Search

Hematopoietic stem cells (HSCs) are used in transplantation therapy to reconstitute the hematopoietic system. Human cord blood (hCB) transplantation has emerged as an attractive alternative treatment option when traditional HSC sources are unavailable, however, the absolute number of hCB HSCs transplanted is significantly lower than bone marrow or mobilized peripheral blood stem cells (MPBSCs). We previously demonstrated that dimethyl-prostaglandin E2 (dmPGE2) increased HSCs in vertebrate models. Here, we describe preclinical analyses of the therapeutic potential of dmPGE2-treatment using human and non-human primate HSCs. dmPGE2 significantly increased total human hematopoietic colony formation in vitro and enhanced engraftment of unfractionated and CD34+ hCB following xenotransplantation. In non-human primate autologous transplantation, dmPGE2-treated CD34+ MPBSCs showed stable multilineage engraftment over one year post-infusion. Together, our analyses indicated that dmPGE2 mediates conserved responses in HSCs from human and non-human primates, and provided sufficient preclinical information to support proceeding to an FDA-approved phase 1 clinical trial., Stem Cell and Regenerative Biology

NRAS mutations are a common oncogenic event in skin cancer, occurring frequently in congenital nevi and malignant melanoma. To study the role of NRAS in zebrafish, a transgenic approach was applied to generate fish that express human oncogenic NRAS(Q61K) under the control of the melanocyte-restricted mitfa promoter. By screening the progeny of the injected animals, two strains stably expressing the NRAS transgene were identified: Tg(mitfa:EGFP:NRAS(Q61K))(1) and Tg(mitfa:EGFP:NRAS(Q61K))(2). Stable expression of this transgene results in hyperpigmented fish displaying a complete ablation of the normal pigment pattern. Although oncogenic NRAS expression alone was found to be insufficient to promote tumor formation, loss of functional p53 was found to collaborate with NRAS expression in the genesis of melanoma. The tumors derived from these animals are variably pigmented and closely resemble human melanoma. Underscoring the pathological similarities between these tumors and human disease and suggesting that common pathways are similar in these models and human disease, gene set enrichment analysis performed on microarray data found that the upregulated genes from zebrafish melanomas are highly enriched in human tumor samples. This work characterizes two zebrafish melanoma models that will be useful tools for the study of melanoma pathogenesis.

Topoisomerases solve the topological problems encountered by DNA throughout the lifetime of a cell. Topoisomerase II alpha, which is highly conserved among eukaryotes, untangles replicated chromosomes during mitosis and is absolutely required for cell viability. A homozygous lethal mutant, can4, was identified in a screen to identify genes important for cell proliferation in zebrafish by utilizing an antibody against a mitosis-specific marker, phospho-histone H3. Mutant embryos have a decrease in the number of proliferating cells and display increases in DNA content and apoptosis, as well as mitotic spindle defects. Positional cloning revealed that the genetic defect underlying these phenotypes was the result of a mutation in the zebrafish topoisomerase II alpha (top2a) gene. top2a was found to be required for decatenation but not for condensation in embryonic mitoses. In addition to being required for development, top2a was found to be a haploinsufficient regulator of adult liver regrowth in zebrafish. Regeneration analysis of other adult tissues, including fins, revealed no heterozygous phenotype. Our results confirm a conserved role for TOP2A in vertebrates as well as a dose-sensitive requirement for top2a in adults.

The zebrafish (Danio rerio) has become an increasingly utilized and relevant model organism in the study of cancer. The use of transgenic and reverse genetic approaches has yielded several strains that model a variety of human neoplasms. In addition to modeling human disease, these strains provide a platform for the analysis of tumor stem cells. Here we describe the basic technique for the isolation and transplantation of tumor tissue in the zebrafish. This technique was designed to study metastasis and invasive potential of zebrafish tumor cells. Additionally, the basic protocol can be modified in order to describe cancer stem cell characteristics, including proliferative capacity, self-renewal, and the minimum number of tumor cells required for engraftment.