Cystic fibrosis is one of the most frequent inherited rare diseases, caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. Apart from symptomatic treatments, therapeutic protocols for curing the disease have not yet been established. The regeneration of genetically corrected, disease-free epithelia in cystic fibrosis patients is envisioned by designing a stem cell/genetic therapy in which patient-derived pluripotent stem cells are genetically corrected, from which target tissues are derived. In this framework, we present an efficient method for seamless correction of pF508del mutation in patient-specific induced pluripotent stem cells by gene edited homologous recombination. Gene edition has been performed by transcription activator-like effector nucleases and a homologous recombination donor vector which contains a PiggyBac transposon-based double selectable marker cassette. This new method has been designed to partially avoid xenobiotics from the culture system, improve cell culture efficiency and genome stability by using a robust culture system method, and optimize timings. Overall, once the pluripotent cells have been amplified for the first nucleofection, the procedure can be completed in 69 days, and can be easily adapted to edit and change any gene of interest.

Use of animal products to culture human stem cells poses risk of pathogens and other contaminations which may render stem cells therapeutically unsuitable. Hence, it is necessary to meet the standards of good manufacturing practices, clinical-grade like and xeno-free conditions. The protocol for culturing clinical grade stem cells needs to be modified so that maximum utilization can be done for stem cell therapies. Modifications can be in the media compositions, additional growth factors, small molecule compounds, and gene manipulations of the required transcription factors or the platform in which stem cells can grow. After the clinical grade standards are met it needs the approval of FDA for use as transplantation therapies. This review provides various methods and inventions for culturing clinical grade stem cells which have been patented. Keywords: Clinical grade, cytokines, embryonic stem cells, growth factors, induced pluripotent stem cells, matrix, mesenchymal stem cells, stem cells, xeno-free.

Gene therapy strategies have become involved in methods for the treatment of inherited or acquired diseases with the aim to correct the faulty genetic code or to modify gene expression. In order to develop safe and effective therapeutic methods for applying in humans, different nucleic acids, termed as vectors, have been engineered, as therapeutic molecules in addition to classical drugs. In this review we included an overview of recent research and patents on therapeutic nucleic acids, designed for use on safe and effective gene therapy protocols for the treatment of diseases, whose results have been obtained in animal models, and which suggests that the use of nucleic acids as therapeutics molecules holds great promise for the future of disease control or cure. Keywords: AAT deficiency, cancer, childhood cerebral adrenoleukodystrophy, gene therapy, haemophilia, inherited retinal degenerations, non-viral vector, nucleic acids, rheumatoid arthritis, viral vector.

Genomic engineering has enormous potential along basic research, drug discovery and cell therapeutics. Many existing methods for targeted gene knockout mutagenesis or integration rely on homologous recombination. The low rate of spontaneous recombination in nearly all mammalian cell types, as well as the scale of screening, effort and time required to isolate the targeted events during genome modification, have hindered progress in this field. The present review has the objective to present latest improvements of technology to develop genetic modification to a clinical grade level so it can be used in human therapy. Keywords: Genomic editing, naive pluripotent stem, patient-specific, pluripotent, stem cell/genetic therapy.

Regenerative medicine seeks to restore normal architecture and function of tissues which have been damaged by disease, trauma and/or ageing. Stem cells are known to be an integral part of any regeneration process. Their isolation or generation, in vitro expansion, recruitment and transplantation to the damaged sites are being heavily pursued. Several applications of pluripotent cell derivatives have arrived at the clinical stage and are offering promising results. Stem cells themselves or their products can exert desired functions in regeneration. The source of stem cells for developing therapies is vast and developments have been made at a very fast pace during the last ten years. Pluripotent stem cells from discarded embryos or induced from postnatal tissues are at the base of these investigations as they can theoretically be permanently cultured in vitro and can be triggered to differentiate any cell in the human body. Induced or directed differentiation of these cells is still being studied and generally the process involves long manipulations and thorough selection, which prevent pluripotent cells from being useful in autologous transplants. So-called adult stem cells or stem cells isolated from postnatal individuals offer the possibility to develop autologous sources for cell therapies. Bone marrow, cord and peripheral blood, fat and oral mucosa are sources of such stem cells. In this case the drawbacks are the availability in sufficient numbers to develop the regenerative protocols and the limited applications of these cells due to their limited differentiation ability. Both pluripotent and adult stem cells are being investigated for the development of clinical trials at the moment. Pluripotent stem cells will be able to be applied widely in cell therapy when directed differentiation can be controlled to the point of producing safe and functional derived cells and tissues. The present review will focus on recent patents on directed differentiation of pluripotent stem cells to derivatives with potential therapeutic value. Keywords: Cell engraftments, directed differentiation, ectoderm, endoderm, functional differentiation, mesoderm, pluripotent derivatives.

Stem cells have the ability to self-replicate in vitro indefinitely under adequate conditions and provided that pluripotent signals are triggered exogenously. Moreover, they are able to differentiate into virtually all human cell types. This vast potential makes stem cells a powerful tool to be used in regenerative medicine and different therapeutic strategies as well as in clinical and basic research, contributing to the generation of novel therapeutic avenues to treat innumerable pathological disorders that today have no known cure. Despite increased disclosures at a high speed pace in terms of benefits, applicability and relevance of stem cell based therapies; more studies in humans are needed to advance in the establishment of safe and effective cell-based treatments. In this review we compile an overview of the latest reports and patents on stem cell accomplishments for the treatment of diseases and injuries from Patent databases as The European Patent Office, WIPO, Fresh patents and Freepatents online, as well as Pubmed references, regarding stem cell therapy. Nevertheless, the vast majority of these promising results have been established in animal models, suggesting that stem cells research must go on prior to transferring therapeutic protocols to the human being although it has much to offer to improve life quality and eliminate disability. Keywords: Cell therapies, engraftment, IPSCs, microvesicles delivered from embryonic stem cells, proliferation stimulating antibodies, stem cell size.

Resumen del poster presentado en el Collaborative Congress of the European Society for Gene and Cell and the Spanish Society for Gene and Cell Therapy celebrado del 25 al 28 de octubre de 2013 en Madrid (Espana)

For the past 12 years, the biology and applications of human embryonic stem cells (hESCs) have received great attention from the scientific community. Derivatives of the first hESC line obtained by J. Thomson's group (Science 282(5391):1145-1147, 1998) have been used in clinical trials in patients with spinal cord injury, and other hESC lines have now been used to generate cells for use in treating blindness (Lancet 379(9817):713-720, 2012). In addition to the classical protocol based on mouse or human feeder layers using open culture methods (In Vitro Cellular & Developmental Biology - Animal 46(3-4):386-394, 2010; Stem Cells 23(9):1221-1227, 2005; Nature Biotechnology 24(2):185-187, 2006; Human Reproduction 21(2):503-511, 2006; Human Reproduction 20(8):2201-2206, 2005; Fertility and Sterility 83(5):1517-1529, 2005), novel hESC lines have been derived xeno-free (without using animal derived reagents) (PLoS One 5 (4):1024-1026, 2010), feeder-free (without supporting cell monolayers) (Lancet 365(9471):1601-1603, 2005), in microdrops under oil (In Vitro Cellular & Developmental Biology - Animal 46(3-4):236-41, 2010) and in suspension with ROCK inhibitor (Nature Biotechnology 28(4):361-4, 2010). Regardless of the culture system, successful hESC derivation usually requires optimization of embryo culture, the careful and timely isolation of its inner cell mass (ICM), and precise culture conditions up to the establishment of pluripotent cell growth during hESC line derivation. Herein we address the crucial steps of the hESC line derivation protocol, and provide tips to apply quality control to each step of the procedure.

The establishment of the first culture of a human embryonic stem cell (hESC) line as immortal and capable of producing virtually every cell of a human body generated high expectations regarding their potential use in regenerative medicine and research applications. The achievement of generating immortal pluripotent cells from adult tissues further raised those expectations, by ideally eliminating ethical and safety concerns regarding the use of human embryonic stem cells. Patient-specific stem cells could be generated, and no human embryo would have been used. After first six years of producing induced pluripotent stem cell lines from somatic tissues, advancements have been made but much research still needs to be conducted in order to produce clinically relevant differentiated cells from induced pluripotent stem cells. The present review outlines the most recent inventions and basic research related to the clinical applications of human pluripotent stem cells. Its main objective is to contribute a concise compilation of the advances in the derivation, culture, directed differentiation and development of application processes of human pluripotent stem cells, in the development of their clinical applications to treat human disease. Registered patents and related bibliography reported mostly from past two years have been considered for inclusion, although basic literature has been included where needed to explain recent developments. Keywords: Derivatives, differentiation, expansion, pluripotent, therapy, stem, Human Embryonic Stem cell, Induced Pluripotent Stem Cells, regenerative therapy, stem cell cultures

We have generated a human feeder cell line from early second trimester Placental Stromal Fibroblasts (ihPSF) stably over-expressing the polycomb protein BMI-1. These feeder cells retain the ability to maintain human Embryonic Stem cells (hESc) over long-term culture whereas hTERT or BMI-1/hTERT immortalised feeder cell lines do not. ihPSFs were able to support the derivation of a new hESc line in near xenofree (free of non-human animal components) conditions and support continued culture of newly derived hESc and human induced Pluripotent Stem (hiPS) cell lines in complete xenofree conditions necessary for clinical use.

We report here the derivation of two new human embryonic stem cell lines, Man-1 and Man-2, and their full characterization as novel pluripotent stem cell lines. Man-1 was derived from an embryo surplus to requirement from routine IVF, while Man-2 was obtained from an oocyte classified as failed to fertilise and subsequently chemically activated. We report the characterisation of pluripotency and the differentiation potential of these lines. Work is in progress to establish novel methods of stem cell derivation and culture, which will avoid the use of xenobiotics and be relevant to clinical production of human embryonic stem cell lines. Both newly derived human embryonic stem cell lines will be available for the research community from the UK Stem Cell Bank (http://www.ukstemcellbank.org.uk).

Derivation and Culture of Human Embryonic Stem Cells at Good Manufacturing Practice standards for clinical transplantation Maria V. Camarasa, Sharon Sneddon, Nicola Bates, Jayne Fitzsimmons, John Aplin, Susan J. Kimber, & Daniel Brison North West Embryonic Stem Cell Centre, Department of Reproductive Medicine, Saint Mary’s Hospital, Manchester M13 OJH; Faculty of Life Sciences, University of Manchester, Core Technology Facility, 46 Grafton St, Manchester M13 9NT and Faculty of Medical and Human Sciences, University of Manchester, Manchester M13 OJH

The role of individual supplements necessary for the self-renewal of human embryonic stem (hES) cells is poorly characterized, and furthermore we have found that previously reported feeder cell- and serum-free culture systems used for individual hES cell lines are unable to maintain HUES7 cells for more than one passage. We have therefore derived a feeder/serum-free culture system that can support the long-term (at least 10 passages) self-renewal of several euploid hES cell lines including MAN1, HUES7, and HUES1 with minimal spontaneous differentiation and without the need for manual propagation. This system contains fibroblast growth factor 2, activin A, neurotrophin 4, and the N2, B27 supplements together with a human fibronectin substrate. We demonstrate that these components exert distinct functions: both FGF2 and activin A were necessary to prevent differentiation of hES cells while NT4 promoted cell survival, FGF2 could not be substituted by IGFII, and the fibronectin substrate supported a rapid rate of hES culture expansion. Inhibition studies showed that β1 integrin-dependent attachment of hES cells to fibronectin was at least partially via the α5 subunit but independent of integrin αV.

A recent approach in anticancer chemotherapy envisages telomerase as a potentially useful target. An attractive strategy deals with the development of compounds able to stabilize telomeric DNA in the G-quadruplex folded structure and, among them, a prominent position is found in the perylenes. With the aim to further investigate the role of drug structure, in view of possible pharmaceutical applications, we synthesized a series of compounds related to PIPER, a well-known perylene-based telomerase inhibitor. We modified the number of condensed aromatic rings and introduced different side chains to modulate drug protonation state and extent of self-aggregation. Effective telomerase inhibition was induced by heptacyclic analogues only, some showing a remarkably wide selectivity index with reference to inhibition of Taq polymerase. G-quadruplex stabilization was monitored by circular dichroism and melting experiments. Cell cytotoxicity measurements indicated a poor short-term cell killing ability for the best G-quartet binders. Besides the presence of a planar seven-condensed ring system, the introduction of a cyclic amine in the side chains critically affects the selectivity window.

It is well established that the effectiveness of anticancer drugs may result from combined cytotoxic and differentiation activities on tumor cells. Also, differentiating agents are able to alter the susceptibility of cancer cells to antineoplastic drug therapy. However, the acquisition and/or development of drug resistance that frequently appears in anticancer treatment can impair these interactions between differentiation agents and cytotoxic drugs. In the present study, we report that the acquisition of resistance to anthracyclines in two humans, promyeolocytic leukemia HL‐60 and eythroleukemia K562 cell lines, results in a restricted maturation process induced by differentiating agents with respect to that exhibited by their corresponding drug‐sensitive counterparts. Interestingly, differentiating agents are able to decrease the overexpression of drug‐efflux pumps as it is the case of MRP1 in the resistant HL‐60 cells, thus increasing the sensitivity of cells to drug treatment. In addition, susceptibility of the drug‐sensitive cells to certain apoptotic stimuli is significantly reduced after differentiation. The results here reported indicate complex interactions between cytotoxic (drug therapy) and non‐cytotoxic (differentiation) cancer treatments, which should be taken into account to improve therapeutic efficiency. © 2004 Wiley‐Liss, Inc.

BACKGROUND, The effectiveness of some chemotherapeutic agents used to treat gastric carcinoma patients may be impaired by the presence of P-glycoprotein (P-gp) and the status of p53. A modulation of P-gp expression by p53 or other alterations during tumorigenesis have been reported. The authors analyzed P-gp expression in relation to p53 and histopathologic features in gastric carcinoma.METHODS. Forty-one resected gastric carcinomas and mucosa distant from the tumor were assessed for P-gp expression by immunohistochemistry with C494 and JSB-1 antibodies. p53 expression was also immunohistochemically assessed by DO7 antibody in tumor samples. P-gp and p53 expression were semiquantitatively analyzed according to the percentage of stained cells. Histologic type, grade, vessel invasion, and stage were also studied.RESULTS. Moderate or high P-gp expression was detected in gastric carcinoma in 29 cases (71%) and in gastric mucosa remote from the tumor in 36 cases (88%). This reduction in P-gp expression was observed in 22% of the carcinomas, all but 1 being p53 immunonegative tumors. Thus, 8 (42%) of the p53 immunonegative carcinomas showed a loss of P-gp expression compared with their distant gastric mucosa. All p53 immunopositive carcinomas coexpressed P-gp. No correlation between P-gp expression and histologic type, grade, vessel invasion, or stage was found.CONCLUSIONS. P-gp expression in gastric carcinomas is frequent and coexpression with p53 is found. The analysis of P-gp expression in carcinomas and distant mucosa show that it is not regulated by p53, but a loss of P-gp detected in some of these carcinomas is mainly associated with a lack of p53 protein accumulation. (C) 2000 American Cancer Society.

AIMS:Most chemotherapeutic regimens used against gastric carcinoma include anthracyclines whose effectiveness can be impaired by the presence of P-glycoprotein. In order to obtain a reliable pattern of P-glycoprotein expression in these tumours an immunohistochemical study using a panel of anti-P-glycoprotein antibodies was performed in frozen and paraffinized tissues. METHODS AND RESULTS:Frozen and paraffinized samples from 25 gastric carcinomas were immunohistochemically analysed using a panel of four anti-P-glycoprotein monoclonal antibodies including C219, MRK16, JSB-1 and C494. Semiquantitative analysis indicated that moderate or high P-glycoprotein levels were detected in 40% to 76% of gastric adenocarcinomas, depending on the anti-P-glycoprotein antibody used. The antibody C494 was the most sensitive in detecting P-glycoprotein in both frozen and paraffinized gastric carcinoma samples. Moreover, C494 showed a pattern of staining exclusively associated with the plasma membrane, in contrast to the cytoplasmic with reinforcement of plasma membrane pattern displayed by the other three antibodies. Significant differences in P-glycoprotein levels were obtained when C494 and MRK16 were used in frozen tissues. Finally, detection of P-glycoprotein in frozen samples did not improve when compared to paraffinized ones. CONCLUSIONS:It appears that P-glycoprotein is frequently expressed in gastric adenocarcinomas, and the use of C494 complemented by JSB-1 is recommended for reliable detection of P-glycoprotein in this neoplasm.