Clinical presentation, treatment and outcome in 23 cats with laryngeal or tracheal lymphoma.

OBJECTIVES: Feline primary laryngeal or tracheal lymphoma (PLTL) is an uncommon extranodal presentation. Information on long-term survival is scarce, although some small case series describe this being achieved with multiagent protocols; an accurate outcome for cats with PLTL is yet to be determined. The aim of this study was to gather information on the clinical presentation, response to treatment and outcome in a large case series of feline PLTL. METHODS: This retrospective multicentre study included cats with a cytological or histopathological confirmation of PLTL. Histopathology samples, when available, were reassessed for grade and immunophenotype. Clinical (age, signalment, retroviral status, presence of anaemia, clinical signs, location and therapy type) and outcome (response, progression-free survival [PFS] and overall survival [OS]) variables were recorded. Survival analyses to assess the impact of variables on PFS and OS were performed. RESULTS: Twenty-three cases were included; cats had a median age of 11 years (range 2-16) and the male:female ratio was 3.6:1. Common clinical signs at presentation included increased respiratory effort (74%) and abnormal upper respiratory tract sounds (48%). Immunophenotyping was performed in 48% of cases and all were B cell. Debulking surgery was performed in 26% of cases. All cats received chemotherapy, COP (cyclophosphamide, vincristine and prednisolone; 39%), CHOP (cyclophosphamide, vincristine, doxorubicin and prednisolone; 44%) and other protocols (17%); 35% had a partial response and 65% a complete response. Median PFS and OS were 909 days (range 23-1484) and 909 days (range 23-2423), respectively. Complete response was associated with longer PFS (P <0.001) and OS (P = 0.012). Pretreatment with steroids was associated with longer OS (P = 0.003). No other variable was found to be significant. CONCLUSIONS AND RELEVANCE: PLTL in cats is mostly of a B-cell phenotype, could be of a low-to-medium grade, and may respond to surgical and medical treatment with a longer survival time than has previously been reported.

Vitrified blastocysts from Preimplantation Genetic Diagnosis (PGD) as a source for human Embryonic Stem Cell (hESC) derivation.

Embryos diagnosed as abnormal in Preimplantation Genetic Diagnosis (PGD) cycles are useful for the establishment of human Embryonic Stem Cells (hESC) lines with genetic disorders. These lines can be helpful for drug screening and for the development of new treatments. Vitrification has proved to be an efficient method to preserve human blastocysts. One hundred and three abnormal or undiagnosed vitrified blastocysts from the PGD programme at Institut Universitari Dexeus were donated for human embryonic stem cell derivation. The overall survival rate after warming was 70.6 %. Our results showed better survival rates when blastocysts have not started the hatching process (initial/expanded 87.8 %, hatching 68.3 % and hatched 27.3 %). Thirty-five blastocysts and 12 partially surviving embryos were seeded. One hESC line with the multiple exostoses type 2 paternal mutation was obtained.

A protocol describing the genetic correction of somatic human cells and subsequent generation of iPS cells.

The generation of patient-specific induced pluripotent stem cells (iPSCs) offers unprecedented opportunities for modeling and treating human disease. In combination with gene therapy, the iPSC technology can be used to generate disease-free progenitor cells of potential interest for autologous cell therapy. We explain a protocol for the reproducible generation of genetically corrected iPSCs starting from the skin biopsies of Fanconi anemia patients using retroviral transduction with OCT4, SOX2 and KLF4. Before reprogramming, the fibroblasts and/or keratinocytes of the patients are genetically corrected with lentiviruses expressing FANCA. The same approach may be used for other diseases susceptible to gene therapy correction. Genetically corrected, characterized lines of patient-specific iPSCs can be obtained in 4-5 months.

Derivation of human embryonic stem cells at the Center of Regenerative Medicine in Barcelona.

We report here the legislative issues related to embryo research and human embryonic stem cell (hESC) research in Spain and the derivation of nine hESC lines at the Center of Regenerative Medicine in Barcelona. You can find the information for obtaining our lines for research purposes at blc@cmrb.eu.

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 and perhaps treating human disease. However, all reprogramming protocols 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 entirely devoid of xenobiotics. We first developed a xeno-free cell culture 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 derived primary cultures of human dermal fibroblasts under strict xeno-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 human iPSC lines were generated, which could be continuously passaged in xeno-free conditions and maintained characteristics indistinguishable from hESCs, including colony morphology and growth behavior, expression of pluripotency-associated markers, and pluripotent differentiation ability in vitro and in teratoma assays. Overall, the results presented here demonstrate that human iPSCs can be generated and 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.

Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells.

The generation of induced pluripotent stem (iPS) cells has enabled the derivation of patient-specific pluripotent cells and provided valuable experimental platforms to model human disease. Patient-specific iPS cells are also thought to hold great therapeutic potential, although direct evidence for this is still lacking. Here we show that, on correction of the genetic defect, somatic cells from Fanconi anaemia patients can be reprogrammed to pluripotency to generate patient-specific iPS cells. These cell lines appear indistinguishable from human embryonic stem cells and iPS cells from healthy individuals. Most importantly, we show that corrected Fanconi-anaemia-specific iPS cells can give rise to haematopoietic progenitors of the myeloid and erythroid lineages that are phenotypically normal, that is, disease-free. These data offer proof-of-concept that iPS cell technology can be used for the generation of disease-corrected, patient-specific cells with potential value for cell therapy applications.

Generation of mouse-induced pluripotent stem cells by transient expression of a single nonviral polycistronic vector.

Induced pluripotent stem (iPS) cells have generated keen interest due to their potential use in regenerative medicine. They have been obtained from various cell types of both mice and humans by exogenous delivery of different combinations of Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28. The delivery of these transcription factors has mostly entailed the use of integrating viral vectors (retroviruses or lentiviruses), carrying the risk of both insertional mutagenesis and oncogenesis due to misexpression of these exogenous factors. Therefore, obtaining iPS cells that do not carry integrated transgene sequences is an important prerequisite for their eventual therapeutic use. Here we report the generation of iPS cell lines from mouse embryonic fibroblasts with no evidence of integration of the reprogramming vector in their genome, achieved by nucleofection of a polycistronic construct coexpressing Oct4, Sox2, Klf4, and c-Myc.