Viability, yield and expansion capability of feline MSCs obtained from subcutaneous and reproductive organ adipose depots.

BACKGROUND: The source of multipotent stromal cells (MSC) can have a significant influence on the health and expansion capacity of the cells. As the applications for allogeneic MSCs in the treatment of feline diseases increase, the location of the initial donor tissue must be analyzed. To date, comparisons have only been made between feline MSCs collected from bone marrow or abdominal fat. This is the first report to compare cells obtained from different adipose depots in the cat with a focus on clinically relevant donor tissues. The tissue was collected from 34 healthy cats undergoing spaying (fat around the ovaries and uterine horn) or subcutaneous fat collected during surgical procedures. RESULTS: The amount of starting material is essential to isolate sufficient MSCs. The total tissue yield from the subcutaneous fat was significantly greater than could be obtained from around the reproductive organs, leading to 3 times more MSCs per donor. However, the concentration of MSCs obtained from reproductive fat was higher than from subcutaneous fat. In addition, the viability of the MSCs from the reproductive fat was significantly higher than the subcutaneous fat. Since most spaying occurs in young cats (under 18 months) reproductive fat was collected from adult cats during spaying, illustrating that age did not alter the yield or viability of the MSCs. When sufficient tissue was collected, it was digested either mechanically or enzymatically. Mechanical digestion further decreased the viability and yield of MSCs from subcutaneous fat compared to enzymatic digestion. Biomarkers of stem cell characterization, expansion capacity and function were detected using qPCR. CD70, CD90 and CD105 were all expressed in high levels in the 3 groups. However, the reproductive fat had higher levels of CD73 with the mechanically digested subcutaneous fat having the least. Gata6 was detected in all samples while Sox2 and Sox17 were also detected with higher quantities found in the enzymatically digested subcutaneous fat. Negative control genes of Gata4 and Pdx1 showed no detection prior to 50 cycles. During the first three passages, age of the donor, location of the donor tissue, or digestion protocol had no effect on cell culture doubling times or cell viability. CONCLUSIONS: While MSCs from reproductive fat had superior cells/tissue weight and initial viability, there were still dramatically fewer cells obtained compared to subcutaneous fat due to the limited amount of tissue surrounding the reproductive organs. Further, in P1-P3 cultures there were no differences noted in doubling time or cell viability between tissue obtained from reproductive or subcutaneous fat depots.

Claudin-2 deficiency associates with hypercalciuria in mice and human kidney stone disease.

The major risk factor for kidney stone disease is idiopathic hypercalciuria. Recent evidence implicates a role for defective calcium reabsorption in the renal proximal tubule. We hypothesized that claudin-2, a paracellular cation channel protein, mediates proximal tubule calcium reabsorption. We found that claudin-2-null mice have hypercalciuria due to a primary defect in renal tubule calcium transport and papillary nephrocalcinosis that resembles the intratubular plugs in kidney stone formers. Our findings suggest that a proximal tubule defect in calcium reabsorption predisposes to papillary calcification, providing support for the vas washdown hypothesis. Claudin-2-null mice were also found to have increased net intestinal calcium absorption, but reduced paracellular calcium permeability in the colon, suggesting that this was due to reduced intestinal calcium secretion. Common genetic variants in the claudin-2 gene were associated with decreased tissue expression of claudin-2 and increased risk of kidney stones in 2 large population-based studies. Finally, we describe a family in which males with a rare missense variant in claudin-2 have marked hypercalciuria and kidney stone disease. Our findings indicate that claudin-2 is a key regulator of calcium excretion and a potential target for therapies to prevent kidney stones.

Altered VEGF Signaling Leads to Defects in Heart Tube Elongation and Omphalomesenteric Vein Fusion in Quail Embryos.

Formation of the endocardial and myocardial heart tubes involves precise cardiac progenitor sorting and tissue displacements from the primary heart field to the embryonic midline-a process that is dependent on proper formation of conjoining great vessels, including the omphalomesenteric veins (OVs) and dorsal aortae. Using a combination of vascular endothelial growth factor (VEGF) over- and under-activation, fluorescence labeling of cardiac progenitors (endocardial and myocardial), and time-lapse imaging, we show that altering VEGF signaling results in previously unreported myocardial, in addition to vascular and endocardial phenotypes. Resultant data show: (1) exogenous VEGF leads to truncated endocardial and myocardial heart tubes and grossly dilated OVs; (2) decreased levels of VEGF receptor 2 tyrosine kinase signaling result in a severe abrogation of the endocardial tube, dorsal aortae, and OVs. Surprisingly, only slightly altered myocardial tube fusion and morphogenesis is observed. We conclude that VEGF has direct effects on the VEGF receptor 2-bearing endocardial and endothelial precursors, and that altered vascular morphology of the OVs also indirectly results in altered myocardial tube formation. Anat Rec, 302:175-185, 2019. © 2018 Wiley Periodicals, Inc.

Live tissue antibody injection: A novel method for imaging ECM in limb buds and other tissues.

Understanding the morphogenesis and differentiation of tissues and organs from progenitor fields requires methods to visualize this process. Despite an ever-growing recognition that ECM plays an important role in tissue development, studies of ECM movement, and patterns in live tissue are scarce. Here, we describe a method in which a living limb bud is immunolabeled prior to fixation using fluorescent antibodies that recognize two ECM constituents, fibronectin and fibrillin 2. The results show that undifferentiated mesenchyme in quail embryos can be distinguished from prechondrogenic cellular condensations, in situ, via ECM antibodies-indicating the developmental transition from naïve mesenchyme to committed skeletal tissue. We conclude that our live tissue injection method is a general approach that allows visualization of the structural characteristics and the distribution pattern of ECM scaffolds, in situ. With slight modifications, the method will produce robust fluorescence images of ECM scaffolds in any suitable tissue mass and allow multiple kinds of optical analyses including virtual 3D reconstructions.

Decellularized Wharton's Jelly from human umbilical cord as a novel 3D scaffolding material for tissue engineering applications.

In tissue engineering, an ideal scaffold attracts and supports cells thus providing them with the necessary mechanical support and architecture as they reconstruct new tissue in vitro and in vivo. This manuscript details a novel matrix derived from decellularized Wharton's jelly (WJ) obtained from human umbilical cord for use as a scaffold for tissue engineering application. This decellularized Wharton's jelly matrix (DWJM) contained 0.66 ± 0.12 µg/mg sulfated glycosaminoglycans (GAGs), and was abundant in hyaluronic acid, and completely devoid of cells. Mass spectroscopy revealed the presence of collagen types II, VI and XII, fibronectin-I, and lumican I. When seeded onto DWJM, WJ mesenchymal stem cells (WJMSCs), successfully attached to, and penetrated the porous matrix resulting in a slower rate of cell proliferation. Gene expression analysis of WJ and bone marrow (BM) MSCs cultured on DWJM demonstrated decreased expression of proliferation genes with no clear pattern of differentiation. When this matrix was implanted into a murine calvarial defect model with, green fluorescent protein (GFP) labeled osteocytes, the osteocytes were observed to migrate into the matrix as early as 24 hours. They were also identified in the matrix up to 14 days after transplantation. Together with these findings, we conclude that DWJM can be used as a 3D porous, bioactive and biocompatible scaffold for tissue engineering and regenerative medicine applications.

Extracellular matrix motion and early morphogenesis.

For over a century, embryologists who studied cellular motion in early amniotes generally assumed that morphogenetic movement reflected migration relative to a static extracellular matrix (ECM) scaffold. However, as we discuss in this Review, recent investigations reveal that the ECM is also moving during morphogenesis. Time-lapse studies show how convective tissue displacement patterns, as visualized by ECM markers, contribute to morphogenesis and organogenesis. Computational image analysis distinguishes between cell-autonomous (active) displacements and convection caused by large-scale (composite) tissue movements. Modern quantification of large-scale 'total' cellular motion and the accompanying ECM motion in the embryo demonstrates that a dynamic ECM is required for generation of the emergent motion patterns that drive amniote morphogenesis.

In vivo time-lapse imaging reveals extensive neural crest and endothelial cell interactions during neural crest migration and formation of the dorsal root and sympathetic ganglia.

During amniote embryogenesis the nervous and vascular systems interact in a process that significantly affects the respective morphogenesis of each network by forming a "neurovascular" link. The importance of neurovascular cross-talk in the central nervous system has recently come into focus with the growing awareness that these two systems interact extensively both during development, in the stem-cell niche, and in neurodegenerative conditions such as Alzheimer's Disease and Amyotrophic Lateral Sclerosis. With respect to the peripheral nervous system, however, there have been no live, real-time investigations of the potential relationship between these two developing systems. To address this deficit, we used multispectral 4D time-lapse imaging in a transgenic quail model in which endothelial cells (ECs) express a yellow fluorescent marker, while neural crest cells (NCCs) express an electroporated red fluorescent marker. We monitored EC and NCC migration in real-time during formation of the peripheral nervous system. Our time-lapse recordings indicate that NCCs and ECs are physically juxtaposed and dynamically interact at multiple locations along their trajectories. These interactions are stereotypical and occur at precise anatomical locations along the NCC migratory pathway. NCCs migrate alongside the posterior surface of developing intersomitic vessels, but fail to cross these continuous streams of motile ECs. NCCs change their morphology and migration trajectory when they encounter gaps in the developing vasculature. Within the nascent dorsal root ganglion, proximity to ECs causes filopodial retraction which curtails forward persistence of NCC motility. Overall, our time-lapse recordings support the conclusion that primary vascular networks substantially influence the distribution and migratory behavior of NCCs and the patterned formation of dorsal root and sympathetic ganglia.

A transgenic quail model that enables dynamic imaging of amniote embryogenesis.

Embryogenesis is the coordinated assembly of tissues during morphogenesis through changes in individual cell behaviors and collective cell movements. Dynamic imaging, combined with quantitative analysis, is ideal for investigating fundamental questions in developmental biology involving cellular differentiation, growth control and morphogenesis. However, a reliable amniote model system that is amenable to the rigors of extended, high-resolution imaging and cell tracking has been lacking. To address this shortcoming, we produced a novel transgenic quail that ubiquitously expresses nuclear localized monomer cherry fluorescent protein (chFP). We characterize the expression pattern of chFP and provide concrete examples of how Tg(PGK1:H2B-chFP) quail can be used to dynamically image and analyze key morphogenetic events during embryonic stages X to 11.

Identification of emergent motion compartments in the amniote embryo.

The tissue scale deformations (≥ 1 mm) required to form an amniote embryo are poorly understood. Here, we studied ∼400 µm-sized explant units from gastrulating quail embryos. The explants deformed in a reproducible manner when grown using a novel vitelline membrane-based culture method. Time-lapse recordings of latent embryonic motion patterns were analyzed after disk-shaped tissue explants were excised from three specific regions near the primitive streak: 1) anterolateral epiblast, 2) posterolateral epiblast, and 3) the avian organizer (Hensen's node). The explants were cultured for 8 hours-an interval equivalent to gastrulation. Both the anterolateral and the posterolateral epiblastic explants engaged in concentric radial/centrifugal tissue expansion. In sharp contrast, Hensen's node explants displayed Cartesian-like, elongated, bipolar deformations-a pattern reminiscent of axis elongation. Time-lapse analysis of explant tissue motion patterns indicated that both cellular motility and extracellular matrix fiber (tissue) remodeling take place during the observed morphogenetic deformations. As expected, treatment of tissue explants with a selective Rho-Kinase (p160ROCK) signaling inhibitor, Y27632, completely arrested all morphogenetic movements. Microsurgical experiments revealed that lateral epiblastic tissue was dispensable for the generation of an elongated midline axis- provided that an intact organizer (node) is present. Our computational analyses suggest the possibility of delineating tissue-scale morphogenetic movements at anatomically discrete locations in the embryo. Further, tissue deformation patterns, as well as the mechanical state of the tissue, require normal actomyosin function. We conclude that amniote embryos contain tissue-scale, regionalized morphogenetic motion generators, which can be assessed using our novel computational time-lapse imaging approach. These data and future studies-using explants excised from overlapping anatomical positions-will contribute to understanding the emergent tissue flow that shapes the amniote embryo.

Embryogenesis of the first circulating endothelial cells.

Prior to this study, the earliest appearance of circulating endothelial cells in warm-blooded animals was unknown. Time-lapse imaging of germ-line transformed Tie1-YFP reporter quail embryos combined with the endothelial marker antibody QH1 provides definitive evidence for the existence of circulating endothelial cells - from the very beginning of blood flow. Blood-smear counts of circulating cells from Tie1-YFP embryos showed that up to 30% of blood-borne cells are Tie1 positive; though cells expressing low levels of YFP were also positive for benzidine, a hemoglobin stain, suggesting that these cells were differentiating into erythroblasts. Electroporation-based time-lapse experiments, exclusively targeting the intra-embryonic mesoderm were combined with QH1 immunostaining. The latter antibody marks quail endothelial cells. Together the optical data provide conclusive evidence that endothelial cells can enter blood flow from vessels of the embryo proper, as well as from extra-embryonic areas. When Tie1-YFP positive cells and tissues are transplanted to wild type host embryos, fluorescent cells emigrate from such transplants and join host vessels; subsequently a few YFP cells are shed into circulation. These data establish that entering circulation is a commonplace activity of embryonic vascular endothelial cells. We conclude that in the class of vertebrates most closely related to mammals a normal component of primary vasculogenesis is production of endothelial cells that enter circulation from all vessels, both intra- and extra-embryonic.

Time-lapse microscopy of macrophages during embryonic vascular development.

BACKGROUND: Macrophages are present before the onset of blood flow, but very little is known about their function in vascular development. We have developed a technique to concurrently label both endothelial cells and macrophages for time-lapse microscopy using co-injection of fluorescently conjugated acetylated low-density lipoprotein (AcLDL) and phagocytic dye PKH26-PCL. RESULTS: We characterize double-labeled cells to confirm specific labeling of macrophages. Double-labeled cells circulate, roll along the endothelium, and extravasate from vessels. Most observed macrophages are integrated into the vessel wall, showing an endothelial-like morphology. We used transgenic quail that express a fluorescent protein driven by the endothelial-specific promoter Tie1 in conjugation with the phagocytic dye to analyze these cells. Circulating PKH26-PCL-labeled cells are mostly Tie1-, but those which have integrated into the vessel wall are largely Tie1+. The endothelial-like phagocytic cells were generally stationary during normal vascular development. We, therefore, induced vascular remodeling and found that these cells could be recruited to sites of remodeling. CONCLUSIONS: The active interaction of endothelial cells and macrophages support the hypothesis that these cells are involved in vascular remodeling. The presence of phagocytic endothelial-like cells suggests either a myeloid-origin to certain endothelial cells or that circulating endothelial cells/hematopoietic stem cells have phagocytic capacity.

Convective tissue movements play a major role in avian endocardial morphogenesis.

Endocardial cells play a critical role in cardiac development and function, forming the innermost layer of the early (tubular) heart, separated from the myocardium by extracellular matrix (ECM). However, knowledge is limited regarding the interactions of cardiac progenitors and surrounding ECM during dramatic tissue rearrangements and concomitant cellular repositioning events that underlie endocardial morphogenesis. By analyzing the movements of immunolabeled ECM components (fibronectin, fibrillin-2) and TIE1 positive endocardial progenitors in time-lapse recordings of quail embryonic development, we demonstrate that the transformation of the primary heart field within the anterior lateral plate mesoderm (LPM) into a tubular heart involves the precise co-movement of primordial endocardial cells with the surrounding ECM. Thus, the ECM of the tubular heart contains filaments that were associated with the anterior LPM at earlier developmental stages. Moreover, endocardial cells exhibit surprisingly little directed active motility, that is, sustained directed movements relative to the surrounding ECM microenvironment. These findings point to the importance of large-scale tissue movements that convect cells to the appropriate positions during cardiac organogenesis.

Dynamic analysis of vascular morphogenesis using transgenic quail embryos.

BACKGROUND: One of the least understood and most central questions confronting biologists is how initially simple clusters or sheet-like cell collectives can assemble into highly complex three-dimensional functional tissues and organs. Due to the limits of oxygen diffusion, blood vessels are an essential and ubiquitous presence in all amniote tissues and organs. Vasculogenesis, the de novo self-assembly of endothelial cell (EC) precursors into endothelial tubes, is the first step in blood vessel formation. Static imaging and in vitro models are wholly inadequate to capture many aspects of vascular pattern formation in vivo, because vasculogenesis involves dynamic changes of the endothelial cells and of the forming blood vessels, in an embryo that is changing size and shape. METHODOLOGY/PRINCIPAL FINDINGS: We have generated Tie1 transgenic quail lines Tg(tie1:H2B-eYFP) that express H2B-eYFP in all of their endothelial cells which permit investigations into early embryonic vascular morphogenesis with unprecedented clarity and insight. By combining the power of molecular genetics with the elegance of dynamic imaging, we follow the precise patterning of endothelial cells in space and time. We show that during vasculogenesis within the vascular plexus, ECs move independently to form the rudiments of blood vessels, all while collectively moving with gastrulating tissues that flow toward the embryo midline. The aortae are a composite of somatic derived ECs forming its dorsal regions and the splanchnic derived ECs forming its ventral region. The ECs in the dorsal regions of the forming aortae exhibit variable mediolateral motions as they move rostrally; those in more ventral regions show significant lateral-to-medial movement as they course rostrally. CONCLUSIONS/SIGNIFICANCE: The present results offer a powerful approach to the major challenge of studying the relative role(s) of the mechanical, molecular, and cellular mechanisms of vascular development. In past studies, the advantages of the molecular genetic tools available in mouse were counterbalanced by the limited experimental accessibility needed for imaging and perturbation studies. Avian embryos provide the needed accessibility, but few genetic resources. The creation of transgenic quail with labeled endothelia builds upon the important roles that avian embryos have played in previous studies of vascular development.

Down modulation of IFN-gamma signaling in alveolar macrophages isolated from smokers.

The master cytokine, IFN-gamma possesses a wide spectrum of biological effects and is crucial for development of the highly activated macrophage phenotype characteristically found during inflammation. However, no data exists regarding the potential influence of cigarette smoke on the status of the expression of the cell surface receptor for IFN-gamma (IFN-gammaR) on alveolar macrophages (AM) of smokers. Here in, we report reduction in the expression of the IFN-gammaR alpha-chain on AM of cigarette smokers, when compared with non-smokers. Ensuing from the loss of receptor expression on the AM of smokers there was a decrease in IFN-gamma-mediated cell signaling. This included a decrease in the phosphorylation of signal transducer and activator of transcription (STAT)-1 and induction of interferon regulatory factor (IRF)-1. Further, diminished activation/induction of transcription factors did not appear to result from induction of known members of the 'suppressors of cytokine signaling (SOCS)' family. Decreased IFN-gamma signal transduction in AM from smokers may have an important implication regarding the use of therapeutic IFN-gamma in the lungs of patients that develop respiratory disorders as a result of tobacco use.

Extracellular matrix macroassembly dynamics in early vertebrate embryos.

This chapter focuses on the in vivo macroassembly dynamics of fibronectin and fibrillin-2--two prominent extracellular matrix (ECM) components, present in vertebrate embryos at the earliest stages of development. The ECM is an inherently dynamic structure with a well-defined position fate: ECM filaments are not only anchored to and move with established tissue boundaries, but are repositioned prior to the formation of new anatomical features. We distinguish two ECM filament relocation processes-each operating on different length scales. First, ECM filaments are moved by large-scale tissue motion, which rearranges major organ primordia within the embryo. The second type of motion, on the scale of the individual ECM filaments, is driven by local motility and protrusive activity of nearby cells. The motion decomposition is made practically possible by recent advances in microscopy and high-resolution particle image velocimetry algorithms. We demonstrate that both kinds of motion contribute substantially to the establishment of normal ECM structure, and both must be taken into account when attempting to understand ECM macroassembly during embryonic morphogenesis. The tissue-scale motion changes the local amount (density) and the tissue-level structure (e.g., orientation) of ECM fibers. Local reorganization includes filament assembly and the segregation of ECM into specific patterns. Local reorganization takes place most actively at Hensen's node and around the primitive streak. These regions are also sites of active cell migration, where fibrillin-2 and fibronectin are often colocalized in ECM globules, and new fibrillin-2 foci are deposited. During filament assembly, the globular patches of ECM are joined into larger linear structures in a hierarchical process: increasingly larger structures are created by the aggregation of smaller units. A future understanding of ECM assembly thus requires the study of the complex interactions between biochemical assembly steps, local cell action, and tissue motion.

Dynamic imaging of cell, extracellular matrix, and tissue movements during avian vertebral axis patterning.

Vertebrate axis patterning depends on cell and extracellular matrix (ECM) repositioning and proper cell-ECM interactions. However, there are few in vivo data addressing how large-scale tissue deformations are coordinated with the motion of local cell ensembles or the displacement of ECM constituents. Combining the methods of dynamic imaging and experimental biology allows both cell and ECM fate-mapping to be correlated with ongoing tissue deformations. These fate-mapping studies suggest that the axial ECM components "move" both as a composite meshwork and as autonomous particles, depending on the length scale being examined. Cells are also part of this composite, and subject to passive displacements resulting from tissue deformations. However, in contrast to the ECM, cells are self-propelled. The net result of cell and ECM displacements, along with proper ECM-cell adhesion, is the assembly of new tissue architecture. Data herein show that disruption of normal cell-ECM interactions during axis formation results in developmental abnormalities and a disorganization of the ECM. Our goal in characterizing the global displacement patterns of axial cells and ECM is to provide critical information regarding existing strain fields in the segmental plate and paraxial mesoderm. Deducing the mechanical influences on cell behavior is critical, if we are to understand vertebral axis patterning. Supplementary material for this article is available online at http://www.mrw.interscience.wiley.com/suppmat/1542-975X/suppmat/72/v72.266.html.

Histamine induces Toll-like receptor 2 and 4 expression in endothelial cells and enhances sensitivity to Gram-positive and Gram-negative bacterial cell wall components.

Histamine is a major inflammatory molecule released from the mast cell, and is known to activate endothelial cells. However, its ability to modulate endothelial responses to bacterial products has not been evaluated. In this study we determined the ability of histamine to modulate inflammatory responses of endothelial cells to Gram-negative and Gram-positive bacterial cell wall components and assessed the role of Toll-like receptors (TLR) 2 and 4 in the co-operation between histamine and bacterial pathogens. Human umbilical vein endothelial cells (HUVEC) were incubated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or peptidoglycan (PGN) in the presence or absence of histamine, and the expression and release of interleukin-6 (IL-6), and NF-kappaB translocation were determined. The effect of histamine on the expression of mRNA and proteins for TLR2 and TLR4 was also evaluated. Incubation of HUVEC with LPS, LTA and PGN resulted in marked enhancement of IL-6 mRNA expression and IL-6 secretion. Histamine alone markedly enhanced IL-6 mRNA expression in HUVEC, but it did not stimulate proportional IL-6 release. When HUVEC were incubated with LPS, LTA, or PGN in the presence of histamine marked amplification of both IL-6 production and mRNA expression was noted. HUVEC constitutively expressed TLR2 and TLR4 mRNA and proteins, and these were further enhanced by histamine. The expression of mRNAs encoding MD-2 and MyD88, the accessory molecules associated with TLR signalling, were unchanged by histamine treatment. These results demonstrate that histamine up-regulates the expression of TLR2 and TLR4 and amplifies endothelial cell inflammatory responses to Gram-negative and Gram-positive bacterial components.

Low responsiveness to IFN-gamma, after pretreatment of mouse macrophages with lipopolysaccharides, develops via diverse regulatory pathways.

We have investigated the mechanisms by which prior exposure of mouse macrophages to lipopolysaccharides (LPS) induces a state of low responsiveness to subsequent exposure to IFN-gamma. We demonstrate that induction of this state requires both de novo gene expression and the suppression of phosphorylation events that lead to activation of transcription factor Stat1 alpha. These observations are mechanistically consistent with the known induction of suppressors of cytokine signaling (SOCS)-1 and SOCS-3 proteins by LPS. In this regard, we demonstrate that overexpression of either SOCS protein suppresses induction of the mouse inducible nitric oxide synthase (iNOS) gene promoter: apparently by suppressing interactions between Stat1 alpha and IFN-gamma activated sites present in both the iNOS, and interferon regulatory factor-1, gene promoters. The induction of SOCS-1 and SOCS-3 by LPS or IFN-beta (an autocrine/paracrine mediator of LPS-induced SOCS-1 mRNA synthesis)occurs by way of multiple protein kinase pathways that include protein tyrosine kinases, protein kinase C, and mitogen-activated protein kinases. These results provide insight that may allow discrimination between LPS-induced inhibition of macrophage functions that are detrimental to the host (e.g. continued exposure to LPS) versus those that might potentially be beneficial (e.g. exposure to subsequent agonists that induce more specific macrophage functions).