Establishment of callus and cell suspension cultures from Gypsophila paniculata leaf segments and study of the attachment of host cells by Erwinia herbicola pv. gypsophilae

Callus and cell suspension cultures were initiated from leaf segments of G. paniculata. Fresh and dry weights measurements of callus showed that callus growth was optimal on MS medium supplemented with 1.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg l^–1 benzyladenin (BA). Calli cultured on this medium, showed a two-fold increase in fresh weight by the fourth week of incubation. The initiated hard green callus was repeatedly subcultured on MS medium containing increasing concentrations of 2,4-D in order to increase its friability. The friable callus was then used for establishment of a cell suspension culture. Maximum growth of the suspension culture was on medium supplemented with 1.0 mg l^–1 2,4-D and 0.2 mg l^–1 BA.The suspension culture was used for studying plant host attachment in both electron and light microscopy. Upon infection with E. herbicola, plant cells showed aggregate formation within 24 h of infection. In the presence of the pathogenic Ehg,the number of aggregates formed was 342 aggregates ml^–1, in the presence of the non-pathogenic Ehg154 aggregates ml^–1 and in the control 115 aggregates ml^–1. These results show that the pathogenic strain causes formation of cell aggregates 5.8 times greater than the non-pathogenic one. Based on these results, it can be hypothesized that bacterial cells of the pathogenic strains bind to the plant cells and may form a bridge for attachment of plant cells to one another. Observations by electron microscope show that bacterial cells do attach to plant cells and that this attachment might be via formation of a bridge between the bacteria and the plant cell.     

New bioengineering insights into human neural precursor cell expansion in culture

Understanding initial cell growth, interactions associated with the process of expansion of human neural precursor cells (hNPCs), and cellular events pre- and postdifferentiation are important for developing bioprocessing protocols to reproducibly generate multipotent cells that can be used in basic research or the treatment of neurodegenerative disorders. Herein, we report the in vitro responses of telencephalon hNPCs grown in a serum-free growth medium using time-lapse live imaging as well as cell-surface marker, aggregate size, and immunocytochemical analyses. Time-lapse analysis of hNPC initial expansion indicated that cell-surface attachment in stationary culture and the frequency of cell-cell interaction in suspension conditions are important for subsequent aggregate formation and hNPC growth. In the absence of cell-surface attachment in low-attachment stationary culture, large aggregates of cells were formed and expansion was adversely affected. The majority of the telencephalon hNPCs expressed CD29, CD90, and CD44 (cell surface markers involved in cell-ECM and cell-cell interactions to regulate biological functions such as proliferation), suggesting that cell-surface attachment and cell-cell interactions play a significant role in the subsequent formation of cell aggregates and the expansion of hNPCs. Before differentiation, about 90% of the cells stained positive for nestin and expressed two neural precursor cells surface markers (CD133 and CD24). Upon withdrawal of growth cytokines, hNPCs first underwent cell division and then differentiated preferentially towards a neuronal rather than a glial phenotype. This study provides key information regarding human NPC behavior under different culture conditions and favorable culture conditions that are important in establishing reproducible hNPC expansion protocols.     

Tandem events in myoblast fusion

Myoblasts, derived from primary chick pectoral muscle explants and grown on collagencoated culture dishes in a low calcium medium, are harvested with EDTA and are gently agitated in suspension. In the presence of calcium, the cells rapidly form easily dissociable aggregates which exclude fibroblasts. The apparent strength of adhesion increases with time until, under appropriate conditions, the myoblasts fuse in suspension to form multinucleate cells. The calcium-dependent dissociable aggregation shows optima for pH, temperature, calcium concentration, and culture age that closely parallel those observed for myotube formation measured with cells attached to tissue culture plates. We conclude from this marked correlation between the effects of these variables on myoblast aggregation and myotube formation that cell-cell adhesion is an integral part of myoblast fusion. Furthermore, we suggest that the formation of multinucleate cells is the result of a sequence of steps beginning with cell-cell recognition and adhesion, progressing to membrane union, and ultimately ending after subsequent morphologic changes producing the morphologies characteristic of multinucleate cells both in suspension and on tissue culture plates.     

EphA3 is up-regulated by epidermal growth factor and promotes formation of glioblastoma cell aggregates

EphA3, a member of the Eph family of receptor tyrosine kinases, has been reported to be overexpressed in some human cancers including glioblastoma. Here, we found that expression of EphA3 is up-regulated in response to epidermal growth factor (EGF) stimulation and promotes formation of cell aggregates in suspension culture of glioblastoma cells. Suppression of EphA3 expression by short hairpin RNA-mediated knockdown or CRISPR/Cas9-mediated gene deletion inhibited EGF-induced promotion of cell aggregate formation, whereas overexpression of EphA3 promoted formation of cell aggregates in suspension culture. EGF-induced EphA3 expression and promotion of cell aggregate formation required Akt activity. Furthermore, N-cadherin, whose expression was regulated by EGF and EphA3, contributed to the formation of cell aggregates in suspension culture. These results suggest that the regulation of EphA3 expression plays a critical role in glioblastoma cell growth in non-adherent conditions.     

Embryogenic callus formation,growth and regeneration in callus and suspension cultures of Miscanthus x ogiformis Honda Giganteus' as affected by proline

The effects of proline additions to culture systems of Miscanthus x ogiformis Honda Giganteus' were investigated. Proline was added in concentrations of 0, 12.5, 25, 50, 100 or 300 mM to the callus induction and suspension culture media containing either Murashige and Skoog or N6 basal salts and 22.6 μM 2,4-dichlorophenoxyacetic acid. Shoot apices and leaves from in vitro-propagated shoots, and immature inflorescences from greenhouse-grown plants were used as explants for callus induction and formation. Suspension cultures initiated from embryogenic callus of immature inflorescences were used to test the effect of proline in suspension cultures. The proline additions affected the formation of embryogenic callus and the growth of suspension cultures. Improvements depended on the proline concentration and the basal salts of the medium. Addition of 12.5 to 50 mM proline to callus induction medium with Murashige and Skoog salts increased embryogenic callus formation on shoot apices and leaf explants while proline had no effect on embryogenic callus formation in medium with N6 salts. Increased growth with increasing proline concentration was obtained in suspension aggregates grown in medium with N6 salts, whereas proline only increased growth of suspension aggregates grown in medium with Murashige and Skoog salts at concentrations of 12.5 or 25 mM. A stimulating effect of proline on plant regeneration was observed in short-term cultures of callus as well as in long-term cultures of suspension aggregates. An optimum proline concentration for plant regeneration was found at 12.5 mM. This revised version was published online in June 2006 with corrections to the Cover Date.     

Embryo development from discrete cell aggregates inIpomoea Batatas (L.) Lam. in response to structural polarity

Summary High numbers of embryos are difficult to obtain in liquid cultures of sweet potato (Ipomoea batatas (L.) Lam.) because discrete cell aggregates, produced through calli fragmentation, do not support embryo growth. In an effort to demonstrate that embryo development is possible from discrete cell aggregates, we compared embryo formation from cell aggregates 250–355 μm in diameter cultured either in suspension in liquid medium, on agar solidified medium, or immobilized on alginate beads floated in liquid medium. Embryos were initiated but remained arrested in their globular stage on cell aggregates cultured in suspension. Embryos developed to the torpedo stage from cell aggregates cultured on solidified medium and from cell aggregates anchored on alginate beads. Thus, embryos continued to develop beyond the globular stage when a structural polarity, which led probably to the establishment of a physiological polarity, was created. The production of sweet potato embryos in liquid culture can be improved by using alginate beads or culture conditions and protocols leading to the release during calli fragmentation of polarized individual cell aggregates. This work was supported in part by a IFAS/Gas Research Institute cooperative grant. Florida Agriculture Experiment Station Journal Series 9297     

What controls the position,number, size,and distribution of neuromuscular junctions on rat muscle fibers?

This review focuses on mechanisms that determine the position, number, size, and distribution of neuromuscular junctions (NMJs) on skeletal muscle fibers. Most of the data reviewed derive from studies of ectopic NMJ formation on soleus (SOL) muscle fibers in adult rats, which recapitulates essential aspects of NMJ formation in normal development. Transplanted axons induce acetylcholine receptor (AChR) aggregates, which are multiple and irregularly distributed initially but subsequently undergo massive reorganization such that one or a few winners survive and reach a certain size while the rest are eliminated (the losers). Results obtained by blocking nerve activity early and stimulating the SOL electrically show that evoked muscle impulse activity is responsible for the growth of winners to a given size and the creation of refractory zones, about 0.75 long, on each side of the winners, in which the elimination of losers occurs. Consequently, when two or more aggregates or NMJs survive on one fiber, they are, on average, at least 1.5 mm apart. Locally applied neural agrin induces comparable aggregation of AChRs and other postsynaptic proteins on denervated SOL fibers and such aggregates undergo similar activity-dependent selection for survival or elimination in refractory zones. In a dose-dependent way, neural agrin alone also induces expression of ε-AChR subunits and stabilizes AChRs to a half-life of 10 days, as found at normal NMJs. It is argued that signs of prepatterning of innervation sites by intrinsic muscle mechanisms may refer to epiphenomena that play no important role in NMJ formation. The conclusion is that neural agrin initiates and then maintains NMJs where motor axons happen to contact receptive muscle fibers and that evoked muscle impulse activity then ensures that the NMJs reach their appropriate size, efficiency and spatial distribution along each fiber.     

Scalable GMP compliant suspension culture system for human ES cells

Suspension bioreactors are an attractive alternative to static culture of human embryonic stem cells (hESCs) for the generation of clinically relevant cell numbers in a controlled system. In this study, we have developed a scalable suspension culture system using serum-free defined media with spinner flasks for hESC expansion as cell aggregates. With optimized cell seeding density and splitting interval, we demonstrate prolonged passaging and expansion of several hESC lines with overall expansion, yield, viability and maintenance of pluripotency equivalent to adherent culture. Human ESCs maintained in suspension as aggregates can be passaged at least 20 times to achieve over 1×10(13) fold calculated expansion with high undifferentiation rate and normal karyotype. Furthermore, the aggregates are able to differentiate to cardiomyocytes in a directed fashion. Finally, we show that the cells can be cryopreserved in serum-free medium and thawed into adherent or suspension cultures to continue passaging and expansion. We have successfully used this method under cGMP or cGMP-equivalent conditions to generate cell banks of several hESC lines. Taken together, our suspension culture system provides a powerful approach for scale-up expansion of hESCs under defined and serum-free conditions for clinical and research applications.     

Suspension culture reveals a morphogenetic property of a thyroid epithelial cell line

It is known that freshly dissociated thyroid cell clusters form follicles in suspension culture. Thyroid epithelial cell lines, grown for many generations in vitro, fail to show colloid-containing lumina when cultured as monolayers. Several thyroid cell lines, some transformed, have been tested with respect to their ability to form extracellular lumina when transferred from monolayer to suspension culture. One cell line in particular, the T78 cell line, showed this property when cultured in suspension. Lumina formed within 3 days even in the absence of added thyrotropin (TSH). The ultrastructure of lumina within cell aggregates resembled that of the thyroid follicle in vivo. The ability to undergo morphogenesis may therefore be an intrinsic property of thyroid epithelial cells which is retained for a large number of generations in vitro and is revealed by proper culture conditions. The shift from monolayer to suspension culture may thus lead to the expression of a thyroid differentiated function such as the formation of follicle-like structures.     

The Mrj co-chaperone mediates keratin turnover and prevents the formation of toxic inclusion bodies in trophoblast cells of the placenta

Defects in protein-folding and -degradation machinery have been identified as a major cause of intracellular protein aggregation and of aggregation-associated diseases. In general, it remains unclear how these aggregates are harmful to normal cellular function. We demonstrate here that, in the developing placenta of the mouse, the absence of the Mrj (Dnajb6) co-chaperone prevents proteasome degradation of keratin 18 (K18; Krt18) intermediate filaments, resulting in the formation of keratin inclusion bodies. These inclusions in chorionic trophoblast cells prevent chorioallantoic attachment during placental development. We show further that keratin-deficient embryos undergo chorioallantoic attachment and that, by genetically reducing keratin expression in Mrj(-/-) conceptuses, chorioallantoic attachment was rescued. Therefore, the chorioallantoic attachment phenotype in Mrj mutants is not due to a deficiency of the normal keratin cytoskeleton, but rather is cytotoxicity caused by keratin aggregates that disrupt chorion trophoblast cell organization and function.     

Microenvironment Modulates Osteogenic Cell Lineage Commitment in Differentiated Embryonic Stem Cells

Background

Due to their self-renewal, embryonic stem cells (ESCs) are attractive cells for applications in regenerative medicine and tissue engineering. Although ESC differentiation has been used as a platform for generating bone in vitro and in vivo, the results have been unsatisfactory at best. It is possible that the traditional culture methods, which have been used, are not optimal and that other approaches must be explored.

Methodology/Principal Findings

ESCs were differentiated into osteoblast lineage using a micro-mass approach. In response to osteogenic differentiation medium, many cells underwent apoptosis, while others left the micro-mass, forming small aggregates in suspension. These aggregates were cultured in three different culture conditions (adhesion, static suspension, and stirred suspension), then examined for osteogenic potential in vitro and in vivo. In adhesion culture, ESCs primed to become osteoblasts recommitted to the adipocyte lineage in vitro. In a static suspension culture, resulting porous aggregates expressed osteoblasts markers and formed bone in vivo via intermembranous ossification. In a stirred suspension culture, resulting non-porous aggregates suppressed osteoblast differentiation in favor of expanding progenitor cells.

Conclusions/Significance

We demonstrate that microenvironment modulates cell fate and subsequent tissue formation during ESC differentiation. For effective tissue engineering using ESCs, it is important to develop optimized cell culture/differentiation conditions based upon the influence of microenvironment.     

Constitutive myc expression impairs hypertrophy and calcification in cartilage.

The myc oncogene is expressed by proliferating quail embryo chondrocytes (QEC) grown as adherent cells and is repressed in QEC maintained in suspension culture. To investigate the interference of myc expression during chondrocyte differentiation, QEC were infected with a retrovirus carrying the v-myc oncogene (QEC-v-myc). Uninfected or helper virus-infected QEC were used as control. In adherent culture, QEC-v-myc displayed a chondrocytic phenotype and synthesized type II collagen and Ch21 protein, while control chondrocytes synthesized type I and type II collagen with no Ch21 protein detected as long as the attachment to the plastic was kept. In suspension culture, QEC-v-myc readily aggregated and within 1 week the cell aggregates released small single cells; still they secreted only type II collagen and Ch21 protein. In the same conditions control cell aggregates released hypertrophic chondrocytes producing type II and type X collagens and Ch21 protein. In the appropriate culture conditions, QEC-v-myc reconstituted a tissue defined as nonhypertrophic, noncalcifying cartilage by the high cellularity, the low levels of alkaline phosphatase enzymatic activity, and the absence of type X collagen synthesis and of calcium deposition. We conclude that the constitutive expression of the v-myc oncogene keeps chondrocytes in stage I (active proliferation and synthesis of type II collagen) and prevents these cells from reconstituting hypertrophic calcifying cartilage.     

Divergent differentiation during embryonal histogenesis of skeletal muscle tissue

Morphometric analysis of the developmental processes of the satellite cells and myosimplasts has been performed in embryonal histogenesis of the skeletal muscle tissue in 17 human fetuses 8-27 weeks of the intrauterine development. The sequence of death of some myoblasts in embryonal histogenesis is described in details. Basing on the data obtained, a conception on existance of muscular-proliferative units (MPU) in composition of the skeletal muscles is put forward. The amount of the MPU determines the whole number of muscle fibers in the muscle. The anlage of the MPU occurs as a result of divergent differentiation of the stem myogenic cells at early stages of myogenesis (myosimplasts and myotubes) from the cells commited to mutual fusion. The fund of these cells is determined by the number of myogenic elements that are at the state of the proliferative rest. One of the mechanisms regulating the number of the resting cells is the growth rate of the simplast lengthwise. The resting cells, appearing at late stages of myogenesis (of the muscle fibers), are the sources for development of the myosatellites in mature muscle fibers. In dying myotubes there is a sharp disturbance in growth processes lengthwise, in biosynthesis of contractile proteins, in correlation between the number of nuclei in the satellite cells and those of simplasts.     

钙离子对293细胞结团和生长的影响

分别在有血清和无血清条件下、方瓶和转瓶中考察了Ca²⁺ 对2 93细胞结团和生长的影响。通过实验发现,Ca²⁺ 浓度在0 1～1 0mmol L范围内对2 93细胞的贴壁和结团性质有显著影响,而对生长影响不大。结果表明:有血清贴壁培养时,较高的Ca²⁺ 浓度有利于细胞贴壁;无血清悬浮培养中,Ca²⁺ 浓度越高,细胞结团越严重,细胞结团达到平衡后的平均粒径(D ,μm)与Ca²⁺ 浓度(c,mmol L)在0.1～0.5mmol L范围内可用一次函数D =58.65c +16.96描述,细胞结团尺寸是可调控的;而细胞在不同的Ca²⁺ 浓度下有相似的生长规律。     

Polarity of three-dimensional structures derived from isolated hog thyroid cells in primary culture

When cultured in polystyrene dishes subjected to previous treatment and supplied with a serum-containing medium, hog thyroid cells form monolayers displaying dome-like arrangements after three to four days. Cells involved in formation of "domes" are morphologically polarized; the apical microvilli of these cells point toward the culture medium. When the tissue is cultured in untreated polystyrene dishes, thyroid cells remain in suspension; their aggregates swell progressively and form hollow spheres encompassed by a single layer of cells. The polarity of the cells forming such spheres is inverse in comparison to the condition characteristic of the intact thyroid gland. When culture medium is supplemented with TSH, PGE1, PGE2 or dBC, structures resembling true follicles are formed in both types of cultures. Gelatin, added to suspension cultures, is also capable of promoting follicle formation. Cultured thyroid cells regularly form an epithelial layer as a result of the interaction of cellular processes. However, the polarization of this layer depends on culture conditions. Thus, structures with either a normal follicle-like polarization of their cells or showing an inverted type of polarization can be obtained.     

Growth characteristics and nutrient depletion of Miscanthus × ogiformis Honda ‘Giganteus’ suspension cultures

The growth characteristics and nutrient depletion in suspension cultures of Miscanthus × ogiformis Honda ‘Giganteus’ grown in media containing either Murashige and Skoog or N6 basal nutrient salts were studied during a culture period of 15 days. Proline was added to both media in concentrations from 0 to 300 mM. The fresh and dry weights of the suspension aggregates and the concentrations of ammonium, nitrate, proline and sugar remaining in the medium were measured at different points in time during the culture period. The results showed an almost total depletion of ammonium but a limited nitrate depletion of only 15 mM in both media. Proline at 12.5 to 50 mM was totally depleted during the culture period whereas approximately half the concentration remained in media with 100 to 300 mM proline. Higher fresh weight increases were obtained by the proline additions to the N6 as compared to the MS suspension cultures. Sucrose was hydrolysed into its monosaccharide components in the culture medium. Glucose was depleted faster than fructose indicating a preference for glucose as a carbohydrate source of the M. × ogiformis cultures. The high water uptake by the suspension aggregates 12 to 15 days after subculture indicate that the cultures should be subcultured more frequently to prevent the formation of nonembryogenic cells in the cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Thrombocyte adhesion and aggregation on surfaces coated with human type-I, -III, -IV and -V collagens

Human collagens of type I, III, IV, and V (CI, CIII, CIV, and CV) can be localized in different anatomic structures of the vessel wall. To investigate the role of vascular collagenous components in mural thrombus formation, the authors studied platelet adhesion to the wells of Falcon culture plates coated with: a) monomeric CI, CIII, CIV, and CV; b) fibrillar CI and CIII, and c) amorphous CIV and CV. On monomeric and amorphous CV, only initial attachment takes place, i.e. platelets bind to the surface without subsequent spreading. Platelet adhesion on monomeric and amorphous CIV proceeds more actively: the total level of adhesion is substantially higher than on CV, with up to 75% of adherent platelets spread out and single unspread platelets from suspension attached to the upper surface of spread platelets. On monomeric and fibrillar CI/CIII, formation of large multi-layer (thrombi-like) aggregates, with spread platelets at the basis, takes place along with processes characteristic for adhesion on CIV/CV. On the contrary, only fibrillar but not monomeric CI and CIII induce platelet aggregation in suspension. The data suggest that the ability of CI and CIII to induce platelet aggregation is fully conditioned by the genetic type of collagen and requires a simultaneous multivalent platelet-collagen interaction, which can be achieved by surface immobilization of collagen or formation of fibrillar structures in suspension.     

Morphologic alterations of blood cells in the impedance aggregometer

The scanning (SEM) and transmission (TEM) electron microscopic appearance of blood cells was studied in correlation with the aggregometer tracing recorded after activation of whole blood samples by collagen or ADP. Early morphologic alterations of platelets characterized by the formation of marginal pseudopods and bulbous protrusions were not indicated by the aggregometer. The initial increase in impedance was caused by the attachment of platelets displaying typical shape change morphology at the surface of the electrode wires joint with collagenous fibrils in collagen activated specimens. During further increase in impedance, aggregates were detectable in the blood suspension and at the electrode, the number and size of which increased up to the maximal extension of the aggregometer tracing. Using low doses of ADP (2-3 microM), dissociation of aggregates in the blood suspension was detectable by SEM, which was not recorded by the aggregometer tracing indicating further limitation of the impedance aggregometer. In collagen activated samples, platelet aggregates were covered by PMN and monocytes that in TEM displayed distinct phagocytosis of platelet fragments and fibrin masses. In ADP specimens, activation of leukocytes was only rarely detectable. The detection of mixed aggregates may be important for further employment of the impedance aggregometer in the diagnosis of hematologic diseases.