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At least 307 records · Page 17

Stem Cell-Based Regenerative Health in Microgravity

The research of Dr. Almeida's Lab at NASA Ames focuses on the effects of microgravity mechanical unloading on stem-cell-based tissue regeneration. The research focuses on testing the broad hypothesis that mechanical load from gravity at 1 g is required for normal adult stem cell proliferation and differentiation during tissue regeneration. Research from the lab has provides evidence supporting the hypothesis in various biological models, including newt tail regeneration, mouse bone and bone marrow in whole animals, and mouse embryonic stem cells all exposed to microgravity during spaceflight. Using these models, the lab has identified overexpression of the cell cycle inhibitor p21/CDKN1a as a candidate molecular mediator mechanism of bone marrow mesenchymal and hematopoietic lineage tissue regenerative arrest in microgravity. The labis currently focused on using p21/CDKN1a null mouse and cell culture approaches in microgravity to test the gravity tissue regeneration hypothesis.

stem cells microgravity regeneration↗

Development of an Autonomous, Dual Chamber Bioreactor for the Growth of 3-Dimensional Epithelial-Stromal Tissues in Microgravity

We are developing a novel, autonomous bioreactor that can provide for the growth and maintenance in microgravity of 3‐D organotypic epithelial‐stromal cultures that require an air‐liquid interface. These complex 3‐D tissue models accurately represent the morphological features, differentiation markers, and growth characteristics observed in normal human epithelial tissues, including the skin, esophagus, lung, breast, pancreas, and colon. However, because of their precise and complex culture requirements, including that of an air‐liquid interface, these 3‐D models have yet to be utilized for life sciences research aboard the International Space Station. The development of a bioreactor for these cultures will provide the capability to perform biological research on the ISS using these realistic, tissue‐like human epithelial‐stromal cell models and will contribute significantly to advances in fundamental space biology research on questions regarding microgravity effects on normal tissue development, aging, cancer, and other disease processes. It will also allow for the study of how combined stressors, such as microgravity with radiation and nutritional deficiencies, affect multiple biological processes and will provide a platform for conducting countermeasure investigations on the ISS without the use of animal models. The technology will be autonomous and consist of a cell culture chamber that provides for air‐liquid, liquid‐liquid, and liquid‐air exchanges within the chambers while maintaining the growth and development of the biological samples. The bioreactor will support multiple tissue types and its modular design will provide for incorporation of add‐on capabilities such as microfluidics drug delivery, media sampling, and in situ biomarker analysis. Preliminary flight testing of the hardware will be conducted on a parabolic platform through NASA's Flight Opportunities Program.

Patel, Zarana S.↗

Whole Genome Sequencing Links Mycobacterium bovis From Cattle, Cheese and Humans in Baja California, Mexico

Mycobacterium bovis causes tuberculosis (TB) in cattle, which in turn can transmit the pathogen to humans. Tuberculosis in dairy cattle is of particular concern where the consumption of raw milk and dairy products is customary. Baja California (BCA), Mexico, presents high prevalence of TB in both cattle and humans, making it important to investigate the molecular epidemiology of the disease in the region. A long-term study was undertaken to fully characterize the diversity of M. bovis genotypes circulating in dairy cattle, cheese and humans in BCA by whole-genome sequencing (WGS). During a 2-year period, 412 granulomatous tissue samples were collected from local abattoirs and 314 cheese samples were purchased from local stores and vendors in BCA and sent to the laboratory for mycobacterial culture, histology, direct PCR and WGS. For tissue samples M. bovis was recovered from 86.8%, direct PCR detected 90% and histology confirmed 85.9% as mycobacteriosis-compatible. For cheese, M. bovis was recovered from 2.5% and direct PCR detected 6% of the samples. There was good agreement between diagnostic tests. Subsequently, a total of 345 whole-genome SNP sequences were obtained. Phylogenetic analysis grouped these isolates into 10 major clades. SNP analysis revealed putative transmission clusters where the pairwise SNP distance between isolates from different dairies was ≤3 SNP. Also, human and/or cheese isolates were within 8.45 (range 0–17) and 5.8 SNP (range 0–15), respectively, from cattle isolates. Finally, a comparison between the genotypes obtained in this study and those reported previously suggests that the genetic diversity of M. bovis in BCA is well-characterized, and can be used to determine if BCA is the likely source of M. bovis in humans and cattle in routine epidemiologic investigations and future studies. In conclusion, WGS provided evidence of ongoing local transmission of M. bovis among the dairies in this high-TB burden region of BCA, as well as show close relationships between isolates recovered from humans, cheese, and cattle. This confirms the need for a coordinated One Health approach in addressing the elimination of TB in animals and humans. Overall, the study contributes to the knowledge of the molecular epidemiology of M. bovis in BCA, providing insight into the pathogen's dynamics in a high prevalence setting.

59 BASIC BIOLOGICAL SCIENCES↗

Techniques for studying the effects of microgravity on model particle/cell systems

In an effort to learn more about the effects of a simulated low gravity environment on skeletal muscle, skeletal muscle cell cultures were grown within the lumen of XM-80 hollow fibers (i.d. = 0.5 mm) in a Clinostat rotating at 100 rpm. Cells were isolated from the thigh muscle tissue of 12 day embryos and were cultured for up to 14 days in the hollow fiber environment. Cells proliferated to confluency within several days, and fusion into multinucleated myotubes was then apparent. Fibers were stretched by a built-in spring mechanism to hold the fiber tightly at the center of rotation, and sections of the fiber were removed at 3, 7 and 14 days for electron microscopic analysis. When the Clinostat is rotated in the horizontal position, the gravity vector approaches zero and the cells are in an environment that simulates microgravity. Control experiments consist of one fiber rotated in the vertical position in the clinostat and another fiber that is held in a horizontal configuration in a comparable sized tube that is not rotated at all. Examination of skeletal muscle cells by electron microscopy revealed that myoblast fusion and myofibril accumulation were extensive. Two general conclusions were apparent. First, muscle cells undergo the normal progression of proliferation, fusion, and myofibril assembly in the presence of simulated microgravity for the first week in culture. After 14 days, however, many muscle fibers undergo degeneration such that myofibrillar structures are not extensive or well organized. Second, although no major abnormalities in myofibril assembly were detected in Clinostat-rotated cultures in comparison to controls that were not rotated in a Clinostat, the myofibrils in non-rotated controls tend to be more highly organized than those in either horizontally or vertically rotated Clinostat samples.

Young, Ronald B.↗

Lymphocyte trafficking and HIV infection of human lymphoid tissue in a rotating wall vessel bioreactor

The pathogenesis of HIV infection involves a complex interplay between both the infected and noninfected cells of human lymphoid tissue, the release of free viral particles, the de novo infection of cells, and the recirculatory trafficking of peripheral blood lymphocytes. To develop an in vitro model for studying these various aspects of HIV pathogenesis we have utilized blocks of surgically excised human tonsils and a rotating wall vessel (RWV) cell culture system. Here we show that (1) fragments of the surgically excised human lymphoid tissue remain viable and retain their gross cytoarchitecture for at least 3 weeks when cultured in the RWV system; (2) such lymphoid tissue gradually shows a loss of both T and B cells to the surrounding growth medium; however, this cellular migration is reversible as demonstrated by repopulation of the tissue by labeled cells from the growth medium; (3) this cellular migration may be partially or completely inhibited by embedding the blocks of lymphoid tissue in either a collagen or agarose gel matrix; these embedded tissue blocks retain most of the basic elements of a normal lymphoid cytoarchitecture; and (4) both embedded and nonembedded RWV-cultured blocks of human lymphoid tissue are capable of productive infection by HIV-1 of at least three various strains of different tropism and phenotype, as shown by an increase in both p24 antigen levels and free virus in the culture medium, and by the demonstration of HIV-1 RNA-positive cells inside the tissue identified by in situ hybridization. It is therefore reasonable to suggest that gel-embedded and nonembedded blocks of human lymphoid tissue, cocultured with a suspension of tonsillar lymphocytes in an RWV culture system, constitute a useful model for simulating normal lymphocyte recirculatory traffic and provide a new tool for testing the various aspects of HIV pathogenesis.

Tissue Culture/instrumentation/methods↗

Monoclonal antibodies directed against surface molecules of multicell spheroids

The objective of this project is to generate a library of monoclonal antibodies (MAb's) to surface molecules involved in the cell-cell interactions of mammalian cells grown as multicell spheroids (MCS). MCS are highly organized 3-dimensional multicellular structures which exhibit many characteristics in vivo tissues not found in conventional monolayer or suspension culture. They also provide a functional assay for surface adhesion molecules. In brief, MCS combine the relevance of organized tissues with the accuracy of in vitro methodology. Further, one can manipulate these MCS experimentally to discern important information about their biology.

Martinez, Andrew O.↗

Growing Three-Dimensional Corneal Tissue in a Bioreactor

Spheroids of corneal tissue about 5 mm in diameter have been grown in a bioreactor from an in vitro culture of primary rabbit corneal cells to illustrate the production of optic cells from aggregates and tissue. In comparison with corneal tissues previously grown in vitro by other techniques, this tissue approximates intact corneal tissue more closely in both size and structure. This novel three-dimensional tissue can be used to model cell structures and functions in normal and abnormal corneas. Efforts continue to refine the present in vitro method into one for producing human corneal tissue to overcome the chronic shortage of donors for corneal transplants: The method would be used to prepare corneal tissues, either from in vitro cultures of a patient s own cells or from a well-defined culture from another human donor known to be healthy. As explained in several articles in prior issues of NASA Tech Briefs, generally cylindrical horizontal rotating bioreactors have been developed to provide nutrient-solution environments conducive to the 30 NASA Tech Briefs, October 2003 growth of delicate animal cells, with gentle, low-shear flow conditions that keep the cells in suspension without damaging them. The horizontal rotating bioreactor used in this method, denoted by the acronym "HARV," was described in "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662), NASA Tech Briefs, Vol. 16, No. 5 (May, 1992), page 150.

Spaulding, Glen F.↗

Tissue Engineering Using Transfected Growth-Factor Genes

A method of growing bioengineered tissues includes, as a major component, the use of mammalian cells that have been transfected with genes for secretion of regulator and growth-factor substances. In a typical application, one either seeds the cells onto an artificial matrix made of a synthetic or natural biocompatible material, or else one cultures the cells until they secrete a desired amount of an extracellular matrix. If such a bioengineered tissue construct is to be used for surgical replacement of injured tissue, then the cells should preferably be the patient s own cells or, if not, at least cells matched to the patient s cells according to a human-leucocyteantigen (HLA) test. The bioengineered tissue construct is typically implanted in the patient's injured natural tissue, wherein the growth-factor genes enhance metabolic functions that promote the in vitro development of functional tissue constructs and their integration with native tissues. If the matrix is biodegradable, then one of the results of metabolism could be absorption of the matrix and replacement of the matrix with tissue formed at least partly by the transfected cells. The method was developed for articular chondrocytes but can (at least in principle) be extended to a variety of cell types and biocompatible matrix materials, including ones that have been exploited in prior tissue-engineering methods. Examples of cell types include chondrocytes, hepatocytes, islet cells, nerve cells, muscle cells, other organ cells, bone- and cartilage-forming cells, epithelial and endothelial cells, connective- tissue stem cells, mesodermal stem cells, and cells of the liver and the pancreas. Cells can be obtained from cell-line cultures, biopsies, and tissue banks. Genes, molecules, or nucleic acids that secrete factors that influence the growth of cells, the production of extracellular matrix material, and other cell functions can be inserted in cells by any of a variety of standard transfection techniques.

Madry, Henning↗

Myocardialization of the cardiac outflow tract

During development, the single-circuited cardiac tube transforms into a double-circuited four-chambered heart by a complex process of remodeling, differential growth, and septation. In this process the endocardial cushion tissues of the atrioventricular junction and outflow tract (OFT) play a crucial role as they contribute to the mesenchymal components of the developing septa and valves in the developing heart. After fusion, the endocardial ridges in the proximal portion of the OFT initially form a mesenchymal outlet septum. In the adult heart, however, this outlet septum is basically a muscular structure. Hence, the mesenchyme of the proximal outlet septum has to be replaced by cardiomyocytes. We have dubbed this process "myocardialization." Our immunohistochemical analysis of staged chicken hearts demonstrates that myocardialization takes place by ingrowth of existing myocardium into the mesenchymal outlet septum. Compared to other events in cardiac septation, it is a relatively late process, being initialized around stage H/H28 and being basically completed around stage H/H38. To unravel the molecular mechanisms that are responsible for the induction and regulation of myocardialization, an in vitro culture system in which myocardialization could be mimicked and manipulated was developed. Using this in vitro myocardialization assay it was observed that under the standard culture conditions (i) whole OFT explants from stage H/H20 and younger did not spontaneously myocardialize the collagen matrix, (ii) explants from stage H/H21 and older spontaneously formed extensive myocardial networks, (iii) the myocardium of the OFT could be induced to myocardialize and was therefore "myocardialization-competent" at all stages tested (H/H16-30), (iv) myocardialization was induced by factors produced by, most likely, the nonmyocardial component of the outflow tract, (v) at none of the embryonic stages analyzed was ventricular myocardium myocardialization-competent, and finally, (vi) ventricular myocardium did not produce factors capable of supporting myocardialization. Copyright 1999 Academic Press.

NASA Discipline Cardiopulmonary↗

Multilineage differentiation of rhesus monkey embryonic stem cells in three-dimensional culture systems

In the course of normal embryogenesis, embryonic stem (ES) cells differentiate along different lineages in the context of complex three-dimensional (3D) tissue structures. In order to study this phenomenon in vitro under controlled conditions, 3D culture systems are necessary. Here, we studied in vitro differentiation of rhesus monkey ES cells in 3D collagen matrixes (collagen gels and porous collagen sponges). Differentiation of ES cells in these 3D systems was different from that in monolayers. ES cells differentiated in collagen matrixes into neural, epithelial, and endothelial lineages. The abilities of ES cells to form various structures in two chemically similar but topologically different matrixes were different. In particular, in collagen gels ES cells formed gland-like circular structures, whereas in collagen sponges ES cells were scattered through the matrix or formed aggregates. Soluble factors produced by feeder cells or added to the culture medium facilitated ES cell differentiation into particular lineages. Coculture with fibroblasts in collagen gel facilitated ES cell differentiation into cells of a neural lineage expressing nestin, neural cell adhesion molecule, and class III beta-tubulin. In collagen sponges, keratinocytes facilitated ES cell differentiation into cells of an endothelial lineage expressing factor VIII. Exogenous granulocyte-macrophage colony-stimulating factor further enhanced endothelial differentiation. Thus, both soluble factors and the type of extracellular matrix seem to be critical in directing differentiation of ES cells and the formation of tissue-like structures. Three-dimensional culture systems are a valuable tool for studying the mechanisms of these phenomena.

Pluripotent Stem Cells/cytology/physiology↗

Thioredoxin VdTrx1, an unconventional secreted protein, is a virulence factor in Verticillium dahliae

Understanding how plant pathogenic fungi adapt to their hosts is of critical importance to securing optimal crop productivity. In response to pathogenic attack, plants produce reactive oxygen species (ROS) as part of a multipronged defense response. Pathogens, in turn, have evolved ROS scavenging mechanisms to undermine host defense. Thioredoxins (Trx) are highly conserved oxidoreductase enzymes with a dithiol-disulfide active site, and function as antioxidants to protect cells against free radicals, such as ROS. However, the roles of thioredoxins in Verticillium dahliae , an important vascular pathogen, are not clear. Through proteomics analyses, we identified a putative thioredoxin (VdTrx1) lacking a signal peptide. VdTrx1 was present in the exoproteome of V. dahliae cultured in the presence of host tissues, a finding that suggested that it plays a role in host-pathogen interactions. We constructed a VdTrx1 deletion mutant Δ VdTrx1 that exhibited significantly higher sensitivity to ROS stress, H 2 O 2 , and tert -butyl hydroperoxide ( t -BOOH). In vivo assays by live-cell imaging and in vitro assays by western blotting revealed that while VdTrx1 lacking the signal peptide can be localized within V. dahliae cells, VdTrx1 can also be secreted unconventionally depending on VdVps36, a member of the ESCRT-II protein complex. The Δ VdTrx1 strain was unable to scavenge host-generated extracellular ROS fully during host invasion. Deletion of VdTrx1 resulted in higher intracellular ROS levels of V. dahliae mycelium, displayed impaired conidial production, and showed significantly reduced virulence on Gossypium hirsutum , and model plants, Arabidopsis thaliana and Nicotiana benthamiana . Thus, we conclude that VdTrx1 acts as a virulence factor in V. dahliae .

Tian, Li↗

Inhibition of intracellular proteolysis in muscle cultures by multiplication-stimulating activity

The effects of the insulin-like growth factor, multiplication-stimulating activity (MSA), on chick myotube cultures are studied. The results indicate that MSA is an effective anabolic agent regulating protein metabolism and amino acid uptake, but not sugar transport. Similar size effects on protein metabolism and amino acid uptake in serum-free media were observed in parallel studies with insulin, although insulin levels well in excess of the normal physiological range are required to produce significant effects. It is suggested that there is a generally low insulin sensitivity in cultured chick myotubes relative to adult tissues.

Janeczko, Richard A.↗

Characterization of atrial natriuretic peptide receptors in brain microvessel endothelial cells

Atrial natriuretic peptide (ANP) binding and ANP-induced increases in cyclic guanosine monophosphate (cGMP) levels have been observed in brain microvessels (Chabrier et al., 1987; Steardo and Nathanson, 1987), suggesting that this fluid-regulating hormone may play a role in the fluid homeostasis of the brain. This study was initiated to characterize the ANP receptors in primary cultures of brain microvessel endothelial cells (BMECs). The apparent equilibrium dissociation constant, Kd, for ANP increased from 0.25 nM to 2.5 nM, and the number of ANP binding sites as determined by Scatchard analysis increased from 7,100 to 170,000 sites/cell between 2 and 10 days of culture following monolayer formation. Time- and concentration-dependent studies on the stimulation of cGMP levels by ANP indicated that guanylate cyclase-linked ANP receptors were present in BMECs. The relative abilities of ANP, brain natriuretic peptide (BNP), and a truncated analog of ANP containing amino acids 5-27 (ANP 5-27) to modulate the accumulation of cGMP was found to be ANP greater than BNP much greater than ANP 5-27. Affinity cross-linking with disuccinimidyl suberate and radiolabeled ANP followed by gel electrophoresis under reducing conditions demonstrated a single band corresponding to the 60-70 kD receptor, indicating the presence of the nonguanylate cyclase-linked ANP receptor. Radiolabeled ANP binding was examined in the presence of various concentrations of either ANP, BNP, or ANP 5-27 and suggested that a large proportion of the ANP receptors present in blood-brain barrier endothelial cells bind all of these ligands similarly. These data indicate both guanylate cyclase linked and nonguanylate cyclase linked receptors are present on BMECs and that a higher proportion of the nonguanylate cyclase linked receptors is expressed. This in vitro culture system may provide a valuable tool for the examination of ANP receptor expression and function in blood-brain barrier endothelial cells.

Endothelium, Vascular/chemistry↗

The Biotechnology Facility for International Space Station

The primary mission of the Cellular Biotechnology Program is to advance microgravity as a tool in basic and applied cell biology. The microgravity environment can be used to study fundamental principles of cell biology and to achieve specific applications such as tissue engineering. The Biotechnology Facility (BTF) will provide a state-of-the-art facility to perform cellular biotechnology research onboard the International Space Station (ISS). The BTF will support continuous operation, which will allow performance of long-duration experiments and will significantly increase the on-orbit science throughput.

NASA Center JSC↗

Growth Stimulation of Biological Cells and Tissue by Electromagnetic Fields and Uses Thereof

The present invention provides systems for growing two or three dimensional mammalian cells within a culture medium facilitated by an electromagnetic field, and preferably, a time varying electromagnetic field. The cells, and culture medium are contained within a fixed or rotating culture vessel, and the electromagnetic field is emitted from at least one electrode. In one embodiment, the electrode is spaced from the vessel. The invention further provides methods to promote neural tissue regeneration by means of culturing the neural cells in the claimed system. In one embodiment, neuronal cells are grown within longitudinally extending tissue strands extending axially along and within electrodes comprising electrically conductive channels or guides through which a time varying electrical current is conducted, the conductive channels being positioned within a culture medium.

Wolf, David A.↗

Growth stimulation of biological cells and tissue by electromagnetic fields and uses thereof

The present invention provides systems for growing two or three dimensional mammalian cells within a culture medium facilitated by an electromagnetic field, and preferably, a time varying electromagnetic field. The cells and culture medium are contained within a fixed or rotating culture vessel, and the electromagnetic field is emitted from at least one electrode. In one embodiment, the electrode is spaced from the vessel. The invention further provides methods to promote neural tissue regeneration by means of culturing the neural cells in the claimed system. In one embodiment, neuronal cells are grown within longitudinally extending tissue strands extending axially along and within electrodes comprising electrically conductive channels or guides through which a time varying electrical current is conducted, the conductive channels being positioned within a culture medium.

Wolf, David A.↗

Leptin modulates gene expression in the heart, cardiomyocytes and the adipose tissue thus mitigating LPS-induced damage

Highlights: • LPS enhanced expression of the leptin gene in mouse tissues and cardiomyocytes. • Leptin downregulated pro-inflammatory genes in vivo and in cardiomyocytes after LPS treatment. • Leptin upregulated expression of antioxidant genes in vivo and in cardiomyocytes after LPS treatment. • Leptin reduced ROS levels in cardiomyocytes after LPS treatment. • Leptin acts to counteract LPS-induced damage in the heart and cardiomyocytes by modulating gene expression. • Anti-inflammatory effects of leptin in transgenic aMUPA mice overexpressing leptin surpassed that of wild type mice. Leptin is an adipokine of pleiotropic effects linked to energy metabolism, satiety, the immune response, and cardioprotection. We have recently shown that leptin causally conferred resistance to myocardial infarction-induced damage in transgenic αMUPA mice overexpressing leptin compared to their wild type (WT) ancestral mice FVB/N. Prompted by these findings, we have investigated here if leptin can counteract the inflammatory response triggered after LPS administration in tissues in vivo and in cardiomyocytes in culture. The results have shown that LPS upregulated in vivo and in vitro all genes examined here, both pro-inflammatory and antioxidant, as well as the leptin gene. Pretreating mice with leptin neutralizing antibodies further upregulated the expression of TNFα and IL-1β in the adipose tissue of both mouse types, and in the αMUPA heart. The antibodies also increased the levels of serum markers for cell toxicity in both mouse types. These results indicate that under LPS, leptin actually reduced the levels of these inflammatory-related parameters. In addition, pretreatment with leptin antibodies reduced the levels of HIF-1α and VEGF mRNAs in the heart, indicating that under LPS leptin increased the levels of these mRNAs. In cardiomyocytes, pretreatment with exogenous leptin prior to LPS reduced the expression of both pro-inflammatory genes, enhanced the expression of the antioxidant genes HO-1, SOD2 and HIF-1α, and lowered ROS staining. In addition, results obtained with leptin antibodies and the SMLA leptin antagonist indicated that endogenous and exogenous leptin can inhibit leptin gene expression. Together, these findings have indicated that under LPS, leptin concomitantly downregulated pro-inflammatory genes, upregulated antioxidant genes, and lowered ROS levels. These results suggest that leptin can counteract inflammation in the heart and adipose tissue by modulating gene expression.

60 APPLIED LIFE SCIENCES↗

Improved method for HPLC analysis of polyamines, agmatine and aromatic monoamines in plant tissue

The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucas carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues.

Non-NASA Center↗