Identification of very small cancer stem cells expressing hallmarks of pluripotency in B16F10 melanoma cells and their reoccurrence in B16F10-derived clones.

Melanoma is characterized by high heterogeneity and plasticity, most likely due to the presence of mutated melanocyte stem cells or immature progenitor cells in the skin that serves as precursors to melanoma. In the present study, for the first time, we identified rare cells in the murine melanoma B16F10, and human A2058 and SK-MEL-28 cell lines that express pluripotency markers, including Oct4, Nanog, Sox2 and a marker of melanoma cancer cells (ALDH1/2). These cells are very small with round morphology and they grow onto melanoma cells, thereby demonstrating feeder layer dependence similar to that of other pluripotent cells. These cells underwent self-renewal, symmetric and asymmetric division. We called these cells murine very small cancer stem cells (VSCSC). VSCSC were also found in B16F10-derived clones after 3-5 consecutive passages, where they occur as single cells or as small colonies, nevertheless, always using melanoma cells as feeders. These cells formed melanospheres enriched with Oct4-and ALDH1/2-positive cells. We also evaluated the possible effect of VSCSC that presented in the parental cell line (B16F10) and in clones based on their functional characteristics. We found that VCSCS present in the B16F10 cell line reappearing in their clones were required for continuous tumor growth and were responsible for melanoma cell heterogeneity and plasticity rather than directly affecting functional characteristics of melanoma cells. Our data, together with those of previous reports suggested the existence of melanoma-competent melanocyte stem cells, which corroborate the hypothesis of the existence of tumor-initiating cells and cancer stem cell hierarchies, at least in melanoma.

In vitro heterogeneity of porcine adipose tissue-derived stem cells.

Tissue-specific adult stem cells (ASC) are heterogeneous and characterized by a mix of progenitor cells that produce cells at various stages of differentiation, and ultimately different terminally differentiated cells. Understanding the heterogeneity of ASCs may lead to the development of improved protocols of cell isolation and optimized cell therapy clinical protocols. Using a combination of enzymatic and explant culture protocols, we obtained pADSC population, which is composed by two distinct morphologies: fibroblast-like cells (FLCs) and endothelial-like cells (ELCs). Both cell sub-types efficiently formed colonies, expressed CD90+/CD105+/CD44+, and differentially expressed such markers such as Nestin, Vimentin, Fibronectin, Cytokeratin, Connexin 43, CD31, CD34 and CD146 as well as the pluripotent stem cell markers Oct-4, Nanog and Sox2. Mixed populations of pADSCs did not lose their multipotentiality and the cells were able to undergo osteogenic, chondrogenic, adipogenic and myogenic differentiation. Furthermore, the mixed population spontaneously formed capillary tube structures. Our findings suggest that different subpopulations can be isolated from adipose tissue and that the ADSCs need to be better evaluated using a wide panel of different markers related to cell differentiation, which is important for stem cell therapy and regenerative medicine, particularly for advanced stem cells therapies - products that are currently under investigation or even use.

Antiproliferative and antiangiogenic effect of Amblyomma sculptum (Acari: Ixodidae) crude saliva in endothelial cells in vitro.

Neovascularization, a process that includes vasculogenesis and angiogenesis, may be a physiological or pathologic event, but in any cases the phenomenon is related to the formation of vascular net and sprouting of endothelial cells from preexisting blood vessel. The tumor environment, which counts on the tumor cell proliferation, is plenty of proangiogenic factors, such as angiogenin, TGF (α and ß), FGF, VEGF, all of them playing a crucial role in angiogenesis, an important hallmark of cancer frequently related to a poor prognosis. Therefore, therapies focusing the inhibition of cancer neovasculogenesis have become an interesting strategy for the development of antitumor therapies. In this work, we investigate the effect of tick saliva on the human endothelial cells, in order to understand its inhibitory effects on angiogenesis. To this end, the HUVEC cells were used as model of angiogenesis in vitro and the anti-proliferative, anti-migratory, cytotoxicity was evaluated. Our data depicts that saliva impairs cell development by causing structural changes while precludes cell proliferation and migration, that are crucial events related to angiogenesis. Aiming the identification of the bioactive components related to antiangiogenic activity, saliva was analyzed through the Mass Spectrometry and among all molecules identified, disintegrins and cathepsin L seems to be primarily responsible for the antiangiogenic effects of saliva.