Botryllus schlosseri,an emerging model for the study of aging,stem cells,and mechanisms of regeneration

The decline of tissue regenerative potential with the loss of stem cell function is a hallmark of mammalian aging. We study Botryllus schlosseri, a colonial chordate which exhibits robust stem cell-mediated regeneration capacities throughout life. Larvae, derived by sexual reproduction and chordate development, metamorphose to clonal founders that undergo weekly formation of new individuals by budding from stem cells. Individuals are transient structures which die through massive apoptosis, and successive buds mature to replicate an entire new body. As a result, their stem cells, which are the only self-renewing cells in a tissue, are the only cells which remain through the entire life of the genotype and retain the effects of time. During aging, a significant decrease in the colonies’ regenerative potential is observed and both sexual and asexual reproductions will eventually halt. When a parent colony is experimentally separated into a number of clonal replicates, they frequently undergo senescence simultaneously, suggesting a heritable factor that determines lifespan in these colonies. The availability of the recently published B. schlosseri genome coupled with its unique life cycle features promotes the use of this model organism for the study of the evolution of aging, stem cells, and mechanisms of regeneration.     

In vitro culture of tissue from the tunicateStyela clava

Summary Pharyngeal explants and circulatory hemocytes from the tunicateStyela clava were cultured in a medium containing tunicate plasma, artificial seawater, RPMI 1640, and antibiotics. Pharnngeal tissue remained viable and proliferated for up to 72 d in vitro. Proliferative activity maintained the pool of hemocytes within explants and facilitated the migration of pharyngeal hemocytes from explants into culture supernatants. The diversity of morphologically distinct cell types within the hemocyte pool of pharyngeal cultures indicated that cell division was followed by regulated differentiation. In contrast to pharyngeal cultures, suspensions of circulatory hemocytes did not survive for prolonged periods in vitro. Proliferative activity could not be detected in circulatory hemocyte cultures. These results are discussed in terms of the differentiation state of hemocytes and the efficacy of culture conditions. This study was supported by the National Science Foundation, Washington, DC (grant DCB 85 19848) and by BRSG funds from UCLA Schools of Medicine and Dentistry. Flow cytometric facilities were sponsored in part by a Johnson Cancer Center Core Grant (CA 16042). David A. Raftos is a Fulbright Postdoctoral Fellow and recipient of a Frederik B. Bang Scholarship in Marine Invertebrate Immunology administered by the American Association of Immunologist. Dan L. Stillman was supported by an REU supplement from the National Science Foundation.     

Polar Constituents of the Tunicate Botryllus schlosseri

Eighteen compounds were identified by GC–MS of their trimethylsilyl derivatives in n-butanolic extract from the biomass of Botryllus schlosseri. Three of them, 5-oxoproline, 5-hydroxyhydantoin, and kinurenic acid, were found in marine invertebrates for the first time. In addition to cellulose, the biomass was also shown to contain complex water-soluble sulfated polysaccharides. These were extracted and fractionated, and sulfate content and monosaccharide composition were determined in the fractions; fucose, xylose, galactose, mannose, glucose, glucosamine, galactosamine, and uronic acids were found. Unlike several other tunicate species, Botryllus schlosseri does not seem to contain any simple galactan sulfate.     

Isolation and culture of vascular smooth muscle cells from rat placenta

We developed a new separation method for isolating placental vascular smooth muscle cells (PVSMCs) from a rat in this study. Our method used the magnetic force between a magnet and ferrous ferric oxide (Fe₃O ₄) to make the separation and extraction processes easier and more efficient. From the first to sixth generation, the cells isolated using this protocol were identified as smooth muscle cells (SMCs) by their immunoreactivity to the SMC markers and by the “hill and valley” morphology. PVSMCs were exposed to angiotensin II (1 μmol/L) and resulted in sharply increased intracellular Ca ²⁺ concentration. Furthermore, activation of protein kinase C (PKC) increased concomitantly with a decrease in calponin expression. These results indicate that the isolated cells had biological activity. Our method of isolating PVSMCs from rat leads to isolation of cultured cells with activity and high purity. The approach will be useful in research studies on placental vascular diseases.     

Involution of the caudal musculature during metamorphosis in the ascidian,Botryllus schlosseri

Summary The caudal musculature of the free-swimming tadpole of the ascidian, B. schlosseri consists of cylindrical mononucleated cells connected in longitudinal rows flanking the axial notochord. During resorption of the larval tail, which is apparently induced by the contraction of the epidermis, muscle cells are dissociated and pushed into the body cavity where most of them are rapidly engulfed by phagocytes. In the initial stages of tail withdrawal muscle cells display surface alterations due to the disruption of intercellular junctions and disarrangement of myofibrils. Extensive degenerative changes, with shrinkage of mitochondria and disintegration of the contractile material are subsequently observed. Lysosomes and autophagic vacuoles are rarely seen and appear to play a secondary role in the degradation of the muscle cells, which occurs predominantly within the phagocytes. Myofilaments and myofibrils have never been observed within autophagic vacuoles. Clumps of muscle fragments and degenerated phagocytes undergo eventual dissolution in the blood lacunae, concomitantly with the differentiation of the young oozooid.This investigation was supported in part by a grant from the Muscular Dystrophy Associations of America and by CNR contract No. 7100396/04115542 from the Istituto di Biologia del Mare, Venice. We gratefully acknowledge the skillful assistance of Mr. G. Gallian, Mr. M. Fabbri and Mr. G. Tognon. We also thank the staff of the Stazione Idrobiologica at Chioggia for collecting the colonies.     

In vitro culture of mesenchymal lineage cells established from the colonial tunicate Botryllus primigenus

Body trunks were isolated from juvenile zooids of the Japanese colonial tunicate Botryllus primigenus and cultured in vitro to establish tissue-specific cell lines. Epidermal cells from some explants spread and formed a flat sheet consisting of vacuolated cells. They then dissociated into single cells, and their growth stopped within two weeks. Continuously proliferating cells were established from four explants. After the 20th implantation, nuclear and mitochondrial DNAs were extracted from these cells. The nucleotide sequences of proliferating cell nuclear antigen (PCNA) and mitochondrial large ribosomal RNA (mtlrRNA) completely matched the PCNA and mtlrRNA taken from living colonies of B. primigenus; this shows that the four independently proliferating cells were indeed of the Botryllus origin. One cell line (Bp0306E10) comprised round-shaped cells with a diameter of 8-10 microm. These cells have been cultured in vitro with a doubling time of approximately 24 hours since June, 2003. The BrdU labeling index was approximately 2%. Monoclonal antibodies raised against the cultured cells recognized a 28 kDa polypeptide and stained free mesenchymal cells in vivo. G418-resistant subclonal cells could be established by introducing a tunicate retrotransposon loaded with the neomycin resistance gene into the cells by electroporation. This study is the first to succeed in producing a sustainable cell culture of Botryllus.     

Long-term culture of porcine bladder epithelial cells

Epithelial cells from normal pig bladders proliferated when cocultured with lethally irradiated feeder cells of the LA7 rat mammary tumor line. When the bladder cells and feeders were plated together at a confluent density, the bladder cells proliferated as the feeder cells died, resulting in a confluent culture of bladder cells. The bladder cells were successfully subcultured by plating with freshly irradiated LA7 feeder cells. In this way, bladder cells from five pigs were carried to confluency in passages 1, 4, 7, 7, and 13, amounting to at least 6, 18, 24, 26, and 45 doublings in culture, respectively, and none showed signs of slowed proliferation at the time of culture termination. Fibroblasts never became a prominent feature of these cultures, and their frequency was determined to be about 26 fibroblasts per 10(5) cells in passage 9. Pig bladder cells in 0.5% serum doubled in number in slightly over 3 d, whereas cells in 5.0% serum doubled in about 6 d. In fresh medium without feeder cells only minimal proliferation of bladder cells occurred. In LA7-conditioned medium the bladder cell numbers decreased, leading to the conclusion that the stimulus from LA7 cells is mechanically or physically transmitted. The bladder cells reacted with antibodies to keratins 7 and 18 but not to keratin 14 or vimentin. Tight junctions, visualized with an antibody to the ZO1 protein, connected all the cells to their neighbors. Most cells in passage 9 carried the diploid chromosome number of 38.     

In vitro cell culture pO2 is significantly different from incubator pO2

Continuous noninvasive monitoring of peri‐cellular liquid phase pO₂ in adherent cultures is described. For neurons and astrocytes, this approach demonstrates that there is a significant difference between predicted and observed liquid phase pO₂. Particularly at low gas phase pO₂s, cell metabolism shifts liquid phase pO₂ significantly lower than would be predicted from the O₂ gas/air equilibrium coefficient, indicating that the cellular oxygen uptake rate exceeds the oxygen diffusion rate. The results demonstrate the need for direct pO₂ measurements at the peri‐cellular level, and question the widely adopted current practice of relying on setting the incubator gas phase level as means of controlling pericellular oxygen tension, particularly in static culture systems that are oxygen mass transfer limited. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Primary culture of gill epithelial cells from the sea bassDicentrarchus Labrax

Summary We have developed the first explant technique that allows the in vitro study of gill physiology and biochemistry in marine species. Gill fragments were cultured at 17° C, in atmospheric Pco₂, with nutrient medium (Leibovitz L15), pH 7.8, supplemented with 10% fetal bovine serum and adjusted to the osmolarity of fish plasma (350 mOsm/liter). Coating plates with collagen, gelatin, or polylysin did not improve our results. Decrease in osmotic pressure, removal of bovine serum, or its replacement by fish serum inhibited growth from the explants. Approximately 50% of the explants produced cell growth, and after 4 days of culture a monolayer of contiguous cells was formed. This technique is rapid and does not require the use of enzymes. The cells appeared flat and thin with an epithelioid shape. They looked polygonal with a maximum length of 10 to 50 μm. Evidence that they are unique gill cells is the presence of polymorphic surface crenelations (microplicae), prominent Golgi apparatus, tight junctions and desmosomes. Comparison with in vivo tissue showed them to be epithelial cells having differentiated in a homogeneous population of respiratorylike (pavement) cells. They are polarized with their apical surface facing the culture medium. The development of this culture system represents a new tool for cellular approaches to determine precisely the functions and transport mechanism of gill cells.     

Different characteristics of endothelial cells from central and peripheral human cornea in primary culture and after subculture

Summary Several methods for isolation and cultivation of human corneal endothelial cells have been described during the last few decades. In contrast to the situation in vivo, the cultured cells show mitogenic activity but often lose their typical morphological appearance. In this paper, we describe a technique to isolate and cultivate morphologically unchanged endothelium from the human cornea. This method revealed different characteristics of endothelial cells according to their position within the human cornea. Endothelial cells isolated from the central part have a morphology similar to that of cells in vivo (i.e., they are densely packed and show no mitogenic activity). In contrast, endothelial cells derived from the peripheral part of the cornea are characterized by mitogenic activity but their cell-to-cell attachment seems to be less tight than in vivo. The significance of these two different endothelial cell types for wound healing in the human cornea is discussed.     

Expansion and long-term culture of differentiated normal rat urothelial cells in vitro

The objective of this study is to establish a reliable cell culture system for the long-term culture of rat urothelial cells (RUC), in which the cells multiply in vitro and form stratified polarized urothelium. Urothelial cells were harvested by the enzymatic digestion of the urothelium exposed by the eversion of resected rat bladders. Primary cultures were initiated in keratinocyte serum-free medium (KSFM) for selective proliferation of urothelial cells. Subsequently, the cells were propagated in a mixture of conditioned medium (CM) derived from Swiss 3T3 cell culture supernatant and KSFM (CM-KSFM). Mean population doubling time was 13.8 +/- 0.9 h. RUC were successfully maintained for 18 passages over a period of 4-5 mo. Detailed investigations of culture conditions showed that CM-KSFM yielded a differentiated multilayer structure. The stratified urothelial sheets measuring 4 x 6 cm2 could be formed and then detached using dispase. Cytokeratin pattern in both the cultured urothelial monolayer and engineered stratified layers was similar to those seen in vivo, as assessed with monoclonal antibody against cytokeratin 17. Ultrastructural morphology showed microvilli, basal cell layer, and desmosomes between adjacent cells in the stratified urothelium.     

In vitro immunization can elicit the expansion of diverse repertoire of B cells from peripheral blood mononuclear cells

We previously developed an in vitro immunization (IVI) protocol of human peripheral blood mononuclear cells (PBMC) for generating antigen-specific human antibodies. In order to clarify whether IVI protocolinduces antigen-specific B cell responses in PBMC, we analyzed family gene usage and sequence of the variable region gene of immunoglobulin heavy chain (VH gene) of the antibody produced from the in vitro immunized PBMC. Sequence homology analyses of VH gene demonstrated that a larger repertoire of B cells can be sensitized with mite-extract than with cholera toxin B subunit and rice allergen. Further, antigen-specific B cells were efficiently expanded by using CpG oligodeoxynucleotide as adjuvant. These results suggest that appropriate combination of sensitizing antigen and adjuvant is primarily important for expansion of antigen-specific B cells in IVI protocol.     

GTSF-2: A new, versatile cell culture medium for diverse normal and transformed mammalian cells

Summary The aim of this study was to test the versatility of a new basal cell culture medium, GTSF-2. In addition to traditional growth-factors, GTSF-2 contains a blend of three sugars (glucose, galactose, and fructose) at their physiological levels. For these studies, we isolated normal endothelial cells from human, bovine, and rat large blood vessels and microvessels. In addition, GTSF-2 was also tested as a replacement for high-glucose-containing medium for PC12 pheochromocytoma cells and for other, transformed cell lines. The cell growth characteristics were assessed with a novel cell viability and proliferation assay, which is based on the bioreduction of the fluorescent dye, Alamar Blue. After appropriate calibration, the Alamar Blue assay was found to be equivalent to established cell proliferation assays. Alamar Blue offers the advantage that cell proliferation can be measured in the same wells over an extended period of time. For some of the cell types (e.g., endothelial cells isolated from the bovine aorta, the rat adrenal medulla, or the transformed cells), proliferation in unmodified GTSF-2 was equivalent to that in the original culture media. For others cell types (e.g., human umbilical vein endothelial cells and PC12 cells), GTSF-2 proved to be a superior growth medium, when supplemented with simple additives, such as endothelial cell growth supplement (bFGF) or horse serum. Our results suggest that GTSF-2 is a versatile basal medium that will be useful for studying organ-specific differentiation in heterotypic coculture studies.     

Human macrovascular endothelial cells: Optimization of culture conditions

Summary The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage.     

Isolation and culture of smooth muscle cells from human umbilical cord arteries

Summary A short method is described for obtaining a large number of pure vascular smooth muscle cells in culture. The smooth muscle cells were isolated from human umbilical cord arteries digested twice by an enzyme mixture of collagenase, trypsin, elastase, and DNAase with addition of α-tosyl-lysyl chloromethane. Primary cell culture and first subculture were not contaminated by endothelial cells, no Factor VIII being produced. The cultures consisted of smooth muscle cells as appeared from phase contrast and electron microscopy. Part of this study was supported by a scholarship from the Dutch Ministry of Education and Science and by the Leyden University Foundation.     

Cell culture density affects the proliferation activity of human adipose tissue stem cells

In this study, we investigated the effect of cell density on the proliferation activity of human mesenchymal stem cells (MSCs) derived from adipose tissue (AT‐MSCs) over time in culture. Passage #4 (P4) and #12 (P12) AT‐MSCs from two donors were plated at a density of 200 (culture condition 1, CC1) or 5000 (culture condition 2, CC2) cells cm^?2. After 7 days of incubation, P4 and P12 AT‐MSCs cultured in CC1 were thin and spindle‐shaped, whereas those cultured in CC2 had extensive cell‐to‐cell contacts and an expanded cell volume. In addition, P4 and P12 AT‐MSCs in CC1 divided more than three times, while those in CC2 divided less than once on average. Flow cytometric analysis using 5(6)‐carboxyfluorescein diacetate N‐succinimidyl ester dye showed that the fluorescence intensity of AT‐MSCs was lower in CC1 than in CC2. Furthermore, expression of proliferation‐associated genes, such as CDC45L, CDC20A and KIF20A, in P4 AT‐MSCs was higher in CC1 than in CC2, and this difference was also observed in P12 AT‐MSCs. These data demonstrated that cell culture density affects the proliferation activity of MSCs, suggesting that it is feasible to design a strategy to prepare suitable MSCs using specific culture conditions. Copyright © 2016 John Wiley & Sons, Ltd.