The art of cobbling a running pump—Will human embryonic stem cells mend broken hearts?

The heart is one of the least regenerative organs in the body, and highly vulnerable to the increasing incidence of cardiovascular diseases in an aging world population. Cell-based approaches aimed at cardiac repair have recently caused great public excitement. But clinical trials of patients’ own skeletal myoblasts or bone marrow cells for transplantation have been disappointing. Human embryonic stem cells (hESCs) form bona fide cardiomyocytes in vitro which are readily generated in mass culture and are being tested in animal models of heart damage. The early results, while encouraging, underscore that much remains to be done. This review focuses on the many challenges that remain before hESCs-mediated repair of the human heart becomes a reality.     

Optimization of biotin and thiamine requirements for somatic embryogenesis of date palm (Phoenix dactylifera L.)

Summary This study was conducted to examine the effect of biotin and thiamine concentrations on callus growth and somatic embryogenesis of date palm (Phoenix dactylifera L.). Embryogenic callus derived from offshoot tip explants was cultured on hormone-free MS medium containing biotin at 0, 0.1, 1, or 2 mg l⁻¹ combined with thiamine at 0.1, 0.5, 2, or 5 mg l⁻¹. Embryogenic callus weight, number of resultant embryos, and embryo length were significantly influenced by thiamine and biotin concentration. The optimum callus growth treatment consisted of 0.5 mg l⁻¹ thiamine and 2 mg l⁻¹ biotin. This treatment also gave the highest number of embryos. Embryo elongation was greatest at 0.5 or 2 mg l⁻¹ thiamine combined with 1 mg l⁻¹ biotin. Embryos from all treatments germinated and regenerants exhibited normal growth in soil. This study provides an insight into the importance of optimizing various culture medium components to overcome in vitro recalcitrace of date palm.     

In vitro rooting of the apple rootstock M 26 in adult and juvenile growth phases and acclimatization of the plantlets

In order to obtain optimum conditions for in vitro propagation of the apple rootstock M 26 ( Malus pumila Mill.) in adult and juvenile growth phases, several rooting experiments were performed. Supraoptimal concentrations of indole-3-butyric acid (IBA) added to the rooting media resulted in profuse callus formation. Since extensive callus production is detrimental to the survival of the plantlets, modified culture conditions were established to reduce callus formation. A reduction of the time of exposure to IBA to 5 days and, thereafter, transfer to a hormone-free medium did not eliminate callus production. Exposure to darkness during the root initiation phase increased rooting. When the rooting medium was based on the Lepoivre formula instead of the Murashige and Skoog formula, callus formation was reduced. Optimum conditions for rooting were obtained at much lower concentration than earlier reported, being 1.25 μM for the juvenile and 0.5 μM for the adult growth phase in the range of IBA concentrations tested. Anatomical studies revealed that root initials are formed after 5 days of IBA-treatment. Therefore, we transferred shoots directly to non-sterile conditions after the root-inducing phase. This resulted in a 90% survival of the plantlets. Subculture on hormone-free medium can thus be eliminated when the optimum auxin concentration is known.     

Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.     

Differential callus type formation by auxins and cytokinin in in vitro cultures of pepper (Capsicum annuum L.)

ABSTRACT

Two types of callus were produced by pepper explants cultured in various media containing auxins, the cytokinin 6-benzylaminopurine (BAP) and the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA). Callus produced on media containing auxins alone was friable, grey-green or green-orange in colour and more compact, whereas when BAP was added to culture media with a low concentration of auxin or when the medium contained TIBA alone, the callus produced was white and very hard. This type of callus was also produced in cultures of older tissues and of young tissues cultured on hormonefree medium. Results are discussed in relation to the role of cytokinins in wounding, phenylpropanoid metabolism and lignin biosynthesis.     

Human Pluripotent Stem Cells Have a Novel Mismatch Repair-dependent Damage Response

Human pluripotent stem cells (PSCs) are presumed to have robust DNA repair pathways to ensure genome stability. PSCs likely need to protect against mutations that would otherwise be propagated throughout all tissues of the developing embryo. How these cells respond to genotoxic stress has only recently begun to be investigated. Although PSCs appear to respond to certain forms of damage more efficiently than somatic cells, some DNA damage response pathways such as the replication stress response may be lacking. Not all DNA repair pathways, including the DNA mismatch repair (MMR) pathway, have been well characterized in PSCs to date. MMR maintains genomic stability by repairing DNA polymerase errors. MMR is also involved in the induction of cell cycle arrest and apoptosis in response to certain exogenous DNA-damaging agents. Here, we examined MMR function in PSCs. We have demonstrated that PSCs contain a robust MMR pathway and are highly sensitive to DNA alkylation damage in an MMR-dependent manner. Interestingly, the nature of this alkylation response differs from that previously reported in somatic cell types. In somatic cells, a permanent G₂/M cell cycle arrest is induced in the second cell cycle after DNA damage. The PSCs, however, directly undergo apoptosis in the first cell cycle. This response reveals that PSCs rely on apoptotic cell death as an important defense to avoid mutation accumulation. Our results also suggest an alternative molecular mechanism by which the MMR pathway can induce a response to DNA damage that may have implications for tumorigenesis.     

Structural and metabolic properties of green tissue cultures from a CAM plant, Kalanchoë blossfeldiana hybr. Montezuma

Abstract Crassulacean acid metabolism (CAM) was studied in mixotrophic callus tissue cultures of Kalanchoë blossfeldiana hybr. Montezuma and compared with plants propagated from the calli. The ultrastructural properties of the green callus cells are similar to mesophyll cells of CAM plants except that occasionally abnormal mitochondria were observed. There was permanent net CO₂ output by the calli in light and darkness, which was lower in darkness than in light. The calli exhibited a diurnal rhythm of malic acid, with accumulation during the night and depletion during the day. ¹⁴C previously incorporated by dark CO₂ fixation into malate was transferred upon subsequent illumination into end products of photosynthesis. All these data indicate that CAM operates in the calli tissue. The results revealed that the capacity for CAM is obviously lower in the calli compared with plantlets developing from the calli, or with ‘adult’ plants. The data suggest also that CAM in the calli was not limited by the activities of CAM enzymes.     

Relation between electrokinetic potentials and growth in callus cultures ofTrigonella foenum-graecum

Callus cultures ofTrigonella foenum-graecum were initiated from radicle or cotyledon portions of seedlings and young leaves and maintained on modified 1-B5 medium. The callus mass was disaggregated by mechanical agitation and the discrete cells thus obtained were used to measure their electrokinetic potential. Studies pertaining to the effects of ageing on electrokinetic potential and growth index revealed a relationship between these two parameters. Thus, the rate of change of electrokinotie potential with age could be employed as a parameter to study the growth kinetics of cells in callus cultures.     

Quantitative Development and Molecular Forms of Acetyl-and Butyrylcholinesterase During Morphogenesis and Synaptogenesis of Chick Brain and Retina

The embryonic development of total specific activities as well as of molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and of butyrylcholinesterase (BChE, EC 3.1.1.8) have been studied in the chick brain. A comparison of the development in different brain parts shows that cholinesterases first develop in diencephalon, then in tectum and telencephalon; cholinesterase development in retina is delayed by about 2-3 days; and the development in rhombencephalon [not studied until embryonic day 6 (E6)] and cerebellum is last. Both enzymes show complex and independent developmental patterns. During the early period (E3-E7) first BChE expresses high specific activities that decline rapidly, but in contrast AChE increases more or less constantly with a short temporal delay. Thereafter the developmental courses approach a late phase (E14-E20), during which AChE reaches very high specific activities and BChE follows at much lower but about parallel levels. By extraction of tissues from brain and retina in high salt plus 1% Triton X-100, we find that both cholinesterases are present in two major molecular forms, AChE sedimenting at 5.9S and 11.6S (corresponding to G2 and G4 globular forms) and BChE at 2.9S and 10.3S (G1 and G4, globular). During development there is a continuous increase of G4 over G2 AChE, the G4 form reaching 80% in brain but only 30% in retina. The proportion of G1 BChE in brain remains almost constant at 55%, but in retina there is a drastic shift from 65% G1 before E5 to 70% G4 form at E7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of a liquid flow-through system to inhibit browning in callus cultures of guayule (Parthenium argentatum Gray)

Calli of P. argentatum were grown on a newly designed liquid nutrient flow-through system which facilitated the subculturing of calli and delayed browning for 6 weeks. Friable calli were obtained on half-strength Gamborg B5-medium supplemented with 0.05 mgl⁻¹ 2,4-dichlorophenoxyacetic acid. Shoots developed on media supplemented with 0.2 mgl⁻¹ benzylaminopurine but lacking 2,4-dichlorophenocyacetic acid.