Reprogramming of human fibroblasts to induced pluripotent stem cells under xeno-free conditions

The availability of induced pluripotent stem cells (iPSCs)has created extraordinary opportunities for modeling andperhaps treating human disease. However, all reprogrammingprotocols used to date involve the use of products of animal origin. Here, we set out to develop a protocol to generate and maintain human iPSC that would be entirelydevoid of xenobiotics. We first developed a xeno-free cellculture media that supported the long-term propagation of human embryonic stem cells (hESCs) to a similar extent as conventional media containing animal origin products or commercially available xeno-free medium. We also derivedprimary cultures of human dermal fibroblasts under strictxeno-free conditions (XF-HFF), and we show that they can be used as both the cell source for iPSC generation as well as autologous feeder cells to support their growth. We also replaced other reagents of animal origin trypsin, gelatin, matrigel) with their recombinant equivalents. Finally, we used vesicular stomatitis virus G-pseudotyped retroviral particles expressing a polycistronic construct encoding Oct4, Sox2, Klf4, and GFP to reprogram XF-HFF cells under xeno-free conditions. A total of 10 xeno-free humaniPSC lines were generated, which could be continuously passaged in xeno-free conditions and aintained characteristics indistinguishable from hESCs, including colonymorphology and growth behavior, expression of pluripotency-associated markers, and pluripotent differentiationability in vitro and in teratoma assays. Overall, the resultspresented here demonstrate that human iPSCs can be generatedand maintained under strict xeno-free conditions and provide a path to good manufacturing practice (GMP) applicability that should facilitate the clinical translation of iPSC-based therapies.

Pharmacoeconomics of Cyclamen europaeum in the management of Acute Rhinosinusitis

Objective. To carry out a pharmacoeconomic analysis of Cyclamen europaeum (CE) in the management of acute rhinosinusitis (ARS) in Spain using data from the PROSINUS study. Study Design. This was a prospective observational study to compare the effectiveness and cost-effectiveness between therapies including CE vs. other therapies in the management of ARS. Methods. The study was carried out as a secondary analysis of the PROSINUS, combining healthcare resource use, productivity loses, and health outcomes from the observational study with costs representative of the Spanish Health System. Results. CE given as monotherapy appears to be more effective (cure rate) than other monotherapies (15.3% higher, p<0.05) and combination (10.3% higher, p<0.05) therapies. The addition of CE to other single-drug or combination therapies showed a statistically significant improvement in terms of cure rates when adding CE to 2-drug combinations (93.9% vs. 76.5%; p<0.05), and no significant effect when added to combinations of three or more drugs (81.1% vs. 79.8; NS). CE based therapies generally showed lower indirect costs, although only the comparison of CE alone vs. other monotherapies, with a net cost savings of 101 per patient, reached statistical significance (331 vs. 432 , p<0.05). In addition, CE-based therapies show lower cost per cured patient in all comparisons except when CE was used in combination with three or more other drugs. Conclusions. The use of Cyclamen europaeum may be associated to better clinical outcomes at no additional cost for the healthcare system, respect to treatments commonly used for ARS in clinical practice.

Growth factor- and cytokine-driven pathways governing liver stemness and differentiation.

Liver is unique in its capacity to regenerate in response to injury or tissue loss. Hepatocytes and other liver cells are able to proliferate and repopulate the liver. However, when this response is impaired, the contribution of hepatic progenitors becomes very relevant. Here, we present an update of recent studies on growth factors and cytokine-driven intracellular pathways that govern liver stem/progenitor cell expansion and differentiation, and the relevance of these signals in liver development, regeneration and carcinogenesis. Tyrosine kinase receptor signaling, in particular, c-Met, epidermal growth factor receptors or fibroblast growth factor receptors, contribute to proliferation, survival and differentiation of liver stem/progenitor cells. Different evidence suggests a dual role for the transforming growth factor (TGF)-β signaling pathway in liver stemness and differentiation. On the one hand, TGF-β mediates progression of differentiation from a progenitor stage, but on the other hand, it contributes to the expansion of liver stem cells. Hedgehog family ligands are necessary to promote hepatoblast proliferation but need to be shut off to permit subsequent hepatoblast differentiation. In the same line, the Wnt family and β-catenin/T-cell factor pathway is clearly involved in the maintenance of liver stemness phenotype, and its repression is necessary for liver differentiation during development. Collectively, data indicate that liver stem/progenitor cells follow their own rules and regulations. The same signals that are essential for their activation, expansion and differentiation are good candidates to contribute, under adequate conditions, to the paradigm of transformation from a pro-regenerative to a pro-tumorigenic role. From a clinical perspective, this is a fundamental issue for liver stem/progenitor cell-based therapies.