improving-reproducibility-to-meet-minimal-information-for-studies

Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many ...

Kansas State University College of Veterinary Medicine

The organization of spinal motor circuitry to the kidney is not well-characterized and changes in renal innervation have been associated with disease states such as hypertension found in the spontaneously hypertensive rat or renal hypertension. Here, we describe the segmental and intra-segmental organization of the spinal motor circuitry that was resolved after neurotropic viral injection into the kidney and retrograde transneuronal transport to the spinal cord. In the first experiment, the serial reconstruction of infected neurons in the thoracolumbar spinal cord from T8-L1 was performed following injection of pseudorabies virus (PRV, Bartha strain) into either the cranial pole, the caudal pole or both the cranial and caudal poles of the left kidney in male rats. In the second experiment, rats received injections of two different PRV strains that were genetically engineered to express unique reporter molecules; one of the engineered strains was injected into the cranial pole and the other was injected into the caudal pole. Either 3- or 4-day post-infection, the animals were anesthetized and sacrificed by transcardial perfusion. PRV-infected neurons were located by immunocytochemistry against either PRV itself (experiment 1) or the unique marker proteins (experiment 2). After injection of both poles of the kidney, the majority of the infected neurons were found in the ipsilateral intermediolateral cell column (IML) from T10 to T12 with the mode at T11. Infected neurons were found in discrete neuron clusters in the intermediolateral cell column along the longitudinal axis in a repeating pattern of high and low density that has been called "beading". Three observations indicated a topographic distribution of renal sympathetic preganglionic neurons (SPN). First, after injection into either the cranial or caudal poles of the kidney, the mode of infected cells was located in segments T11 and T12, respectively. The one spinal segment shift in the mode suggested a topographic distribution. Second, in spinal segments T8-L1, comparison of the distributions of the neurons innervating each pole of the left kidney revealed an overlap in the distribution, except in the T11 segment. In the T11 segment, the neurons projecting to each pole tended to segregate into separate populations. Third, in rats that received injections of two PRV strains that were genetically engineered to express unique markers into opposite poles of the kidney, a segregation of neurons projecting to the cranial and caudal poles of the kidney was noted again in the T11 spinal segment and the segregation at adjacent spinal levels was obvious. The analysis of the distribution of infected neurons within each spinal cord segment (intra-segmental distribution) revealed three different patterns along the cranial-caudal dimension. In segments T8-T10, >60% of the infected neurons were located in the caudal half of the spinal segment. In segments T12-L1, >60% of the infected neurons were located in the cranial half of the spinal segment. In segment T11, the neurons were more evenly distributed throughout the segment. These intra-segmental distribution patterns were found after both 3- or 4-day survival periods post-infection and were found in most animals. The distribution of clusters of neurons revealed a similar intra-segmental pattern. Thus, as was described previously for the sympathetic postganglionic neurons that innervate the kidney, the present work indicates a topographic organization in the second-order neurons in the renal sympathetic efferent pathway. The physiological significance of this anatomical organization remains to be determined.

Distribution of sympathetic preganglionic neurons innervating the kidney in the rat: PRV transneuronal tracing and serial reconstruction.

Stem cells are the next frontier in medicine. Stem cells are thought to have great therapeutic and biotechnological potential. This will not only to replace damaged or dysfunctional cells, but also rescue them and/or deliver therapeutic proteins after they have been engineered to do so. Currently, ethical and scientific issues surround both embryonic and fetal stem cells and hinder their widespread implementation. In contrast, stem cells recovered postnatally from the umbilical cord, including the umbilical cord blood cells, amnion/placenta, umbilical cord vein, or umbilical cord matrix cells, are a readily available and inexpensive source of cells that are capable of forming many different cell types (i.e., they are "multipotent"). This review will focus on the umbilical cord-derived stem cells and compare those cells with adult bone marrow-derived mesenchymal stem cells.

Stem cells in the umbilical cord.

Here, we describe a focused microarray for screening rat embryonic stem cells (ESCs) and provide validation data that this array can distinguish undifferentiated rat ESCs from rat trophoblast stem (TS) cells, rat extraembryonic endoderm cells, mouse embryonic fibroblast feeder cells, and differentiated rat ESCs. Using this tool, genuine rat ESC lines, which have been expanded in a conventional rat ESC medium containing two inhibitors (2i), for example, glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase (MEK) inhibitors, and leukemia inhibitory factor, and genuine rat ESCs, which have been expanded in rat ESC medium containing four inhibitors (4i), for example, GSK3, MEK, Alk5, and Rho-associated kinase inhibitors were compared; as were genuine rat ESCs from 4 different strains of rats. Expression of Cdx2, a gene associated with trophoblast determination, was observed in genuine, undifferentiated rat ESCs from 4 strains and from both 2i and 4i ESC derivation medium. This finding is in contrast to undifferentiated mouse ESCs that do not express Cdx2. The rat ESC focused microarray described in this report has utility for rapid screening of rat ESCs. This tool will enable optimization of culture conditions in the future.

A focused microarray for screening rat embryonic stem cell lines.

A1 One health advances and successes in comparative medicine and translational researchCheryl StroudA2 Dendritic cell-targeted gorilla adenoviral vector for cancer vaccination for canine melanomaIgor Dmitriev, Elena Kashentseva, Jeffrey N. Bryan, David T. CurielA3 Viroimmunotherapy for malignant melanoma in the companion dog modelJeffrey N. Bryan, David Curiel, Igor Dmitriev, Elena Kashentseva, Hans Rindt, Carol Reinero, Carolyn J. HenryA4 Of mice and men (and dogs!): development of a commercially licensed xenogeneic DNA vaccine for companion animals with malignant melanomaPhilip J. BergmanA5 Successful immunotherapy with a recombinant HER2-expressing Listeria monocytogenes in dogs with spontaneous osteosarcoma paves the way for advances in pediatric osteosarcomaNicola J. Mason, Josephine S. Gnanandarajah, Julie B. Engiles, Falon Gray, Danielle Laughlin, Anita Gaurnier-Hausser, Anu Wallecha, Margie Huebner, Yvonne PatersonA6 Human clinical development of ADXS-HER2Daniel O'ConnorA7 Leveraging use of data for both human and veterinary benefitLaura S. TremlA8 Biologic replacement of the knee: innovations and early clinical resultsJames P. StannardA9 Mizzou BioJoint Center: a translational success storyJames L. CookA10 University and industry translational partnership: from the lab to commercializationMarc JacobsA11 Beyond docking: an evolutionarily guided OneHealth approach to drug discoveryGerald J. Wyckoff, Lee Likins, Ubadah Sabbagh, Andrew SkaffA12 Challenges and opportunities for data applications in animal health: from precision medicine to precision husbandryAmado S. GuloyA13 A cloud-based programmable platform for healthHarlen D. HaysA14 Comparative oncology: One Health in actionAmy K. LeBlancA15 Companion animal diseases bridge the translational gap for human neurodegenerative diseaseJoan R. Coates, Martin L. Katz, Leslie A. Lyons, Gayle C. Johnson, Gary S. Johnson, Dennis P. O'BrienA16 Duchenne muscular dystrophy gene therapyDongsheng DuanA17 Polycystic kidney disease: cellular mechanisms to emerging therapiesJames P. CalvetA18 The domestic cat as a large animal model for polycystic kidney diseaseLeslie A. Lyons, Barbara GandolfiA19 The support of basic and clinical research by the Polycystic Kidney Disease FoundationDavid A. BaronA20 Using naturally occurring large animal models of human disease to enable clinical translation: treatment of arthritis using autologous stromal vascular fraction in dogsMark L. WeissA21 Regulatory requirements regarding clinical use of human cells, tissues, and tissue-based productsDebra A. WebsterA22 Regenerative medicine approaches to Type 1 diabetes treatmentFrancis N. KaranuA23 The zoobiquity of canine diabetes mellitus, man's best friend is a friend indeed-islet transplantationEdward J. RobbA24 One Medicine: a development model for cellular therapy of diabetesRobert J. Harman.

A One Health overview, facilitating advances in comparative medicine and translational research.

Mesenchymal stromal cells (MSCs) hold great promise in the field of regenerative medicine due to their ability to create a variable localized anti-inflammatory effect in injuries such as Crohn's disease and osteoarthritis or by incorporation in tissue engineered constructs. Currently, the MSC literature uses rodents for preclinical disease models. There is growing interest in using naturally occurring disease in large animals for modeling human disease. By review of the canine MSCs literature, it appears that canine MSCs can be difficult to maintain in culture for extended passages and this greatly varies between tissue sources, compared with human and rodent MSCs, and limited lifespan is an obstacle for preclinical investigation and therapeutic use. Research using canine MSCs has been focused on cells derived from bone marrow or adipose tissue, and the differences in manufacturing MSCs between laboratories are problematic due to lack of standardization. To address these issues, here, a stepwise process was used to optimize canine MSCs isolation, expansion, and cryopreservation utilizing canine umbilical cord-derived MSCs. The culture protocol utilizes coating of tissue culture surfaces that increases cellular adherence, increases colony-forming units-fibroblast efficiency, and decreases population doubling times. Canine MSCs isolated with our protocol could be maintained longer than published canine MSCs methods before senescing. Our improved cryopreservation protocols produce on average >90% viable MSCs at thaw. These methods enable master-bank and working-bank scenarios for allogeneic MSC testing in naturally occurring disease in dogs.

A Protocol for the Isolation, Culture, and Cryopreservation of Umbilical Cord-Derived Canine Mesenchymal Stromal Cells: Role of Cell Attachment in Long-Term Maintenance.

Mark L. Weiss

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Kansas State University College of Veterinary Medicine

Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis

Department of Anatomy and Physiology