High Regenerative Potential Research Articles

An ex vivo human cartilage repair model to evaluate the potency of a cartilage cell transplant.

BackgroundCell-based therapies such as autologous chondrocyte implantation are promising therapeutic approaches to treat cartilage defects to prevent further cartilage degeneration. To assure consistent quality of cell-based therapeutics, it is important to be able to predict the biological activity of such products. This requires the development of a potency assay, which assesses a characteristic of the cell transplant before implantation that can predict its cartilage regeneration capacity after implantation. In this study, an ex vivo human cartilage repair model was developed as quality assessment tool for potency and applied to co.don’s chondrosphere product, a matrix-associated autologous chondrocyte implant (chondrocyte spheroids) that is in clinical use in Germany.MethodsChondrocyte spheroids were generated from 14 donors, and implanted into a subchondral cartilage defect that was manually generated in human articular cartilage tissue. Implanted spheroids and cartilage tissue were co-cultured ex vivo for 12 weeks to allow regeneration processes to form new tissue within the cartilage defect. Before implantation, spheroid characteristics like glycosaminoglycan production and gene and protein expression of chondrogenic markers were assessed for each donor sample and compared to determine donor-dependent variation.ResultsAfter the co-cultivation, histological analyses showed the formation of repair tissue within the cartilage defect, which varied in amount for the different donors. In the repair tissue, aggrecan protein was expressed and extra-cellular matrix cartilage fibers were present, both indicative for a cartilage hyaline-like character of the repair tissue. The amount of formed repair tissue was used as a read-out for regeneration capacity and was correlated with the spheroid characteristics determined before implantation. A positive correlation was found between high level of aggrecan protein expression in spheroids before implantation and a higher regeneration potential after implantation, reflected by more newly formed repair tissue.ConclusionThis demonstrated that aggrecan protein expression levels in spheroids before implantation can potentially be used as surrogate potency assay for the cartilage cell transplant to predict its regenerative capacity after implantation in human patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-1065-8) contains supplementary material, which is available to authorized users.

Efficient callus formation and plant regeneration are heritable characters in sugar beet (Beta vulgaris L.).

BackgroundObtaining dedifferentiated cells (callus) that can regenerate into whole plants is not always feasible for many plant species. Sugar beet is known to be recalcitrant for dedifferentiation and plant regeneration. These difficulties were major obstacles for obtaining transgenic sugar beets through an Agrobacterium-mediated transformation procedure. The sugar beet line ‘NK-219mm-O’ is an exceptional line that forms callus efficiently and is easy to regenerate, but the inheritance of these characters was unknown. Another concern was whether these characters could coexist with an annual habitat that makes it possible to breed short life-cycle sugar beet suitable for molecular genetic analysis.FindingsFive sugar beet lines including NK-219mm-O were crossed with each other and subjected to in vitro culture to form callus. F1s with a NK-219mm-O background generally formed callus efficiently compared to the others, indicating that efficient callus formation is heritable. The regeneration potential was examined based on the phenotypes of calli after placement on regeneration medium. Five phenotypes were observed, of which two phenotypes regenerated shoots or somatic embryo-like structures. Vascular differentiation was evident in regenerable calli, whereas non-regenerable calli lacked normally developed vascular tissues. In a half-diallel cross, the callus-formation efficiency and the regeneration potential of reciprocal F1s progeny having a NK-219mm-O background were high. Finally, we crossed NK-219mm-O with an annual line that had a poor in vitro performance. The callus-formation efficiency and the regeneration potential of reciprocal F1 were high. The regenerated plants showed an annual habitat.ConclusionsEfficient callus formation and the high plant regeneration potential of NK-219mm-O were inherited and expressed in the F1. The annual habitat does not impair these high in vitro performances.Electronic supplementary materialThe online version of this article (doi:10.1186/s41065-016-0015-z) contains supplementary material, which is available to authorized users.

Processes and factors that affect regeneration and establishment of the invasive aquatic plant Cabomba caroliniana

Many invasive macrophytes reproduce through fragments that are transported actively or passively to new habitats. On arrival, regeneration of fragments and colonisation of the new environment are critical steps in the invasion process. The effects of environmental and plant factors that potentially affect regeneration and colonisation success of Cabomba caroliniana A. Gray (Cabombaceae; cabomba, fanwort) were studied. C. caroliniana fragments had a very high regeneration potential that was not affected by nutrient availability. However, larger and apical fragments had higher establishment rate and regenerative growth. The coarseness of the substrate did not have any effect on establishment of C. caroliniana. Overall, even a single-node fragment had 50% establishment success when planted in substrate and this increased to 100% for fragments with two or more nodes. In contrast, free-floating propagules had only a 1–30% chance of colonisation, depending on the size of the fragment. It appears that propagule pressure is of less importance for the colonisation of C. caroliniana than the process of physical establishment in the substrate which acts as a bottleneck. Future work should address the specific processes that determine retention and establishment of propagules in the sediment.

The heterodimeric transcription factor complex ERF115-PAT1 grants regeneration competence.

Regeneration of a tissue damaged by injury represents a physiological response for organ recovery1-3. Although this regeneration process is conserved across multicellular taxa, plants appear to display extremely high regenerative capacities, a feature widely used in tissue culture for clonal propagation and grafting4,5. Regenerated cells arise predominantly from pre-existing populations of division-competent cells6,7; however, the mechanisms by which these cells are triggered to divide in response to injury remain largely elusive8. Here, we demonstrate that the heterodimeric transcription factor complex ETHYLENE RESPONSE FACTOR115 (ERF115)-PHYTOCHROME A SIGNAL TRANSDUCTION1 (PAT1) sustains meristem function by promoting cell renewal after stem cell loss. High-resolution time-lapse imaging revealed that cell death promotes ERF115 activity in cells that are in direct contact with damaged cells, triggering divisions that replenish the collapsed stem cells. Correspondingly, the ERF115-PAT1 complex plays an important role in full stem cell niche recovery upon root tip excision, whereas its ectopic expression triggers neoplastic growth, correlated with activation of the putative target gene WOUND INDUCED DEDIFFERENTIATION1 (WIND1)9. We conclude that the ERF115-PAT1 complex accounts for the high regenerative potential of plants, granting them the ability to efficiently replace damaged cells with new ones.

The potential for propagation of the commercial sea cucumber Isostichopus fuscus (Ludwig, 1875) by induced transverse fission

This study evaluated the potential to propagate asexually the brown sea cucumber Isostichopus fuscus by induction of transverse fission, and its ability to survive, grow and regenerate body parts into a whole animal. Two independent experiments were performed. Experiment 1: sixty-two adult animals (18.8 ± 0.2 cm and 368.1 ± 7.2 g) were cut six centimeters from the rear, and during this process they eviscerated. Survival of body-parts (anterior and posterior) of animals and regeneration times were evaluated, until all individuals showed complete regeneration in terms of its morphology (lasted 13-wk). Animals were maintained in starved condition and had high survivorship (100%). Complete regeneration occurred within 84 to 95 days. Experiment 2: 48 completely regenerated posterior body-parts of I. fuscus (with mouth and anus well developed) were used (lasted 13-wk) and animals were fed ad libitum four diets in powder and two controls: diet A with Ascophyllum nodosum and Sargassum spp; diet B was a commercial shrimp feed with a mix of proteins from marine animals and vegetal material; diet C with Padina durvillaei and Sargassum ecuadoreanum; and diet D with a mixture of diet C with calcium citrate and Vitamin D. Two controls were used. Survival was not affected by diet but this significantly affected somatic growth rate in length and weight. The fastest growth rates (in length and weight) were for diet B (0.50 ± 0.10 cm month−1 and 0.57 ± 0.11 g month−1) and the lowest for diet A (0.15 ± 0.10 cm month−1 and 0.11 ± 0.07 g month−1). No growth was detected in controls. I. fuscus had a high potential for regeneration. Our results encourage further research to explore the feasibility of mariculture and/or restoration programs of wild sea cucumber populations in Ecuador, using asexual propagation techniques for I. fuscus.

Improved in vitro culture and multiplication of differentAllium hirtifoliumBioss. ecotypes

Persian shallot (Allium hirtifolium), called as “Mooseer” in Iran belongs to Alliaceae family and is one of the important edible alliums in Iran. It is native to Iran and grows as a wild plant across the Zagross Mountains. Mooseer bulbs and seeds can be used in propagation, but the bulbs are very slow to grow and the seeds have deep dormancy. Taking into the nutraceuticals value of Persian shallot, it is necessary to improve the multiplication rate of Allium hirtifolium and prevent its extinction. Meanwhile in vitro culture technique can be used for large scale production of different species. In this study the responses of six ecotypes of Allium hirtifolium (Esfahan, Hamedan, Lorestan, Sardasht, Zanjan, Yasuj) collected from different climate in Iran, for in vitro shoot multiplication was investigated. Explants from basal plate were cultured in optimized medium for shoot induction composed of Murashige and Skoog (MS) medium with 2 mg L-1 N6-benzyladenine (BA) and 1 mg L-1 Naphthaleneacetic acid (NAA). Shoots produced were then transferred to hormone free medium for root induction. Although shoot multiplication was obtained in all the ecotypes but results showed the significant variability of days to shoot induction, percent of shoot induction, shoot height, shoot number and percent of root induction in different ecotypes. Explants derived from Zanjan ecotype had the highest potential for regeneration, in which about 17.5 shoots were produced per each explants in 12 weeks culture and showed the highest root induction (80%). These developed plantlets would be open possibilities for domestication and creation of new genotypes which leads to preservation of this species.

DEVELOPMENT OF A COMBINATORIAL MICROASSAY TO ENGINEER BIOMIMETIC VASCULAR SCAFFOLDS

Regeneration-promoting vascular substitutes such as pro-healing stents or vascular grafts could minimise the incidence of thrombosis, restenosis and other events leading to device failure. One strategy to promote endogenous repair mechanisms is to functionalise the surfaces of the vascular graft with peptides or proteins that capture circulating endothelial progenitor cells (EPCs). EPCs are circulating CD34+ bone marrow-derived cells that could have a significant impact on endothelialisation due to high proliferative and regenerative potential. Previous studies have tested surface-immobilised antibodies to capture EPCs, as well as surface-immobilised extracellular matrix-derived peptides to promote adhesion and expansion. However, these strategies have not been tested in combination. We hypothesise that the co-immobilisation of peptides and antibodies could maximise endothelialisation by targeting both the EPC capture and expansion steps.

RELATIONSHIP OF MORPHOGENETIC PROCESSES IN WHEAT TISSUE CULTURE

Relationship of different morphogenetic processes in immature embryo cultures from 15 spring bread wheat varieties of different ecological and geographical origin was studied. Embryos (14–16 days post anthesis with 1.3–1.5 mm in size) were placed with the sculletum upwards on a solid agar medium containing the inorganic components of Linsmaier & Skoog (LS), 3 % sucrose, 2.0 mg l -1 2,4-dichlorophenoxyacetic acid (2,4-D). Induced calli were subcultured after 25–30 days interval in fresh medium supplemented with 0.5 mg l -1 2,4-D and 0.5 mg l -1 kinetin. Embryogenic calli were transferred to LS medium containing 0.2 mg l -1 indole-3-acetic acid (IAA). Varietal polymorphism was revealed in relation to callusogenesis, morphogenesis and plant regeneration. The frequency of callusogenesis made 94.3 % with variation from 76.6 % to 100 % depending on a genotype. An active morphogenic process was revealed in 72 % of the varieties tested. The regeneration level depended on the type of morphogenesis (embryoidogenesis, hemmorhizogenesis and rhizogenesis). On average across all varieties it was not high and made 97.9 %; that is one morphogenic line produced about one plant. Organogenesis in 80.2 % of morphogenic calluses did not reach the development stage of the whole plant and stopped with root production. Plant regeneration by embryoido- and hemmorhizogenesis occurred in 19.8 % of morphogenic calluses. For the study of theoretical aspects of embryoido- and organogenesis as well as genetic transformation of plants the varieties with high regeneration potential are proposed as model objects (Spektr, Skala, Leones, and Zhnitsa). Positive correlation of embryoido-, hemmorhizogenesis and plant regeneration was revealed (r = 0.777), and it proves that there is a common genetic system responsible for those processes. When factorial trait shifted by 1 %, the resultant trait (regeneration) increases by 3.59 %. Negative correlation was found between rhizogenesis and regeneration level (r = -0.749). The rise in rhizogenesis frequency by 1 % results in regeneration decrease by 1.1 %. There is no correlation of calluso- and morphogenesis indicating that they are genetically independent. The analysis of multiple correlations between calluso-, morphogenesis and plant regeneration revealed the dominant factors determining regenerant output. They were the processes of the embryoido- and hemmorhizogenesis. Their contribution to the variability of plant regeneration level was about 51 %. The effect of rhizogenesis was about 12 %. Possible reasons of the revealed correlations between different morphogenetic processes are discussed.

Optic nerve input-dependent regulation of neural stem cell proliferation in the optic tectum of adult zebrafish.

Adult neurogenesis attracts broad attention as a possible cure for neurological disorders. However, its regulatory mechanism is still unclear. Therefore, they have been studying the cell proliferation mechanisms of neural stem cells (NSCs) using zebrafish, which have high regenerative potential in the adult brain. The presence of neuroepithelial-type NSCs in the optic tectum of adult zebrafish has been previously reported. In the present study, it was first confirmed that NSCs in the optic tectum decrease or increase in proportion to projection of the optic nerves from the retina. At 4 days after optic nerve crush (ONC), BrdU-positive cells decreased in the optic tectum's operation side. In contrast, at 3 weeks after ONC, BrdU-positive cells increased in the optic tectum's operation side. To study the regulatory mechanisms, they focused on the BDNF/TrkB system as a regulatory factor in the ONC model. It was found that bdnf was mainly expressed in the periventricular gray zone (PGZ) of the optic tectum by using in situ hybridization. Interestingly, expression level of bdnf significantly decreased in the optic tectum at 4 days after ONC, and its expression level tended to increase at 3 weeks after ONC. They conducted rescue experiments using a TrkB agonist and confirmed that decrease of NSC proliferation in the optic tectum by ONC was rescued by TrkB signal activation, suggesting stimuli-dependent regulation of NSC proliferation in the optic tectum of adult zebrafish. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419-437, 2017.

CO2 decomposition using metal ferrites prepared by co-precipitation method

To catalytically decompose the greenhouse gas, CO2, spinel structure M-ferrites (M=Co, Ni, Cu, Zn) were synthesized by chemical co-precipitation using metal salts and sodium hydroxide as starting materials. The crystallite size of the newly-prepared M-ferrites increased and the BET surface area decreased with increasing calcination temperature. A thermal analysis of the reduction and reoxidation of M-ferrites indicated that substitution of divalent transition metals (i.e., Cu, Ni and Co) into Fe3O4 improved the reduction kinetics in the order of Cu>Ni>Co. ZnFe2O4 was the most difficult compound to completely reduce due to its stable structure. Commercial samples of the reduced Fe3O4, CoFe2O4 and ZnFe2O4 showed an increase in mass through the reoxidation process, but it was much more difficult for oxygen atoms to enter the structure of the reduced samples of NiFe2O4 and CuFe2O4. The M-ferrites in a batch type reactor showed better efficiency than the commercial Fe3O4. Also found was that CoFe2O4 showed a high regeneration potential, although it required a higher critical reaction temperature. NiFe2O4 and CuFe2O4 were excellent candidate materials for CO2 decomposition at lower temperatures.

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation.

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

Chitosan–alunite composite: An effective dye remover with high sorption, regeneration and application potential

This study was undertaken to prepare a novel and environmentally friendly composite for the use in the wastewater treatment process. This composite was produced by immobilizing alunite with a glucosamine biopolymer, chitosan. Batch and dynamic flow mode decolorization potential of the chitosan–alunite composite (CAC) was systematically evaluated in Acid Red 1 (AR1) and Reactive Red 2 (RR2) contaminated media. pH, sorbent dosage, contact time and flow rate were screened through the sorption experiments. Equilibrium sorption experiments indicated that CAC has very high sorption potential for RR2 and AR1 dyes with the maximum sorption capacities of 462.74 and 588.75mgg−1, respectively. Good regeneration and reuse potential in 20 consecutive cycles are other important advantages of this composite. More importantly, CAC could also be used in the treatment of real wastewater without performance decrease. Overall, this study suggests that CAC is a promising sorbent for the removal of anionic dyes from aqueous solutions.

Beyond fibrosis: stellate cells as liver stem cells.

The high regenerative potential of the liver is driven by parenchymal and non-parenchymal cells, which restore the original liver mass after injury by cell proliferation. The contribution of stem- and progenitor cells to liver regeneration is mainly observed when hepatocyte proliferation is impaired. However, the origin of stem/progenitor cells and their effectivity to restore injured liver is currently discussed controversially. Hepatic stellate cells, which are mainly known for their contribution to fibrosis in chronic liver diseases, were recently identified as mesenchymal stem cells (MSC) of the liver. Stellate cells are also involved in liver regeneration and fulfill a dual function by supporting neighboring cells and developing into liver epithelial cells. This demonstrates that stellate cells not only exhibit the same expression profile and differentiation potential but also functional similarities to MSC ofother organs, which are at present intensively studied by many groups for their therapeutic use in liver diseases.

Regeneração natural de espécies arbóreas em diferentes comunidades de um remanescente de floresta ombrófila mista

A diversidade de ambientes encontrados em florestas naturais pode influenciar na regeneração natural e futuramente na dinâmica sucessional. Nesse contexto, objetivou-se avaliar a regeneração natural de espécies arbóreas em diferentes comunidades (grupos florísticos) de uma Floresta Ombrófila Mista. Foram amostradas 69 parcelas com dimensões de 10m x 10m, onde foram medidos os indivíduos com 3,0cm ≤ circunferência à altura do peito (CAP) >30,0cm. Em subparcelas de 3,16m x 3,16m, foram medidos os indivíduos com CAP >3,0cm e altura ≥50cm. As espécies com maior potencial de regeneração natural nas comunidades avaliadas foram Sebastiania brasiliensis e Stillingia oppositifolia (Grupo 1), Myrceugenia cucullata e Allophylus guaraniticus (Grupo 2) e Stillingia oppositifolia e Myrceugenia miersiana (Grupo 3). Destaca-se que as espécies Allophylus edulis, Blepharocalyx salicifolius, Justicia sp., Myrceugenia cucullata e Stillingia oppositifolia estiveram entre as 10 principais espécies nas três comunidades, mostrando elevado potencial de regeneração. As três comunidades favoreceram a ocorrência de elevada riqueza de espécies (Grupo 1=101; Grupo 2=98; Grupo 3=101). Todavia, as espécies secundárias tardias e secundárias iniciais mostraram maior adaptação aos diferentes locais da floresta, com densidade superior às espécies pioneiras.

Hybrid Periportal Hepatocytes Regenerate the Injured Liver without Giving Rise to Cancer

Compensatory proliferation triggered by hepatocyte loss is required for liver regeneration and maintenance but also promotes development of hepatocellular carcinoma (HCC). Despite extensive investigation, the cells responsible for hepatocyte restoration or HCC development remain poorly characterized. We used genetic lineage tracing to identify cells responsible for hepatocyte replenishment following chronic liver injury and queried their roles in three distinct HCC models. We found that a pre-existing population of periportal hepatocytes, located in the portal triads of healthy livers and expressing low amounts of Sox9 and other bile-duct-enriched genes, undergo extensive proliferation and replenish liver mass after chronic hepatocyte-depleting injuries. Despite their high regenerative potential, these so-called hybrid hepatocytes do not give rise to HCC in chronically injured livers and thus represent a unique way to restore tissue function and avoid tumorigenesis. This specialized set of pre-existing differentiated cells may be highly suitable for cell-based therapy of chronic hepatocyte-depleting disorders.

Periostin of human periodontal ligament fibroblasts promotes migration of human mesenchymal stem cell through the αvβ3 integrin/FAK/PI3K/Akt pathway.

The periodontal ligament (PDL) is characterized by rapid turnover, high remodeling capacity and high inherent regenerative potential compared with other connective tissues. Periostin, which is highly expressed in the fibroblasts in the PDL, has been widely discussed in relation to collagen fibrillogenesis in the PDL. Recently, several reports have indicated periostin in cell migration. The aim of this study was to examine whether human PDL fibroblasts (hPDLFs) with high levels of periostin expression promote the migration of human bone marrow mesenchymal stem cells (hMSCs). The migration of hMSCs was examined by transwell chamber migration assay under different conditions: medium alone, hPDLFs, human dermal fibroblasts, recombinant periostin, integrin αvβ3 blocking antibody (anti-CD51/61 antibody) and inhibitors of FAK (PF431396) and PI3K (LY294002). Phosphorylation of FAK and Akt in hMSCs under stimulation of periostin was examined by western blotting. The migration assay revealed that the number of migrated hMSCs by hPDLFs was significantly larger than those by dermal fibroblasts, periostin small interfering RNA hPDLFs and medium alone. Furthermore, recombinant periostin also strongly induced hMSC migration. The addition of anti-CD51/61 antibody, PF431396 and LY294002 caused a significant reduction in the number of migrated hMSCs respectively. The anti-CD51/61 antibody inhibited both FAK and Akt phosphorylations under periostin stimulation. PF431396 inhibited both FAK and Akt phosphorylations. LY294002 inhibited only Akt phosphorylation, and FAK phosphorylation was not influenced under periostin stimulation. Periostin expression in hPDLFs promotes the migration of hMSCs through the αvβ3 integrin/FAK/PI3K/Akt pathway in vitro.

Вплив розміру експлантів та фітогормонального складу живильного середовища на відновлення меристем часнику після кріоконсервування методом вітрифікації

Investigation of the effect of explants dimensions and composition of nutritive medium on morphometric parameters of devitrified garlic meristems will enable a significant optimizing of the cryopreservation techniques intended to establish a collection of its cultivars at low temperature bank. Apexes were subjected to dehydration with plant vitrification solution (1M sucrose + 2M glycerol + 2.5 M ethylene glycol based on Murashige-Skoog salt medium) for 120 min, placed into a polypropylene container and immersed into liquid nitrogen. Thawing was performed in a water bath at 40°C, washing of cryoprotectants was carried-out by a two-fold transfer into the medium with 0.3 Msucrose. It has been shown that the solution under study was non-toxic to garlic meristems and allowed the obtaining of a high level of viability. After cryopreservation by means of vitrification of garlic meristems of ‘Manuylivskyy’ spring variety the number of regenerated plants reached (56.3 ± 11.2)%, and ‘Duchess’ winter variety did (44.8 ± 3.7)%. For the cultivation of the cryopreserved garlic meristems as the phytohormone the kinetin is advisable to be used in concentration of 0.5 mg/l. It has been shown that after cryopreservation by vitrification the garlic meristems of 2–3 mm had the highest viability (60–70%). Larger meristems (3.5–4 mm) had a high regenerative potential but low viability (below 25%). In smaller meristems (1–1.5 mm) the viability rates were reduced (30–47%) as well as a regenerative potential. In the meristems of 0.5 mm a regenerative potential was absent. Probl Cryobiol Cryomed 2015; 25(1): 3-12.

Single epicardial cell transcriptome sequencing identifies Caveolin 1 as an essential factor in zebrafish heart regeneration.

In contrast to mammals, adult zebrafish have a high capacity to regenerate damaged or lost myocardium through proliferation of cardiomyocytes spared from damage. The epicardial sheet covering the heart is activated by injury and aids muscle regeneration through paracrine effects and as a multipotent cell source, and has received recent attention as a target in cardiac repair strategies. Although it is recognized that epicardium is required for muscle regeneration and itself has high regenerative potential, the extent of cellular heterogeneity within epicardial tissue is largely unexplored. Here, we performed transcriptome analysis on dozens of epicardial lineage cells purified from zebrafish harboring a transgenic reporter for the pan-epicardial gene tcf21. Hierarchical clustering analysis suggested the presence of at least three epicardial cell subsets defined by expression signatures. We validated many new pan-epicardial and epicardial markers by alternative expression assays. Additionally, we explored the function of the scaffolding protein and main component of caveolae, caveolin 1 (cav1), which was present in each epicardial subset. In BAC transgenic zebrafish, cav1 regulatory sequences drove strong expression in ostensibly all epicardial cells and in coronary vascular endothelial cells. Moreover, cav1 mutant zebrafish generated by genome editing showed grossly normal heart development and adult cardiac anatomy, but displayed profound defects in injury-induced cardiomyocyte proliferation and heart regeneration. Our study defines a new platform for the discovery of epicardial lineage markers, genetic tools, and mechanisms of heart regeneration.

Hexavalent Chromium Removal and Reduction to Cr (III) by Polystyrene Tris(2-aminoethyl)amine

A commercially available chelating polymer, polystyrene tris(2-aminoethyl)amine, was used for the removal of chromium from aqueous solution. The influence of pH, contact time, adsorbent dosage and initial Cr (VI) concentration on adsorption was studied. The optimum pH for the removal of Cr (VI) was at pH 5, while optimum contact time and adsorbent dosage were 120 minutes and 10 g/L, respectively. Total chromium and Cr (VI) concentrations were analyzed by ICP-MS and UV-Visible. Adsorption isotherms using Langmuir and Freundlich isotherm models revealed that the data fitted Langmuir isotherm model better than Freundlich with a maximum adsorption capacity of 312.27 mg/g. FTIR spectroscopy, Scanning electron microscopy (SEM) and Energy Dispersive Spectrometry (EDS) analyses were performed on the adsorbent before and after binding Cr (VI). All analyses confirmed the complexation of Cr (VI) to the adsorbent. Desorption experiments using KCl solution indicated 89.3% release of chromium, rendering this method of high potential for adsorbent regeneration.