Kannika Siripattarapravat

Polycomb gene expression and histone H3 lysine 27 trimethylation changes during bovine preimplantation development

Trimethylation of histone H3 at lysine 27 (H3K27me3) is established by polycomb group genes and is associated with stable and heritable gene silencing. The aim of this study was to characterize the expression of polycomb genes and the dynamics of H3K27me3 during bovine oocyte maturation and preimplantation development. Oocytes and in vitro-produced embryos were collected at different stages of development. Polycomb gene expression was analyzed by real-time quantitative RT-PCR and immunofluorescence. Global H3K27me3 levels were determined by semiquantitative immunofluorescence. Transcripts for EZH2, EED, and SUZ12 were detected at all stages analyzed, with EZH2 levels being the highest of the three at early stages of development. By the time the embryo reached the blastocyst stage, the level of PcG gene mRNA levels significantly increased. Immunofluorescence staining indicated nuclear expression of EZH2 at all stages while nuclear localized EED and SUZ12 were only evident at the morula and blastocyst stages. Semiquantitative analysis of H3K27me3 levels showed that nuclear fluorescence intensity was the highest in immature oocytes, which steadily decreased after fertilization to reach a nadir at the eight-cell stage, and then increased at the blastocyst stage. These results suggest that the absence of polycomb repressive complex 2 proteins localized to the nucleus of early embryos could be responsible for the gradual decrease in H3K27me3 during early preimplantation development.

Human-Induced Pluripotent Stem Cells Produced Under Xeno-Free Conditions

Induced pluripotent stem cells (iPSCs) have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from adult individuals. While cell differentiation protocols have been successfully developed, and animal models of human disease have proved that these cells have the potential to treat human diseases and conditions produced as a consequence of aging, degeneration, injury, and birth defects, logistical issues still remain unsolved and hamper the possibility of testing these cells in human clinical trials. Among them is the widely spread use of animal products for the generation and culture of iPSCs. We report here a xeno-free iPSC generation system that addresses all the steps of iPSCs production including the isolation and culture of adult skin fibroblasts, and iPSCs generation, expansion, and maintenance. iPSCs generated with a polycistronic lentiviral vector under xeno-free conditions displayed markers of pluripotency and gave rise to embryoid bodies (EBs) displaying indicators of the 3 primary germ layers. Xeno-free iPSCs injected into nude mice produced classic teratomas, and teratoma explants cultured under conditions favoring fibroblastic cells gave rise to cells morphologically indistinguishable from input cells. Protocols here described will facilitate the implementation of new cellular therapies for preclinical and clinical studies, potentially reducing the regulatory burden without compromising the differentiation potential of the cells.

Somatic cell nuclear transfer in zebrafish

We developed a method for somatic cell nuclear transfer in zebrafish using laser-ablated metaphase II eggs as recipients, the micropyle for transfer of the nucleus and an egg activation protocol after nuclear reconstruction. We produced clones from cells of both embryonic and adult origins, although the latter did not give rise to live adult clones.

Increased cellular turnover in response to fluoxetine in neuronal precursors derived from human embryonic stem cells.

Previous reports have shown that antidepressants increase neuronal cell proliferation and enhance neuroplasticity both in vivo and in vitro. This study investigated the direct effects of one such antidepressant, fluoxetine , on cell proliferation and on the production of neurotrophic factors in neuronal precursors derived from human embryonic stem cells (hESCs; H9). Fluoxetine induced the differentiation of neuronal precursors, strongly enhancing neuronal characteristics. The rate of proliferation was higher in fluoxetine -treated cells than in control cells, as determined by MTT [3(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide] assay. The CPDL (cumulative population doubling level) of the fluoxetine-treated cells was significantly increased in comparison to that of control cells (p<.001). Bromodeoxyuridine incorporation and staurosporine-induced apoptosis assays were elevated in fluoxetine-treated cells. Quantitative RT-PCR analysis revealed no significant differences in the expression of neurotrophic factors, brain-derived neurotrophic factor (BDNF);glial-derived neurotrophic factor (GDNF) and cAMP-responsive element-binding protein (CREB) between cells treated with fluoxetine for two weeks and their untreated counterparts. These results may help elucidate the mechanism of action of fluoxetine as a therapeutic drug for the treatment of depression. Data presented herein provide more evidence that, in addition to having a direct chemical effect on serotonin levels, fluoxetine can influence hESC-derived neuronal cells by increasing cell proliferation, while allowing them to maintain their neuronal characteristics.

The influence of donor nucleus source on the outcome of zebrafish somatic cell nuclear transfer

The success of nuclear reprogramming following somatic cell nuclear transfer (SCNT) is thought to depend on factors present in the egg. Little is known about the role - if any - played by the somatic cell type on the outcome of the procedure. We tested whether cells of different lineages might have different capacities for reprogramming following SCNT, comparing cells isolated from five different tissues of transgenic zebrafish for their developmental potential when used as SCNT donor cells. We used transgenic zebrafish lines expressing green fluorescence protein under an endogenous tissue-specific promoter: HGn62A-skin, HGn28A-skin, HGn8E-heart, HG21C-fin and notochord and HGn30A-hatch gland. We analyzed the efficiency of cloning, as measured by reconstructed embryos that developed up to the hatched-fry stage. Specifically, donor cells of fin and notochord origin yielded the best rate of cloned fish production. All of the other cell types used were capable of producing cloned fish, albeit with significantly lower efficiency. These results indicate that the type of zebrafish cells used for SCNT can influence the outcome of the procedure. Future epigenetic analysis of these cells will help determine specific chromatin profiles in somatic cells that have an impact on nuclear reprogramming procedures.

Chicken primordial germ cell motility in response to stem cell factor sensing

Avian primordial germ cells (PGCs) are destined to migrate a long distance from their extra embryonic region via the vascular system to the gonadal ridges where they form the germ cells. Although PGC migration is crucial for a genetic continuation to the next generation, the factors and mechanisms that control their migration remain largely unknown. In the present study the chemotactic effect of stem cell factor (SCF) was examined on chicken blood circulating PGCs (cPGC), employing 3D chemotaxis slides and time-lapsed imaging analyses as an in vitro study model. Upon in vitro exposure to an SCF gradient, 77.1% (54 out of 70) of cPGCs showed a clear response, of which 48.1% (26 out of 54) polarized with the consecutive formation of a persistent membrane protrusion and significant directional migration towards the gradient and the others showed transient membrane protrusions. In contrast, the controls and apparently SCF unresponsive cPGCs and c-kit-negative red blood cells (RBCs) showed only cytoplasmic cycling with random formations of membrane blebbing and no directional migration. Significant (p < 0.05) differences between the SCF-treated and control cPGCs and RBCs were found in the migration parameters of eccentricity, accumulated and Euclidean distances, and migration velocity. The SCF-treated PGCs also revealed a chemotactic response, as judged by their significant displacement of center of mass and Rayleigh test. Complete inhibition of all the SCF-induced responses in PGCs was found following pretreatment of the cPGCs with 10 µM of the c-kit inhibitor, STI57l, prior to SCF exposure. In addition, cPGCs were found to be positive for c-kit expression using a polyclonal goat anti-mouse c-kit primary antibody, suggesting that the cPGCs were capable of SCF sensing and the potential involvement of SCF/c-kit in the chemotactic migration. Therefore, SCF is suggested to function as a chemoattractant in the migration of chicken cPGC.

Genetic diversity analysis of Thai indigenous chickens based on complete sequences of mitochondrial DNA D-loop region

Objective Complete mtDNA D-loop sequences of four Thai indigenous chicken varieties, including Pra-dhu-hang-dam (PD), Leung-hang-khao (LK), Chee (CH), and Dang (DA) were explored for genetic diversity and relationships with their potential ancestor and possible associates to address chicken domestication in Thailand. Methods A total of 220 complete mtDNA D-loop sequences of the four Thai indigenous chicken varieties were obtained by Sanger direct sequencing of polymerase chain reaction amplicons of 1,231 to 1,232 base pair in size. A neighbor-joining dendrogram was constructed with reference complete mtDNA D-loop sequences of Red Junglefowl (RJF) and those different chicken breeds available on National Center for Biotechnology Information database. Genetic diversity indices and neutrality test by Tajima’s D test were performed. Genetic differences both within and among populations were estimated using analysis of molecular variance (AMOVA). Pairwise fixation index (FST) was conducted to evaluated genetic relationships between these varieties. Results Twenty-three identified haplotypes were classified in six haplogroups (A–E and H) with the majority clustered in haplogroup A and B. Each variety was in multiple haplogroups with haplogroups A, B, D, and E being shared by all studied varieties. The averaged haplotype and nucleotide diversities were, respectively 0.8607 and 0.00579 with non-significant Tajima’s D values being observed in all populations. Haplogroup distribution was closely related to that of RJF particularly Gallus gallus gallus (G. g. gallus) and G. g. spadiceus. As denoted by AMOVA, the mean diversity was mostly due to within-population variation (90.53%) while between-population variation (9.47%) accounted for much less. By pairwise FST, LK was most closely related to DA (FST = 0.00879) while DA was farthest from CH (FST = 0.24882). Conclusion All 4 Thai indigenous chickens are in close relationship with their potential ancestor, the RJF. A contribution of shared, multiple maternal lineages was in the nature of these varieties, which have been domesticated under neutral selection.

Method for somatic cell nuclear transfer in zebrafish

Somatic cell nuclear transfer (SCNT) has been a well-known technique for decades and widely applied to generate identical animals, including ones with genetic alterations. The system has been demonstrated successfully in zebrafish. The elaborated requirements of SCNT, however, limit reproducibility of the established model to a few groups in zebrafish research community. In this chapter, we meticulously outline each step of the published protocol as well as preparations of equipments and reagents used in zebrafish SCNT. All describable detailed-tips are elaborated in texts and figures.

Chapter 16 – Somatic Cell Nuclear Transfer in Zebrafish

The use of Zebrafish as a model-organism is growing exponentially. Somatic Cell Nuclear Transfer could potentially enhance the way gene modifications are done and facilitate the selection and preservation of specific strains and mutant lines. However, since first reported in 2002, SCNT failed to be implemented widely. The reason for the lack of widespread use is likely the difficulty to perform the protocol as was described. This chapter addresses all the critical parameters in the procedure including handling of the recipient eggs, donor cells, and care of the reconstructed embryos. We describe a novel protocol that overcomes common hindrances and incorporates a new approach for enucleation and cell transfer, parameters that can greatly influence SCNT success. We predict that the simplicity of this protocol will entice other laboratories to use it.