Use of lectins for probing differentiated human embryonic stem cells for carbohydrates

The carbohydrates present on the surface of differentiated human embryonic stem cells (hESCs) are not yet well established. Here, we have employed a panel of lectins and several anti-carbohydrate antibodies to determine the carbohydrates that are present at day 12 of hESC differentiation as embryoid bodies (EBs). On the basis of staining with fluorescein-labeled lectins, we have determined the presence of both terminal and internally linked alpha-d-mannopyranosyl groups, poly-N-acetyllactosaminyl chains, both alpha2,3- and alpha2,6-linked N-acetylneuraminic acid (Neu5Ac), alpha1,6-linked l-fucosyl, and beta-D-galactosyl groups, and more specifically, the T, Tn, and sialyl-Tn antigens. However, no alpha1,2-linked l-fucosyl, terminal nonreducing alpha-D-galactosyl, N-acetyl-beta-D-glucosaminyl, nor N-acetyl-alpha-D-galactosaminyl groups were found by this approach. We also established the presence of Neu5Acalpha2,3/2,6-Galbeta1,4 GlcNAc-terminated chains on the surfaces of 12-day-old EBs, as indicated by the great enhancement of staining by Erythrina cristagalli agglutinin (ECA) after treatment with neuraminidase. In each case, inhibition of binding by a haptenic sugar or treatment with neuraminidase was used to eliminate the possibility of nonspecific binding of the lectins. A comparison with undifferentiated cell staining revealed an increase in alpha2,3-linked Neu5Ac as well as a change to exclusively alpha1,6-linked l-fucose upon differentiation.     

In vitro analysis of integrin expression in stem cells from bone marrow and cord blood during chondrogenic differentiation

The use of adult mesenchymal stem cells (MSC) in cartilage tissue engineering has been implemented in the field of regenerative medicine and offers new perspectives in the generation of transplants for reconstructive surgery. The extracellular matrix (ECM) plays a key role in modulating function and phenotype of the embedded cells and contains the integrins as adhesion receptors mediating cell-cell and cell-matrix interactions. In our study, characteristic changes in integrin expression during the course of chondrogenic differentiation of MSC from bone marrow and foetal cord blood were compared. MSC were isolated from bone marrow biopsies and cord blood. During cell culture, chondrogenic differentiation was performed. The expression of integrins and their signalling components were analysed with microarray and immunohistochemistry in freshly isolated MSC and after chondrogenic differentiation. The fibronectin-receptor (integrin a5b1) was expressed by undifferentiated MSC, expression rose during chondrogenic differentiation in both types of MSC. The components of the vitronectin/osteopontin-receptors (avb5) were not expressed by freshly isolated MSC, expression rose with ongoing differentiation. Receptors for collagens (a1b1, a2b1, a3b1) were weakly expressed by undifferentiated MSC and were activated during differentiation. As intracellular signalling components integrin linked kinase (ILK) and CD47 showed increasing expression with ongoing differentiation. For all integrins, no significant differences could be found in the two types of MSC. Integrin-mediated signalling seems to play an important role in the generation and maintenance of the chondrocytic phenotype during chondrogenic differentiation. Especially the receptors for fibronectin, vitronectin, osteopontin and collagens might be involved in the generation of the ECM. Intracellularly, their signals might be transduced by ILK and CD47. To fully harness the potential of these cells, future studies should be directed to ascertain their cellular and molecular characteristics for optimal identification, isolation and expansion.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration via somatic embryogenesis through cell suspension cultures of horsegram [<Emphasis Type="Italic">Macrotyloma uniflorum</Emphasis> (Lam.) verdc.]

Summary In vitro regeneration of plants via somatic embryogenesis through cell suspension culture was achieved in horsegram. Embryogenic calluses were induced on leaf segments on solid Murashige and Skoog (MS) medium with 9.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Differentiation of somatic embryos occurred when the embryogenic calluses were transferred to liquid MS medium containing 2,4-D. Maximum frequency (33.2%) of somatic embryos was observed on MS medium supplemented with 7.9 μM 2,4-D. Cotyledonary-torpedo-shaped embryos were transferred to liquid MS medium without growth regulators for maturation and germination. About 5% of the embryos germinated into plants, which grew further on solid MS medium. The plants were hardened and established in soil. Effects of various auxins, cytokinins, carbohydrates, amino acids, and other additives on induction and germination of somatic embryos were also studied. A medium supplemented with 7.9 μM 2,4-D, 3.0% sucrose, 40 mg l⁻¹ L-glutamine, and 1.0 μM abscisic acid was effective to achieve a high frequency of somatic embryo induction, maturation, and further development.     

Differential expression of glucose transporter isoforms during embryonic stem cell differentiation

In mouse blastocysts six facilitative glucose transporter isoforms (GLUT)1-4, 8 and 9 are expressed. We have used the mouse embryonic stem (ES) cell line D3 and spontaneously differentiating embryoid bodies (EB) to investigate GLUT expression and the influence of glucose during differentiation of early embryonic cells. Both ES cells and EBs (2d-20d) expressed GLUT1, 3, and 8, whereas the isoforms 2 and 4 were detectable exclusively in EBs. Differentiation-associated expression of GLUT was analyzed by double staining with stage-specific embryonic antigen (SSEA-1), cytokeratins (CK18, 19), nestin, and desmin. Similar to trophoblast cells in mouse blastocysts the outer cell layer of endoderm-like cells showed a high GLUT3 expression in early EBs. In 20-day-old EBs no GLUT3 protein and only minor GLUT3 mRNA amounts could be detected. A minimal glucose concentration of 5 mM applied during 2 and 8 days of EB culture resulted in up-regulated GLUT4, Oct-4 and SSEA-1 levels and a delay in EB differentiation. We conclude that GLUT expression depends on cellular differentiation and that the expression is modulated by glucose concentration. The developmental and glucose-dependent regulation of GLUT strongly suggests a functional role of glucose and glucose transporters in ES cell differentiation and embryonic development.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Melothria maderaspatana</Emphasis> via indirect organogenesis

An effective protocol was developed for in vitro regeneration of the Melothria maderaspatana via indirect organogenesis in liquid and solid culture systems. Organogenesis was achieved from liquid culture calluses derived from leaf and petiole explants of mature plants. Organogenic calluses (98.2?±?0.36 and 94.8?±?0.71%) were induced from both leaf and petiole explants on Murashige and Skoog (MS) liquid medium containing 6.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 µM thidiazuron (TDZ); and 6.0 µM 2,4-D and 1.0 µM benzyladenine (BA) combinations, respectively. Adventitious shoot regeneration (68.2?±?0.06 shoots per explant) was achieved on MS medium supplemented with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water and 0.06 mM glutamine from leaf-derived calluses. Petiole-derived calluses produced adventitious shoots (45.4?±?0.09 shoots per explant) on MS medium fortified with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water, and 0.08 mM glutamine. Elongation of shoots occurred in MS medium with 2.0 µM gibberellic acid (GA₃). Regenerated shoots (2–3 cm in length) rooted (74.2?±?0.38%) and hardened (85?±?1.24%) when they were transferred to 1/2-MS medium supplemented with 3.0 µM indole-3-butyric acid (IBA) followed by garden soil, vermiculate, and sand (2:1:1 ratio) mixture. The elongated shoots (4–5 cm in length) were exposed simultaneously for rooting as well as hardening (100%) in moistened [(1/8-MS basal salt solution with 5 µM IBA and 100 mg l^?1 Bavistin® (BVN)] garden soil, vermiculate, and sand (2:1:1 ratio) mixture. Subsequently, the plants were successfully established in the field. The survival percentage differed with seasonal variations.     

Effects of low frequency electromagnetic fields on the chondrogenic differentiation of human mesenchymal stem cells

Electromagnetic fields (EMF) have been shown to exert beneficial effects on cartilage tissue. Nowadays, differentiated human mesenchymal stem cells (hMSCs) are discussed as an alternative approach for cartilage repair. Therefore, the aim of this study was to examine the impact of EMF on hMSCs during chondrogenic differentiation. HMSCs at cell passages five and six were differentiated in pellet cultures in vitro under the addition of human fibroblast growth factor 2 (FGF‐2) and human transforming growth factor‐β₃ (TGF‐β₃). Cultures were exposed to homogeneous sinusoidal extremely low‐frequency magnetic fields (5 mT) produced by a solenoid or were kept in a control system. After 3 weeks of culture, chondrogenesis was assessed by toluidine blue and safranin‐O staining, immunohistochemistry, quantitative real‐time polymerase chain reaction (PCR) for cartilage‐specific proteins, and a DMMB dye‐binding assay for glycosaminoglycans. Under EMF, hMSCs showed a significant increase in collagen type II expression at passage 6. Aggrecan and SOX9 expression did not change significantly after EMF exposure. Collagen type X expression decreased under electromagnetic stimulation. Pellet cultures at passage 5 that had been treated with EMF provided a higher glycosaminoglycan (GAG)/DNA content than cultures that had not been exposed to EMF. Chondrogenic differentiation of hMSCs may be improved by EMF regarding collagen type II expression and GAG content of cultures. EMF might be a way to stimulate and maintain chondrogenesis of hMSCs and, therefore, provide a new step in regenerative medicine regarding tissue engineering of cartilage. Bioelectromagnetics 32:283–290, 2011. © 2010 Wiley‐Liss, Inc.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

This study investigated the factors affecting in vitro flowering of Perilla frutescens. The shoots regenerated from cotyledonary and hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and indole-3-acetic acid, each at 0.5 mg l⁻¹, were excised and transferred to MS medium containing 30 g l⁻¹ of sucrose, 8.25 g l⁻¹ of ammonium nitrate, and 1.0 mg l⁻¹ of BA. After 40 d of culture, 86.2% of shoots flowered and most of which self-fertilized in vitro and produced mature fruits with viable seeds. These seeds were germinated and plants were grown to maturity and flowered in soil under greenhouse conditions. The in vitro flowering system reported in this study may facilitate rapid breeding of P. frutescens and offers a model system for studying the physiological mechanism of flowering.     

In Vitro Susceptibility of <Emphasis Type="Italic">C. albicans</Emphasis> and <Emphasis Type="Italic">C. neoformens</Emphasis> to Potential Metabolites from <Emphasis Type="Italic">Streptomycetes</Emphasis>

Forty two Streptomycetes isolates from soils of Kodachadri region in Western ghats were recovered by soil dilution technique. Cross streak method was followed for primary screening of antifungal activity. Positive isolates were subjected to secondary screening by cold extraction of fermentation broth in butanol solvent. Six isolates exhibited broad spectrum antifungal activity against all the tested yeast pathogens like Candida albicans, Candida lipolytica, Cryptococcus neoformens and Saccharomyces cerevisiae. One isolate showed excellent antifungal activity against all test organisms with maximum zone of inhibition 60 mm each incase of C. neoformens and C. albicans. Partial characterization of antifungal metabolite by TLC resulted in a purple spot with an R_f value 0.50. The UV absorption spectra at 218 nm indicated possible chemical nature of the active metabolite as polyene group and purity was assessed by analytical HPLC.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis of <Emphasis Type="Italic">Passiflora alata</Emphasis>

Passiflora alata in vitro organogenesis was studied based on explant type, culture medium composition, and incubation conditions. The results indicated that the morphogenic process occurred more efficiently when hypocotyl segment-derived explants were cultured in media supplemented with cytokinin and AgNO₃ incubated under a 16-h photoperiod. The shoot bud elongation and plant development were obtained by transferring the material to MSM culture medium supplemented with GA₃ and incubated in flasks with vented lids. Histological analyses of the process revealed that the difficulties in obtaining plants could be related to the development of protuberances and leaf primordia structures, which did not contain shoot apical meristem. Roots developed easily by transferring elongated shoots to 1/2 MSM culture medium. Plant acclimatization occurred successfully, and somaclonal variation was not visually detected. The efficiency of this organogenesis protocol will be evaluated for genetic transformation of this species to obtain transgenic plants expressing genes that can influence the resistance to Cowpea aphid borne mosaic virus.     

Long-range chromosomal engineering is more efficient <Emphasis Type="Italic">in vitro</Emphasis> than <Emphasis Type="Italic">in vitro</Emphasis>

Cre/LoxP mediated chromosomal engineering in embryonic stem (ES) cells has a variety of applications, including the creation of model systems for studying aneuploidy. Targeted meiotic recombination (TAMERE) was proposed as a high efficiency in vivo alternative to effect Cre-mediated recombination, in which Cre recombinase under control of the Synaptonemal Complex 1 promoter is expressed during male meiosis in transgenic mice. TAMERE has been successfully used with LoxP sites up to 100 kb apart. We tested TAMERE for a chromosome engineering application in which LoxP sequences were integrated into sites 3.9 Mb apart on the same (cis) or opposite (trans) copies of mouse Chromosome 16 (MMU16). TAMERE was ineffective in generating either a deletion or a translocation in vivo. The TAMERE method may be of limited use for large genomic rearrangements. The desired translocation was achieved with an in vitro method that can be used in any ES cell line. Mice produced from the reciprocal duplication/deletion of MMU16 in a region homologous to human chromosome 21 provide models that are useful in studies of Down syndrome.     

Complexity in the cattle <Emphasis Type="Italic">CD94</Emphasis>/<Emphasis Type="Italic">NKG2</Emphasis> gene families

Natural killer cell responses are controlled to a large extent by the interaction of an array of inhibitory and activating receptors with their ligands. The mostly nonpolymorphic CD94/NKG2 receptors in both humans and mice were shown to recognize a single nonclassical MHC class I molecule in each case. In this paper, we describe the CD94/NKG2 gene family in cattle. NKG2 and CD94 sequences were amplified from cDNA derived from four animals. Four CD94 sequences, ten NKG2A, and three NKG2C sequences were identified in total. In contrast to human, we show that cattle have multiple distinct NKG2A genes, some of which show minor allelic variation. All of the sequences designated NKG2A have two tyrosine-based inhibitory motifs in the cytoplasmic domain and one putative gene has, in addition, a charged residue in the transmembrane domain. NKG2C appears to be essentially monomorphic in cattle. All of the NKG2A sequences are similar apart from NKG2A-01, which, in contrast, shares the majority of its carbohydrate recognition domain with NKG2-C. Most of the genes appear to generate multiple alternatively spliced forms. These findings suggest that the CD94/NKG2A heterodimers in cattle, in contrast to other species, are binding several different ligands. Because NKG2C is not polymorphic, this raises questions as to the combined functional capacity of the CD94/NKG2 gene families in cattle.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the genus <Emphasis Type="Italic">Clivia</Emphasis>

We have developed a protocol for the in vitro propagation of the genus Clivia. Shoots were regenerated when fragments of the peduncle-pedicel junction (PP junction) from young inflorescences were used as explants. The optimal media for PP junction were Murashige and Skoog (MS)-based medium containing 10 M of 6-benzyladenine (BA) and 10 M of 2,4-dichlorophenoxyacetic acid (2,4-D) or MS supplemented with 5 M BA, 10 M -naphthaleneacetic acid (NAA), 250 mg l^-1 glutamine and 500 mg l^–1 casein hydrolysate and their usage depended on the breeding lines. Multiplication from initiations and in vitro seedlings was the best when the explants were cut longitudinally through the meristem and placed on MS plus 44 M BA. Plantlets were transferred on to hormone -free MS medium with charcoal for rooting.     

<Emphasis Type="Italic">In vitro</Emphasis> stem elongation of stemless carline thistle

In vitro culture of stemless carline thistle was established using immature zygotic embryos. A satisfactory bud multiplication was achieved on MS medium supplemented with BAP (1 mg L^–1) and IAA (0.2 mg L^–1). Maximum rooting of buds was induced upon short cultivation (4 or 8 days) on an auxin-supplemented medium. Highest number of roots was obtained with NAA in the medium while the longest roots developed on the IAA-supplemented medium. Plantlets that subsequently developed were in rosette form if grown in light (16/8 h light to darkness photoperiod) and with elongated stems if raised in darkness. Light grown plantlets treated with GA₃ showed dose dependent stem increase in length, reaching maximum at the concentration of 10^–4 M. This was correlated with the length and the average number of internodes. If cultivated in the presence of ancymidol, dark grown plantlets showed reduced stem length. However, the inhibitory effect of the growth retardant on stem elongation was completely overcome by the addition of GA₃.     

<Emphasis Type="Italic">In vitro</Emphasis> culture studies on <Emphasis Type="Italic">Stevia rebaudiana</Emphasis>

Summary Shoot apex, nodal, and leaf explants of Stevia rebaudiana Bertoni can regenerate shoots when cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 8.87 μM) and indole-3-acetic acid (5.71 μM). Rooting of the in vitro-derived shoots could be achieved following subculture onto auxin-containing medium. A survival rate of 70% was recorded at the hardening phase on the substrate cocopeat. The presence of the sweet diterpene glycosides, viz. stevioside and rebaudioside, was confirmed in the in vitro-derived tissues of Stevia using HPTLC techniques. Callus cultured on agar-solidified MS medium supplemented with BA (8.87 μM) and indole-3-butyric acid (9.80 μM) showed the highest sweetener content.