Derivació de línies de cèl·lules mare embrionàries humanes

Les cèl·lules mare embrionàries (CME) representen una font potencial de cèl·lules per a ús terapèutic en algunes malalties produïdes per la pèrdua de la funció cel·lular. Normalment, aquestes cèl·lules procedeixen d'embrions donats per les parelles sotmeses a tècniques de reproducció assistida (TRA). Es presenten els resultats obtinguts en el Banc de Línies Cel·lulars del Centre de Medicina Regenerativa de Barcelona (CMRB), on s'han descongelat 254 embrions donats per parelles sotmeses al programa de fecundació in vitro (FIV) de l'Institut Universitari Dexeus. La taxa de supervivència va ser del 51,9 %. S'han obtingut cinc línies de CME. Tres d'aquestes línies procedeixen d'embrions de mala qualitat i presenten la capacitat d'autorenovació, pluripotència i diferenciació característiques d'aquestes cèl·lules. Els embrions de mala qualitat, sovint descartats en els centres de RA, poden ser una font útil per a la derivació de CME.Human embryonic stem cells (hESC) represent a potential source for cell therapy for many degenerative diseases. Usually hESC lines are derived from surplus embryos donated from couples undergoing In Vitro Fertilisation (IVF). We here present the results obtained in the Stem Cell Bank at the Center of Regenerative Medicine in Barcelona. 254 embryos have been thawed. The embryos were donated from couples from the IVF programme at the Institut Universitari Dexeus. The embryo survival rate was 51.9%. Five ESC lines were obtained. Three of these lines came from poor quality embryos. The cell lines present self-renewal, pluripotency, and differentiation properties characteristic of these cells Poor quality embryos, usually discarded in assisted reproduction centres, could be useful for ESC derivation

Screening for generalized anxiety disorder in Spanish Primary Care Centers with the GAD-7

The aim of the study was to determine the criterion validity of a computerized version of the General Anxiety Disorder-7 (GAD-7) questionnaire to detect general anxiety disorder in Spanish primary care centers. A total of 178 patients completed the GAD-7 and were administered the Composite International Diagnostic Interview (CIDI) for DSM-IV Axis I Disorders, which was used as a reference standard. Sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios were calculated. A cut-off of 10 yielded a sensitivity of .87, a specificity of .78, a positive predictive value of .93, a negative predictive value of .64, a positive likelihood ratio of 3.96 a negative likelihood ratio of .17 and Younden’s Index of .65. The GAD-7 performed very well with a cut-off value of 10, the most frequently used cut-off point. Thus, a computerized version of the GAD-7 is an excellent screening tool for detecting general anxiety disorder in Spanish primary care settings

Development of xenobiotic-free conditions towards the generation and propagation of clinically-safe human pluripotent stem cells

Les cèl·lules mare embrionàries humanes (hESC) i més recentment les cèl·lules de pluripotència induïda (iPSC) representen una oportunitat sense precedents per al desenvolupament de noves estratègies terapèutiques per malalties degeneratives humanes. Així mateix, la possibilitat d'obtenir iPSC específiques de pacient obre la porta per l'establiment de models de malaltia genuïnament humans. Una de les limitacions que dificulta l'aplicació clínica de les cèl·lules pluripotents és que la seva obtenció es du a terme a l'actualitat amb medis i reactius que contenen fonts proteiques d'origen animal (xenobiòtics). Aquesta tesis contribueix al desenvolupament de protocols de derivació i de cultiu de hESC, que les apropa a la seva utilització clínica. A més hem desenvolupat estratègies basades en iPSC més segures i eficaces per al tractament de malalties humanes.Human embryonic stem cells (hESC) and, more recently, induced pluripotent cells (iPSC) represent a new and unprecedented opportunity for the development of new therapeutic strategies for human degenerative diseases. The possibility to derive patient specific iPSC opens the door to he establishment of disease models exquisitely human. One of the limitation that, concretely, limits the clinic application of pluripotent cells is the fact that their derivation, currently, is obtained with media and reagents which contain animal proteic sources (xenobiotics). This thesis contributes to the development of protocols of derivation and culture of hESC that bring them closed to a clinical application. Moreover, we have developed iPSC-based strategies that are both safer and more effective for the treatment of human diseases

Generation of induced pluripotent stem cells from human cord blood cells with only two factors:OCT4 and SOX2

Induced pluripotent stem cells (iPSC ) provide an invaluable resource for regenerative medicine as they allow the generation/nof patient-specific progenitors with potential value for cell therapy. However, in many instances, an off-the-shelf approach is/ndesirable, such as for cell therapy of acute conditions or when the patient’s somatic cells are altered as a consequence of a chronic/ndisease or aging. Cord blood (CB) stem cells appear ideally suited for this purpose as they are young cells expected to carry/nminimal somatic mutations and possess the immunological immaturity of newborn cells; additionally, several hundred thousand/nimmunotyped CB units are readily available through a worldwide network of CB banks. Here we present a detailed protocol for the/nderivation of CB stem cells and how they can be reprogrammed to pluripotency by retroviral transduction with only two factors/n(OCT 4 and SO X2) in 2 weeks and without the need for additional chemical compounds

Generation of cardiomyocytes from new human embrionic stem cell lines derived from poor-quanlity blastocysts

Human embryonic stem (hES) cells represent a potential source for cell replacement therapy of many degenerative diseases. Most frequently, hES cell lines are derived from surplus embryos from assisted reproduction cycles, independent of their quality or morphology. Here, we show that hES cell lines can be obtained from poor-quality blastocysts with the same efficiency as that obtained from good- or intermediate-quality blastocysts. Furthermore, we show that the self-renewal, pluripotency, and differentiation ability of hES cell lines derived from either source are comparable. Finally, we present a simple and reproducible embryoid body-based protocol for the differentiation of hES cells into functional cardiomyocytes. The five new hES cell lines derived here should widen the spectrum of available resources for investigating the biology of hES cells and advancing toward efficient strategies of regenerative medicine

Generation of cardiomyocytes from new human embrionic stem cell lines derived from poor-quanlity blastocysts

Human embryonic stem (hES) cells represent a potential source for cell replacement therapy of many degenerative diseases. Most frequently, hES cell lines are derived from surplus embryos from assisted reproduction cycles, independent of their quality or morphology. Here, we show that hES cell lines can be obtained from poor-quality blastocysts with the same efficiency as that obtained from good- or intermediate-quality blastocysts. Furthermore, we show that the self-renewal, pluripotency, and differentiation ability of hES cell lines derived from either source are comparable. Finally, we present a simple and reproducible embryoid body-based protocol for the differentiation of hES cells into functional cardiomyocytes. The five new hES cell lines derived here should widen the spectrum of available resources for investigating the biology of hES cells and advancing toward efficient strategies of regenerative medicine

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

The availability of induced pluripotent stem cells (iPSCs)/nhas created extraordinary opportunities for modeling and/nperhaps treating human disease. However, all reprogramming/nprotocols 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/ndevoid of xenobiotics. We first developed a xeno-free cell/nculture 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/nprimary cultures of human dermal fibroblasts under strict/nxeno-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/niPSC lines were generated, which could be continuously passaged in xeno-free conditions and aintained characteristics indistinguishable from hESCs, including colony/nmorphology and growth behavior, expression of pluripotency-/nassociated markers, and pluripotent differentiation/nability in vitro and in teratoma assays. Overall, the results/npresented here demonstrate that human iPSCs can be generated/nand 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

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/ndue to their potential use in regenerative medicine. They have/nbeen obtained from various cell types of both mice and humans by/nexogenous delivery of different combinations of Oct4, Sox2, Klf4,/nc-Myc, Nanog, and Lin28. The delivery of these transcription factors/nhas mostly entailed the use of integrating viral vectors (retroviruses/nor lentiviruses), carrying the risk of both insertional mutagenesis/nand oncogenesis due to misexpression of these exogenous/nfactors. Therefore, obtaining iPS cells that do not carry integrated/ntransgene sequences is an important prerequisite for their eventual/ntherapeutic use. Here we report the generation of iPS cell lines/nfrom mouse embryonic fibroblasts with no evidence of integration/nof the reprogramming vector in their genome, achieved by nucleofection/nof a polycistronic construct coexpressing Oct4, Sox2, Klf4,/nand c-My

Derivació de línies de cèl·lules mare embrionàries humanes

Les cèl·lules mare embrionàries (CME) representen una font potencial de cèl·lules per a ús terapèutic en algunes malalties produïdes per la pèrdua de la funció cel·lular. Normalment, aquestes cèl·lules procedeixen d'embrions donats per les parelles sotmeses a tècniques de reproducció assistida (TRA). Es presenten els resultats obtinguts en el Banc de Línies Cel·lulars del Centre de Medicina Regenerativa de Barcelona (CMRB), on s'han descongelat 254 embrions donats per parelles sotmeses al programa de fecundació in vitro (FIV) de l'Institut Universitari Dexeus. La taxa de supervivència va ser del 51,9 %. S'han obtingut cinc línies de CME. Tres d'aquestes línies procedeixen d'embrions de mala qualitat i presenten la capacitat d'autorenovació, pluripotència i diferenciació característiques d'aquestes cèl·lules. Els embrions de mala qualitat, sovint descartats en els centres de RA, poden ser una font útil per a la derivació de CME.Human embryonic stem cells (hESC) represent a potential source for cell therapy for many degenerative diseases. Usually hESC lines are derived from surplus embryos donated from couples undergoing In Vitro Fertilisation (IVF). We here present the results obtained in the Stem Cell Bank at the Center of Regenerative Medicine in Barcelona. 254 embryos have been thawed. The embryos were donated from couples from the IVF programme at the Institut Universitari Dexeus. The embryo survival rate was 51.9%. Five ESC lines were obtained. Three of these lines came from poor quality embryos. The cell lines present self-renewal, pluripotency, and differentiation properties characteristic of these cells Poor quality embryos, usually discarded in assisted reproduction centres, could be useful for ESC derivation

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

The availability of induced pluripotent stem cells (iPSCs)/nhas created extraordinary opportunities for modeling and/nperhaps treating human disease. However, all reprogramming/nprotocols 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/ndevoid of xenobiotics. We first developed a xeno-free cell/nculture 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/nprimary cultures of human dermal fibroblasts under strict/nxeno-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/niPSC lines were generated, which could be continuously passaged in xeno-free conditions and aintained characteristics indistinguishable from hESCs, including colony/nmorphology and growth behavior, expression of pluripotency-/nassociated markers, and pluripotent differentiation/nability in vitro and in teratoma assays. Overall, the results/npresented here demonstrate that human iPSCs can be generated/nand 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