Bcl-2. The model is robust in its ability to simulate cell population dynamics in batch suspension culture and in response to thymidine-induced growth inhibition: 89% of simulated cell concentrations are within two standard deviations of experimental data. Kinetic rate constants in model equations suggest that Bcl-2 over-expression extends culture longevity from 6 days to at least 15 days by suppressing the specific rate of early apoptotic cell formation by more than 6-fold and necrotic cell formation by at least 3-fold, despite nearly a 3-fold decrease in initial cell growth rate and no significant change in the specific rate of late apoptotic cell formation. This computational analysis supports a mechanism in which Bcl-2 is a common mediator of early apoptotic and necrotic events occurring at rates that are dependent on cellular factors accumulating over time. The model has current application to the rational design of cell cultures through metabolic engineering for the industrial production of biopharmaceuticals.

Apoptosis↗

PubMed2005

Recellularization of decellularized allograft scaffolds in ovine great vessel reconstructions.

BACKGROUND: Decellularized allograft tissues have been identified as a potential extracellular matrix (ECM) scaffold on which to base recellularized tissue-engineered vascular and valvular substitutes. Decreased antigenicity and the capacity to recellularize suggest that such constructs may have favorable durability. Detergent/enzyme decellularization methods remove cells and cellular debris while leaving intact structural protein "scaffolds." Allograft pulmonary artery tissues decellularized with an anionic detergent/enzyme methodology were tested in a long-term implantation model that used arterial wall repairs in the great vessels of juvenile sheep. METHODS: Twelve test sheep were implanted (n = 4) for each of three different scaffold protocols that compared traditional dimethylsulfoxide cryopreservation, cryopreservation followed by decellularization, and decellularization of fresh tissue. Four additional sheep served as controls (n = 2 sham, n = 2 fresh tissue). Patches were fashioned and implanted into pulmonary artery and aortic defects. Panel reactive antibodies (PRA) were measured over time (10 to 20 weeks). Explant histopathology determined recellularization morphology as well as calcium, collagen, and elastin distribution within explanted tissue. RESULTS: Unlike traditionally cryopreserved tissues, the decellularized tissues contained no residual cells or cellular debris before implantation, which correlated with measurable reductions in PRA. Decellularized explants demonstrated time-dependent migration of recipient cells through matrix, typically staining positive for alpha-smooth muscle actin with no calcification. CONCLUSIONS: The properties demonstrated seem consistent with characteristics necessary for implantable tissue-engineered scaffolds. The decellularization method described appears to create a biologically suitable ECM scaffold for in vivo migration of phenotypically appropriate cells while avoiding antigenicity and calcification.

Animals↗

PubMed2004

Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells.

Bone marrow mesenchymal stem cells (MSCs) can differentiate into a variety of cell types, including vascular smooth muscle cells (SMCs), and have tremendous potential as a cell source for cardiovascular regeneration. We postulate that specific vascular environmental factors will promote MSC differentiation into SMCs. However, the effects of the vascular mechanical environment on MSCs have not been characterized. Here we show that mechanical strain regulated the expression of SMC markers in MSCs. Cyclic equiaxial strain downregulated SM alpha-actin and SM-22alpha in MSCs on collagen- or elastin-coated membranes after 1 day, and decreased alpha-actin in stress fibers. In contrast, cyclic uniaxial strain transiently increased the expression of SM alpha-actin and SM-22alpha after 1 day, which subsequently returned to basal levels after the cells aligned in the direction perpendicular to the strain direction. In addition, uniaxial but not equiaxial strain induced a transient increase of collagen I expression. DNA microarray experiments showed that uniaxial strain increased SMC markers and regulated the expression of matrix molecules without significantly changing the expression of the differentiation markers (e.g., alkaline phosphatase and collagen II) of other cell types. Our results suggest that uniaxial strain, which better mimics the type of mechanical strain experienced by SMCs, may promote MSC differentiation into SMCs if cell orientation can be controlled. This study demonstrates the differential effects of equiaxial and uniaxial strain, advances our understanding of the mechanical regulation of stem cells, and provides a rational basis for engineering MSCs for vascular tissue engineering and regeneration.

Biomarkers↗

PubMed2006

Adipose tissue engineering by human adipose-derived stromal cells.

Tissue engineering has emerged as a promising alternative approach to current clinical treatments for restoration of soft tissue defects. The purpose of this study was to investigate adipose tissue formation in vitro and in vivo by using human adipose-derived stromal cells (ADSCs) utilizing a gelatin sponge (Gelform) as a scaffold. Adipogenic potentials of human ADSCs were demonstrated by Oil-O-red staining and cellular morphology. After seeding human ADSCs in a density of 3 x 10(6) cells/ml on three-dimensional gelatin sponges, tissue-engineered constructs were exposed to adipogenic differentiation medium for in vitro studies and implanted in the backs of severe combined immunodeficient (SCID) mice for in vivo adipose regeneration. Adipogenesis of ADSC-seeded gelatin sponges was confirmed by Oil-O-red staining after 4 weeks of in vitro incubation. The optical density of the elution from Oil-O-red staining of adipogenic constructs is significantly higher than that of the control group (p < 0.05, n = 4). With short-term in vitro differentiation, adipogenic constructs turned into fat tissue 4 weeks after in vivo implantation, confirmed by biochemical and immunohistochemical examination. No adipogenic-morphological change or fat formation was observed in in vitro or in vivo studies when ADSCs were exposed to a control medium without adipogenic stimulation. These results indicate that engineered adipose tissue can be achieved using human ADSCs and biocompatible and degradable gelatin sponges.

Adipose Tissue↗

PubMed2003

Tissue-engineered heart valve on acellular aortic valve scaffold: in-vivo study.

The feasibility of constructing a tissue-engineered heart valve on an acellular porcine aortic valve leaflet was evaluated. A detergent and enzymatic extraction process was developed to remove the cellular components from porcine aortic valves. The acellular valve leaflets were seeded for 7 days in vitro with cells from canine arterial wall and endothelial cells. The constructs were implanted into the lumens of 6 canine abdominal aortas to assess the reconstruction of the valve leaflets. It was found that all cellular components had been removed from the porcine aortic valves. The valve leaflets were completely reconstructed at the end of the 10th week in vivo. Scanning electron microscopy showed that the valve leaflets were partially covered with endothelial cells. It was concluded that porcine aortic valves can be decellularized by the detergent and enzymatic extraction process and it is feasible to construct a tissue-engineered heart valve in vivo on an acellular valve scaffold.

Animals↗

PubMed2005

Mapping cellular transcriptosomes in autopsied Alzheimer's disease subjects and relevant animal models.

Alzheimer's disease (AD) is a late-onset and progressive neurodegenerative disorder characterized clinically by memory loss, impairment of other cognitive functions, and changes in behavior and personality. The overall aim of this review is to summarize recent advances in studies of AD progression and the use of animal models in gene expression studies of AD progression. Genetic causes of AD are known only for early-onset AD patients. For a majority of late-onset AD patients, causal factors are still unknown. Currently, there are no early detectable biomarkers for late-onset AD, and there is a lack of understanding of AD pathophysiology, particularly at the early stages of disease progression, before pathology develops. Human histopathological and biochemical studies provide valuable information regarding the last stages of AD pathogenesis. However, to understand early cellular changes in AD progression before symptoms develop, animal models are still our only alternative. Several research groups have created genetically engineered animal models, particularly models of the mouse, rat, fly, and worm, which have allowed us to better, understand the initiating events of AD progression. Recently, state-of-the-art methods have helped elucidate gene expression changes in affected and unaffected tissues from postmortem AD brains and from animal models developed for AD studies. These methods allow the investigation of mRNA-based transcriptosomal profiles of brain specimens from AD humans and transgenic animals. The major finding from these studies is that AD progression and pathogenesis involve multiple cellular pathways, which suggests that AD is a complex and heterogeneous disease.

Alzheimer Disease↗

PubMed2003

Dissecting autoimmune diabetes through genetic manipulation of non-obese diabetic mice.

Type 1 diabetes results from a genetically and immunologically complex autoimmune process that is specifically directed against the pancreatic beta cells. Non-obese diabetic mice spontaneously develop a form of autoimmune diabetes closely resembling the disease in humans. This happens because, like human diabetic patients, non-obese diabetic mice have an unfortunate combination of apparently normal alleles at numerous loci associated with Type 1 diabetes. In isolation, each of these allelic variants affords a small degree of susceptibility to diabetes. In combination, however, they set in motion a series of immunological events that lead to islet inflammation and overt diabetes. Type 1 diabetes is associated with defects in self-tolerance and immunoregulation. It involves presentation of beta cell antigens to autoreactive T lymphocytes by professional antigen-presenting cells, the recruitment of antigen-activated T cells into pancreatic islets, and the differentiation of these antigen-activated lymphocytes into beta cell killers. Understanding the precise sequence of events in the pathogenesis of Type 1 diabetes has been, and remains, a challenging task. Much of our understanding of the immunology of the disease stems from studies of genetically engineered, non-obese diabetic mice. These mice provide reductionist systems, with which the contribution of individual cellular elements, molecules or genes to the disease process can be dissected. This review focuses on the lessons that have been learned through studies of these mice.

Animals↗

PubMed2003

Recovery from heart failure with circulatory assist: a working group of the National, Heart, Lung, and Blood Institute.

BACKGROUND: Over the past decade, mechanical circulatory support has been beneficial as a bridge to cardiac transplantation, and anecdotal evidence suggests that heart failure patients fitted with mechanical assist devices experience direct cardiac benefits. Moreover, recent trials on limited numbers and subpopulations of patients--notably the Randomized Evaluation of Mechanical Assistance for the Treatment of Congestive Heart Failure (REMATCH)--support earlier observations of improved cardiac function and point towards the use of assist devices as destination therapy. Methods and results To investigate this phenomenon, on August 2-3, 2001, the National Heart, Lung, and Blood Institute convened the working group, "Recovery from Heart Failure with Circulatory Assist" in Bethesda, Maryland. The team included cardiac surgeons, cardiologists, and experts in experimental research. The goal was to prioritize recommendations to guide future programs in: (1). elucidating the mechanisms leading to reverse remodeling associated with a left ventricular assist device (LVAD); (2). exploring advanced treatments, including novel pharmacologies, tissue engineering, and cell therapies, to optimize recovery with LVAD therapy; and (3). identifying target genes, proteins, and cellular pathways to focus on for production of novel therapies for myocardial recovery and cardiovascular disease. CONCLUSIONS: The working group also made research and clinical recommendations to eventually translate findings into improved therapeutic strategies and device design: (1). support collaborations among clinical and basic scientists with an emphasis on clinical/translational research that might eventually lead to clinical trials; (2). identify candidate patients most likely to benefit from LVAD as a destination therapy; (3). explore potential biomarkers indicating when patients could most successfully be weaned from devices; and (4). promote clinical and experimental study of mechanically assisted organs and the tissue derived from them.

Adrenergic beta-Antagonists↗

PubMed2005

Biomaterial coatings by stepwise deposition of silk fibroin.

A completely aqueous, stepwise deposition process with Bombyx mori silk fibroin for the assembly of nanoscale thin film coatings is reported the first time. The focus of this work was to develop an understanding of the control of this deposition process and to characterize the films formed from a physicochemical perspective. The deposition process was monitored by UV spectrophotometry and research quartz crystal microbalance. Both absorbance and film thickness correlated linearly with the number of silk fibroin layers deposited, analogous to multilayered materials fabricated from conventional polyelectrolytes. The polymer adsorption process was stable and reproducible, with control of a single layer thickness ranging from a few to tens of nanometers, determined by the concentrations of silk fibroin, salt concentration in the dipping solution, and method of rinsing. The driving force for the assembly of silk fibroin onto the substrate was primarily hydrophobic interactions, while some electrostatic interactions were also involved. The difference with this approach from traditional polyelectrolyte layer-by-layer techniques is that an intervening drying step is used to control the structure and stability of the self-assembled silk fibroin. The assembled films were stable under physiological conditions and supported human bone marrow stem cell adhesion, growth, and differentiation. This approach offers new options to engineer biomaterial coatings as well as bulk materials with control of both interfacial properties conducive to specific cellular or tissue responses and the potential to entrap and deliver labile molecules or other components due to the all-aqueous process described.

Bone Marrow Cells↗

PubMed2006

Highly sensitive biomolecular fluorescence detection using nanoscale ZnO platforms.

Fluorescence detection is currently one of the most widely used methods in the areas of basic biological research, biotechnology, cellular imaging, medical testing, and drug discovery. Using model protein and nucleic acid systems, we demonstrate that engineered nanoscale zinc oxide structures can significantly enhance the detection capability of biomolecular fluorescence. Without any chemical or biological amplification processes, nanoscale zinc oxide platforms enabled increased fluorescence detection of these biomolecules when compared to other commonly used substrates such as glass, quartz, polymer, and silicon. The use of zinc oxide nanorods as fluorescence enhancing substrates in our biomolecular detection permitted sub-picomolar and attomolar detection sensitivity of proteins and DNA, respectively, when using a conventional fluorescence microscope. This ultrasensitive detection was due to the presence of ZnO nanomaterials which contributed greatly to the increased signal-to-noise ratio of biomolecular fluorescence. We also demonstrate the easy integration potential of zinc oxide nanorods into periodically patterned nanoplatforms which, in turn, will promote the assembly and fabrication of these materials into multiplexed, high-throughput, optical sensor arrays. These zinc oxide nanoplatforms will be extremely beneficial in accomplishing highly sensitive and specific detection of biological samples involving nucleic acids, proteins and cells, particularly under detection environments involving extremely small sample volumes of ultratrace-level concentrations.

DNA↗

PubMed2003

Modulation of berberine bridge enzyme levels in transgenic root cultures of California poppy alters the accumulation of benzophenanthridine alkaloids.

California poppy (Eschscholzia californica Cham.) root cultures produce a variety of benzophenanthridine alkaloids, such as sanguinarine, chelirubine and macarpine, with potent biological activity. Sense and antisense constructs of genes encoding the berberine bridge enzyme (BBE) were introduced into California poppy root cultures. Transgenic roots expressing BBE from opium poppy (Papaver somniferum L.) displayed higher levels of BBE mRNA, protein and enzyme activity, and increased accumulation of benzophenanthridine alkaloids compared to control roots transformed with a beta-glucuronidase gene. In contrast, roots transformed with an antisense-BBE construct from California poppy had lower levels of BBE mRNA and enzyme activity, and reduced benzophenanthridine alkaloid accumulation, relative to controls. Pathway intermediates were not detected in any transgenic root lines. Suppression of benzophenanthridine alkaloid biosynthesis using antisense-BBE also reduced the growth rate of the root cultures. Two-dimensional 1H-NMR spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic roots lines. However, transformed roots with low levels of benzophenanthridine alkaloids contained larger cellular pools of certain amino acids compared to controls. In contrast, cellular pools of several amino acids were reduced in transgenic roots with elevated benzophenanthridine alkaloid levels relative to controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was only marginally altered in all transgenic root lines; thus, altering metabolic flux through benzophenanthridine alkaloid pathways can affect cellular pools of specific amino acids. Consideration of such interactions is important for the design of metabolic engineering strategies that target benzophenanthridine alkaloid biosynthesis.

Alkaloids↗

PubMed1997

Biologically erodable microspheres as potential oral drug delivery systems.

Biologically adhesive delivery systems offer important advantages over conventional drug delivery systems. Here we show that engineered polymer microspheres made of biologically erodable polymers, which display strong adhesive interactions with gastrointestinal mucus and cellular linings, can traverse both the mucosal absorptive epithelium and the follicle-associated epithelium covering the lymphoid tissue of Peyer's patches. The polymers maintain contact with intestinal epithelium for extended periods of time and actually penetrate it, through and between cells. Thus, once loaded with compounds of pharmacological interest, the microspheres could be developed as delivery systems to transfer biologically active molecules to the circulation. We show that these microspheres increase the absorption of three model substances of widely different molecular size: dicumarol, insulin and plasmid DNA.

Adhesiveness↗

PubMed2026

TogoPhosTAC as a delivery-ready platform for targeted protein dephosphorylation.

Phosphorylation-targeting chimeras (PhosTACs) enable targeted protein dephosphorylation by recruiting phosphatases through induced proximity. However, the direct recruitment of phosphatase subunits or holoenzymes with small molecules remains challenging, as suitable ligands are scarce and often compromise enzymatic activity or cellular function. Here, we present togoPhosTAC, a hybrid modality that integrates a small-molecule PhosTAC, an engineered FKBP12F36V-phosphatase, and a lipid nanoparticle delivery system. This strategy allows delivery of preassembled PhosTAC-FKBP12F36V-phosphatase complexes or PhosTAC-phosphatase mRNA, enabling rapid and efficient intracellular dephosphorylation. We demonstrate that togoPhosTAC can selectively dephosphorylate EGFR, α-synuclein, and tau in biological contexts, providing a versatile strategy that circumvents the need for genetically engineered phosphatases. We also find togoPhosTAC further enhances tau dephosphorylation as well as its disaggregation in cellulo. Importantly, intrahippocampal or intranasal delivery of togoPhosTAC in PS19 tau transgenic male mice leads to a marked reduction in pathological tau phosphorylation across multiple sites (Ser202, Thr205, Thr231, Ser396, and Ser404), decreases pathological tau burden in related brain regions, and improves Alzheimer's disease-related behavioral deficits. Together, these findings establish a versatile and generalizable approach for precise protein dephosphorylation in disease-relevant systems, overcoming key limitations in phosphatase-recruiting drug discovery.

Animals↗

PubMed1992

Design of yeast-secreted albumin derivatives for human therapy: biological and antiviral properties of a serum albumin-CD4 genetic conjugate.

Due to its remarkably long half-life, together with its wide in vivo distribution and its lack of enzymatic or immunological functions, human serum albumin (HSA) represents an optimal carrier for therapeutic peptides/proteins aimed at interacting with cellular or molecular components of the vascular and interstitial compartments. As an example, we designed a genetically engineered HSA-CD4 hybrid aimed at specifically blocking the entry of the human immunodeficiency virus into CD4+ cells. In contrast with CD4, HSA-CD4 is correctly processed and efficiently secreted by Kluyveromyces yeasts. In addition, its CD4 moiety exhibits binding and antiviral in vitro properties similar to those of soluble CD4. Finally, the elimination half-life of HSA-CD4 in a rabbit experimental model is comparable to that of control HSA and 140-fold higher than that of soluble CD4. These results indicate that the genetic fusion of bioactive peptides to HSA is a plausible approach toward the design and recovery of secreted therapeutic HSA derivatives with appropriate pharmacokinetic properties.

Animals↗

PubMed1996

Specific triggering of the Fas signal transduction pathway in normal human keratinocytes.

The epidermis is continually exposed to genotoxic injury and requires an efficient mechanism to eliminate genetically altered cells. The membrane receptor, Fas, initiates apoptosis in many cell types, including keratinocytes. Receptor cross-linking is the vital post-ligand binding step in Fas signal transduction, and we have utilized FK1012, capable of oligomerizing proteins engineered to contain the FK506 binding protein (FKBP), to trigger Fas via FKBP-linked receptor cytoplasmic domains in human keratinocytes. An FKBP chimera containing the Fas cytoplasmic domain targeted to the plasma membrane induced an up to 89% decrease in viability of keratinocytes, as reflected by the activity of constitutive promoters, in response to FK1012. Oligomerization of Fas, either with engineered Fas.FKBP by FK1012 or via antibody cross-linking of full-length Fas-induced cellular changes consistent with apoptosis. The lpr Fas point mutation abolished this effect. A Fas.FKBP construct unlinked to the membrane was fully active in this assay. Early developmental age or pre-treatment of cells with GM-CSF, TGF-beta, EGF, KGF, IFN-gamma, or phorbol ester failed to protect against Fas effects. These findings reveal that the Fas signal transduction pathway is active in keratinocytes, requires no induction, and dominantly overrides growth stimuli.

Apoptosis↗

PubMed2003

Models of breast cancer: quo vadis, animal modeling?

Rodent models for breast cancer have for many decades provided unparalleled insights into cellular and molecular aspects of neoplastic transformation and tumorigenesis. Despite recent improvements in the fidelity of genetically engineered mice, rodent models are still being criticized by many colleagues for not being 'authentic' enough to the human disease. Motives for this criticism are manifold and range from a very general antipathy against the rodent model system to well-founded arguments that highlight physiological variations between species. Newly proposed differences in genetic pathways that cause cancer in humans and mice invigorated the ongoing discussion about the legitimacy of the murine system to model the human disease. The present commentary intends to stimulate a debate on this subject by providing the background about new developments in animal modeling, by disputing suggested limitations of genetically engineered mice, and by discussing improvements but also ambiguous expectations on the authenticity of xenograft models to faithfully mimic the human disease.

Animals↗