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PubMed1983

Cell separation using positive immunoselective techniques.

Positive immunoselection is the direct selection and recovery of cells which express a given specificity from among a heterogeneous group of contaminating cells. A variety of methods are available to effect such separations. The principles of affinity chromatography, using solid-phase matrices or cellular immunoadsorbents, are extensively used. Liquid-phase positive immunoselection can also be performed using either a fluorescence-activated cell sorter or by using 'cellular engineering' to protect a cell from an otherwise noxious environment. The enzyme catalase coupled to specific antibody has been used for this purpose and renders cells resistant to hydrogen peroxide. The various positive immunoselection techniques available are reviewed and evaluated in the following report.

Animals↗

PubMed2003

Induction of neural differentiation by electrically stimulated gene expression of NeuroD2.

Regulation of cell differentiation is an important assignment for cellular engineering. One of the techniques for regulation is gene transfection into undifferentiated cells. Transient expression of NeuroD2, one of neural bHLH transcription factors, converted mouse N1E-115 neuroblastoma cells into differentiated neurons. The regulation of neural bHLH expression should be a novel strategy for cell differentiation. In this study, we tried to regulate neural differentiation by NeuroD2 gene inserted under the control of heat shock protein-70 (HSP) promoter, which can be activated by electrical stimulation. Mouse neuroblastoma cell line, N1E-115, was stably transfected with expression vector containing mouse NeuroD2 cDNA under HSP promoter. Transfected cells were cultured on the electrode surface and applied electrical stimulation. After stimulation, NeuroD2 expression was induced, and transfected cells adopt a neuronal morphology at 3 days after stimulation. These results suggest that neural differentiation can be induced by electrically stimulated gene expression of NeuroD2.

Animals↗

PubMed2001

A multicellular spheroid-based sensor for anti-cancer therapeutics.

The progress in cellular engineering offers novel approaches for anti-cancer therapies. To investigate the effectiveness of potential therapies efficient screening methods are required. We propose an impedance measurement system which enables the use of multicellular spheroid models in bioelectronic screening systems either for non destructive life-time diagnostic or anti-cancer therapies. A biohybrid sensor system is created comprising gene-manipulated T47D clone 11 breast carcinoma spheroids positioned hydrodynamically in a capillary system with electrodes. A novel approach employing an antisense-5'butyrylcholinesterase expression system is probed on reaggregated tumor cells under simulated microgravity, inhibiting the gene transcription and translation of the embryonic proliferation marker butyrylcholinesterase expressed in different tumor types. Alterations in the morphology of cell aggregates e.g. apoptosis or necrosis can be detected by impedance spectroscopy monitoring the electric behavior of membranes and extracellular space with a high resolution and reproducibility. The hydrodynamic positioning of 3D in vitro cell aggregates and the short time for the measurements represent an innovative method for a synchronized multicapillary screening system. The combination of the measuring system with a bioreactor enables cyclic life time recordings of impedance spectra for monitoring the cell aggregate properties for a long period.

Antineoplastic Agents↗

PubMed1999

[Developments in cell therapy in the year 2000].

For the past thirty years, hematology has switched from the concept of bone marrow transplantation to the concept of hematopoietic stem cell (HSC) transplantation, from allograft to autograft, from non-manipulated graft to hyper-selection, from hematopoietic cellular therapy to immunotherapy. Indications of these transplantations are now more clear for malignant diseases and are ongoing for auto-immune diseases. A better knowledge of the HSC allows the control of their proliferation and differentiation, opening the field of ex vivo expansion. Very recently, new stem cells have been identified, establishing that a differentiated cell retain its totipotency: a nervous system cell can differentiate into HSC, which will further give hematopoiesis, mesenchymental cells or hepatocytes. New tools are under development: human ES cells, biomaterials, functionalized materials, opening the field of cellular engineering in the year 2000.

Adult↗

PubMedDate not supplied

Proteomics: theoretical and experimental considerations.

Cellular engineering relies on the ability to decipher the genetic basis of various phenotypes. Emerging technologies for analyzing the biological function of the information encoded in the genome of particular organisms and/or tissues focus on the monitoring of transcription (mRNA) and translation (protein) processes. Elementary theoretical considerations presented in this article strongly suggest that a combination of mRNA and protein expression patterns should be simultaneously considered to fully develop a conceptual understanding of the functional architecture of genomes and gene networks. We propose a framework of experimental and mathematical methods for acquiring and analyzing quantitative proteomic information and discuss recent developments in proteome analytical technology.

Bacterial Proteins↗

PubMed2005

Programming cellular function.

The process of cellular engineering is rapidly accelerating owing to advances in technologies to manipulate DNA and other biomolecules, giving rise to the field of synthetic biology. A meeting was held in August 2005 to present progress in the field and to discuss topics in ethics, safety and security.

Animals↗

PubMed2005

Development of a novel serum-free freezing medium for mammalian cells using the silk protein sericin.

Cryopreservation is a pivotal process in cellular engineering for creating a continuous source of generated functional cell lines and for the convenience of various medical treatments that involve cell culture. FBS (fetal bovine serum) supplemented with 10% (v/v) DMSO is extensively used as a freezing medium for mammalian cells using conventional methods. However, FBS should ideally be avoided, owing to serious concerns regarding bovine spongiform encephalopathy and other infections such as viruses, and an alternative to FBS is eagerly awaited. Furthermore, bio-medicines and cells for transplantation should not be infectious. The present study aimed to develop a novel serum-free freezing medium. For this purpose, we focused on using the silk protein sericin as a cryoprotectant for storage and developed a novel serum-free freezing medium consisting of PBS, 1% (v/w) sericin, 0.5% (v/w) maltose, 0.3% (v/w) proline, 0.3% (v/w) glutamine and 10% DMSO. This novel freezing medium was compared with the conventional FBS supplemented with DMSO and also with three purchased freezing media with respect to cryopreservation of the P 3 U1 myeloma cell line and Chinese-hamster ovary cells. As a result, the constructed medium containing sericin successfully cryopreserved both cell types as efficiently as the conventional medium of FBS containing 10% DMSO and was superior to all three of the purchased media. The constructed medium containing sericin also cryopreserved normal human dermal fibroblasts, human epidermal keratinocytes, the rat phaeochromocytoma cell line PC12 and insect (Spodoptera frugiperda) cell line S f 9 as effectively as the conventional medium of FBS and DMSO.

Animals↗

PubMedDate not supplied

Stimulation of regeneration in mammals: pipe dream or realistic goal?

This article covers a broad spectrum of mammalian regenerative phenomena, including the natural capacity for regeneration of organs and tissues and the classification of mammalian reparative responses. Several broad strategies have been formulated for the stimulation or enhancement of regeneration. Historically, the most common strategy has been to alter the environment surrounding a damaged or regenerating structure. A more contemporary approach to the stimulation of regeneration is the application of cellular engineering principles, which involve strategies such as the implantation of cultured cells, with or without appropriate substrates. Genetic engineering, involving the implantation of genetically engineered cells or the introduction of genes directly into cells in vivo is in the early stages of practical application, although certain laboratory applications have been quite successful.

Animals↗

PubMed2001

Historical and current perspectives on bone marrow transplantation for prevention and treatment of immunodeficiencies and autoimmunities.

Primary immunodeficiency diseases often fully meet the definition of "experiments of nature." Much of the expanding understanding of the lymphoid systems and immunologic functions generated in recent years has been derived from studying patients with primary, generally genetically determined immunodeficiency diseases, as well as other relatively rare secondary immunodeficiency diseases. Increasing knowledge of immunologic defenses, their interacting cellular and molecular components, the evolving details of sequential stages of cellular differentiation, and the nature and control of the cellular and molecular interactions in immunity have now made it possible to define precisely many primary immunodeficiency diseases in full molecular genetic terms. With this wealth of scientific information based on experimental and clinical research, incredible advances have also been made in using bone marrow transplantation (BMT) often as a curative treatment for immunodeficiency, some 60 to 70 other diseases, leukemias, lymphomas, other cancers, and a rapidly expanding constellation of metabolic diseases or enzyme deficiencies. Also, progress in applying allogeneic BMT to prevent, treat, and cure complex autoimmune diseases, primary immunodeficiency diseases and certain forms of cancers, is considered. Further, mixed BMT (syngeneic plus allogeneic) that establishes a form of stable mixed chimerism has also been employed in animal experiments, which revealed that BMT can be used to treat not only immunodeficiency diseases, but also systemic and organ-specific autoimmune diseases, eg, diabetes and erythematous lupus-like diseases. Moreover, performing BMT in conjunction with organ allografts, eg, thymus or pancreatic transplants, has successfully prevented rejection of these allografts, sometimes without recourse to long-term irradiation or toxic chemical immunosuppressive agents. A crucial role for stromal cells in cellular engineering has now also been realized in animal models as a means of preventing graft rejection and promoting full and persistent reconstitution or correction of genetically-based diseases. With all of these achievements, BMT promises continued dramatic and impressive new approaches to clinical and scientific research and reveals an attractive strategy for the treatment and prevention of many currently intractable human diseases. If these achievements can be extended to larger outbred animals and humans, BMT may set the stage for induction of improved immunologic tolerance and for developing treatments for additional intractable human diseases in the 21st century.

Animals↗

PubMed1975

Protection of lethally irradiated mice with allogeneic fetal liver cells: influence of irradiation dose on immunologic reconstitution.

After lethal irradiation long-lived, immunologically vigorous C3Hf mice were produced by treatment with syngeneic fetal liver cells or syngeneic newborn or adult spleen cells. Treatment of lethally irradiated mice with syngeneic or allogeneic newborn thymus cells or allogeneic newborn or adult spleen cells regularly led to fatal secondary disease or graft-versus-host reactions. Treatment of the lethally irradiated mice with fetal liver cells regularly yielded long-lived, immunologically vigorous chimeras. The introduction of the fetal liver cells into the irradiated mice appeared to be followed by development of immunological tolerance of the donor cells. The findings suggest that T-cells at an early stage of differentiation are more susceptible to tolerance induction than are T-lymphocytes at later stages of differentiation. These investigations turned up a perplexing paradox which suggests that high doses of irradiation may injure the thymic stroma, rendering it less capable of supporting certain T-cell populations in the peripheral lymphoid tissue. Alternatively, the higher and not the lower dose of irradiation may have eliminated a host cell not readily derived from fetal liver precursors which represents an important helper cell in certain cell-mediated immune functions, e.g., graft-versus-host reactions, but which is not important in others, e.g., allograft rejections. The higher dose of lethal irradiation did not permit development or maintenance of a population of spleen cells that could initiate graft-versus-host reactions but did permit the development of a population of donor cells capable of achieving vigorous allograft rejection. These observations contribute to understanding of some of the persisting immunodeficiencies that are observed in man after fatal irradiation and bone marrow transplantation. These results should suggest better approaches to more effective cellular engineering for correction of immunodeficiency diseases and for treatment of immunodeficiency diseases and of leukemias and malignancies of man.

Animals↗

PubMed2005

Cross-talk between bone morphogenetic protein and transforming growth factor-beta signaling is essential for exendin-4-induced insulin-positive differentiation of AR42J cells.

A key goal of cellular engineering is to manipulate progenitor cells to become beta-cells, allowing cell replacement therapy to cure diabetes mellitus. As a paradigm for cell engineering, we have studied the molecular mechanisms by which AR42J cells become beta-cells. Bone morphogenetic proteins (BMPs), implicated in a myriad of developmental pathways, have not been well studied in insulin-positive differentiation. We found that the canonical intracellular mediators of BMP signaling, Smad-1 and Smad-8, were significantly elevated in AR42J cells undergoing insulin-positive differentiation in response to exendin-4 treatment, suggesting a role for BMP signaling in beta-cell formation. Similarly, endogenous BMP-2 ligand and ALK-1 receptor (activin receptor-like kinase-1; known to activate Smads 1 and 8) mRNAs were specifically up-regulated in exendin-4-treated AR42J cells. Surprisingly, Smad-1 and Smad-8 levels were suppressed by the addition of BMP-soluble receptor inhibition of BMP ligand binding to its receptor. Here, insulin-positive differentiation was also ablated. BMP-2 ligand antisense also strongly inhibited Smad-1 and Smad-8 expression, again with the abolition of insulin-positive differentiation. These results demonstrate a previously unrecognized key role for BMP signaling in mediating insulin-positive differentiation through the intracellular Smad signaling pathway. In short, BMP signaling may represent a novel downstream target of exendin-4 (glucagon-like peptide 1) signaling and potentially serve as an upstream regulator of transforming growth factor-beta isoform signaling to differentiate the acinar-like AR42J cells into insulin-secreting cells.

Animals↗

PubMed2004

Turning on stem cell cardiogenesis with extremely low frequency magnetic fields.

Modulation of stem cell differentiation is an important assignment for cellular engineering. Embryonic stem (ES) cells can differentiate into cardiomyocytes, but the efficiency is typically low. Here, we show that exposure of mouse ES cells to extremely low frequency magnetic fields triggered the expression of GATA-4 and Nkx-2.5, acting as cardiac lineage-promoting genes in different animal species, including humans. Magnetic fields also enhanced prodynorphin gene expression, and the synthesis and secretion of dynorphin B, an endorphin playing a major role in cardiogenesis. These effects occurred at the transcriptional level and ultimately ensued into a remarkable increase in the yield of ES-derived cardiomyocytes. These results demonstrate the potential use of magnetic fields for modifying the gene program of cardiac differentiation in ES cells without the aid of gene transfer technologies and may pave the way for novel approaches in tissue engineering and cell therapy.

Cell Differentiation↗

PubMedDate not supplied

Improved patency of an elastomeric vascular graft by hybridization.

A newly devised hybrid graft with high antithrombogenicity for small caliber vascular grafts was developed. The design concept was based on the incorporation of a compliant open cell structured graft, autogenous endothelial cells (ECs), and artificial basement membrane. The latter, a gel complex of type I collagen and dermatan sulfate that showed enhanced adhesion and growth of ECs but reduced platelet adhesion, was coated onto a microporous polyurethane graft (internal diameter, 3 mm; length, 4.5 cm), with near-natural compliance. Ten seeded grafts were implanted bilaterally into the carotid arteries of dogs; anticoagulant or antiplatelet therapy was not administered. This hybrid graft showed a marked improvement in patency at 1 month compared with that of simply preclotted grafts (control specimens). This result was explained by the almost complete endothelialization when the graft was implanted, a high degree of adherent strength resistance to shear stress, and a high proliferative potential. Thus, this approach of combining biomechanical and cellular engineering designs may lead to an important functional small caliber graft.

Animals↗

PubMed2005

Kinetic studies and biochemical pathway analysis of anaerobic poly-(R)-3-hydroxybutyric acid synthesis in Escherichia coli.

Poly-(R)-3-hydroxybutyric acid (PHB) was synthesized anaerobically in recombinant Escherichia coli. The host anaerobically accumulated PHB to more than 50% of its cell dry weight during cultivation in either growth or nongrowth medium. The maximum specific PHB production rate during growth-associated synthesis was approximately 2.3 +/- 0.2 mmol of PHB/g of residual cell dry weight/h. The by-product secretion profiles differed significantly between the PHB-synthesizing strain and the control strain. PHB production decreased acetate accumulation for both growth and nongrowth-associated PHB synthesis. For instance under nongrowth cultivation, the PHB-synthesizing culture produced approximately 66% less acetate on a glucose yield basis as compared to a control culture. A theoretical biochemical network model was used to provide a rational basis to interpret the experimental results like the fermentation product secretion profiles and to study E. coli network capabilities under anaerobic conditions. For example, the maximum theoretical carbon yield for anaerobic PHB synthesis in E. coli is 0.8. The presented study is expected to be generally useful for analyzing, interpreting, and engineering cellular metabolisms.

Anaerobiosis↗

PubMed1998

ErbB tyrosine kinases and the two neuregulin families constitute a ligand-receptor network.

The recently isolated second family of neuregulins, NRG2, shares its primary receptors, ErbB-3 and ErbB-4, and induction of mammary cell differentiation with NRG1 isoforms, suggesting functional redundancy of the two growth factor families. To address this possibility, we analyzed receptor specificity of NRGs by using an engineered cellular system. The activity of isoform-specific but partly overlapping patterns of specificities that collectively activate all eight ligand-stimulatable ErbB dimers was revealed. Specifically, NRG2-alpha [corrected], like NRG1-beta [corrected], emerges as a narrow-specificity ligand, whereas NRG2-beta [corrected] is a pan-ErbB ligand that binds with different affinities to all receptor combinations, including those containing ErbB-1, but excluding homodimers of ErbB-2. The latter protein, however, displayed cooperativity with the direct NRG receptors. Apparently, signaling by all NRGs is funneled through the mitogen-activated protein kinase (MAPK). However, the duration and potency of MAPK activation depend on the identity of the stimulatory ligand-receptor ternary complex. We conclude that the NRG-ErbB network represents a complex and nonredundant machinery developed for fine-tuning of signal transduction.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed2006

The Escherichia coli proteome: past, present, and future prospects.

Proteomics has emerged as an indispensable methodology for large-scale protein analysis in functional genomics. The Escherichia coli proteome has been extensively studied and is well defined in terms of biochemical, biological, and biotechnological data. Even before the entire E. coli proteome was fully elucidated, the largest available data set had been integrated to decipher regulatory circuits and metabolic pathways, providing valuable insights into global cellular physiology and the development of metabolic and cellular engineering strategies. With the recent advent of advanced proteomic technologies, the E. coli proteome has been used for the validation of new technologies and methodologies such as sample prefractionation, protein enrichment, two-dimensional gel electrophoresis, protein detection, mass spectrometry (MS), combinatorial assays with n-dimensional chromatographies and MS, and image analysis software. These important technologies will not only provide a great amount of additional information on the E. coli proteome but also synergistically contribute to other proteomic studies. Here, we review the past development and current status of E. coli proteome research in terms of its biological, biotechnological, and methodological significance and suggest future prospects.

Bacterial Proteins↗

PubMed1998

Reactions of cells to topography.

Though contact guidance has been known since the very early days of cell culture very little quantitative examination of the reaction of cells to topography has been made. Exceptions to this subjective approach are given prominence below. Yet if we are to understand how cells react and if we are to be able to design ideal substrata for particular cells we need this information. Precision and quantitation are required both of the methods of examination of the cells but also in the definition of that topography. Recently it has become clear that the these reactions occur at the nanometric scale and have importance for use in cellular engineering and tissue repair. Topography appears to provide a set of very powerful signals for cells.

Animals↗

PubMed2004

Separating antiviral and GVHD activities of donor T cells prior to bone marrow transplantation.

Approaches to speed immune reconstitution following bone marrow transplantation (BMT) or peripheral-blood hematopoietic stem-cell transplantation (HSCT) could markedly reduce morbidity and mortality, particularly following partially major histocompatibility complex (MHC)-matched related donor (PMRD) transplants. However, it is critical to simultaneously eliminate the subpopulation of donor T cells that are alloreactive with the recipient and may produce graft-vs-host disease (GVHD). In this article, we discuss a number of promising cellular engineering approaches that could be applied to this problem, including the use of veto cells, regulatory T-cell subsets, and psoralen-treated donor lymphocytes. Emphasis is placed on whether these approaches can simultaneously transfer broad-spectrum immunity to the recipient without producing GVHD.

Adoptive Transfer↗