A protocol describing the genetic correction of somatic human cells and subsequent generation of IPS cell

The generation of patient-specific induced pluripotent stem cells (iPSCPSCPSCs) offers unprecedented opportunities for modeling and treating human disease. In combination with gene therapy, the iPSCPSCPSC 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 iPSCPSCPSCs 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 iPSCPSCPSCs can be obtained in 4–5 months.

Disease-corrected haematopoietic progenitors from Fanconi anemia induced pluripotent stem cells

The generation of induced pluripotent stem (iPS) cells has enabled the derivation of patient-specific pluripotent cells andprovided valuable experimental platforms to model human disease. Patient-specific iPS cells are also thought to hold greattherapeutic 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 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 generationof patient-specific progenitors with potential value for cell therapy. However, in many instances, an off-the-shelf approach isdesirable, such as for cell therapy of acute conditions or when the patient’s somatic cells are altered as a consequence of a chronicdisease or aging. Cord blood (CB) stem cells appear ideally suited for this purpose as they are young cells expected to carryminimal somatic mutations and possess the immunological immaturity of newborn cells; additionally, several hundred thousandimmunotyped CB units are readily available through a worldwide network of CB banks. Here we present a detailed protocol for thederivation of CB stem cells and how they can be reprogrammed to pluripotency by retroviral transduction with only two factors(OCT 4 and SO X2) in 2 weeks and without the need for additional chemical compounds.

Redirecció de l'especificitat de les cél·lules T contra epítops del VIH restringit per la HLA-A2 mitjaçant transferència genética

Existeixen creixents evidències què la resposta dels limfòcits T CD8+ alpha beta citotòxics (CTLs) és un element fonamental en la infecció produïda pel VIH. Les CTLs VIH especifiques es consideren molt importants en la reducció de la càrrega viral i en la contenció de la infecció. Encara que la combinació dels antiretrovirals (HAART) ha suposat una millora considerable en la lluita contra el VIH induint una important reducció de la càrrega viral i augmentant el nombre de cèl•lules T CD4+, diverses complicacions han fet ressaltar la necessitat de noves alternatives terapèutiques. Les complicacions inclouen: manca de recuperació d’una resposta immune sòlida contra el VIH, toxicitat a llarg termini de la teràpia i el descobriment que les cèl•lules T CD4+ constitueixen un reservori pel virus. Les noves alternatives controlaran la replicació viral i reconstituiran la immunitat. L’eficàcia de la immunoteràpia cel•lular amb transferència adoptiva de CTLs virals específics s’ha provat en diferents infeccions virals humanes, incloent el VIH. Proposem una modificació de la immunoteràpia adoptiva redirigint l’especificitat de les cèl•lules T contra el VIH mitjançant la transfecció dels gens del TCR. En aquest assaig preclínic, ens aprofitarem de la tecnologia dels animals transgènics per les molècules de HLA, amb la finalitat de generar TCRs d’alta afinitat dirigits contra epitops del VIH restringits per la molècula HLA. Aquests TCRs seran induïts in vivo i seleccionats in vitro. Les cadenes alpha i beta dels TCRs VIH específics procedents de les CTLs seran clonades mitjançant tècniques de biologia molecular. Aquests TCRs VIH específics seran transferits a cèl•lules T CD8+ humanes i la seva especificitat i capacitat citolítica contra cèl•lules diana que presentin antígens de VIH-1 s’estudiaran mitjançant la combinació de diverses tècniques noves (FCC, transfecció mitjançant Nucleoefector). Finalment, una construcció retroviral adient per la seva transducció en cèl•lules T humanes s’establirà amb un TCR òptim seleccionat.

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.

The curcumin analog DM-1 induces apoptotic cell death in melanoma

The main difficulty in the successful treatment of metastatic melanoma is that this type of cancer is known to be resistant to chemotherapy. Chemotherapy remains the treatment of choice, and dacarbazine (DTIC) is the best standard treatment. The DM-1 compound is a curcumin analog that possesses several curcumin characteristics, such as antiproliferative, antitumor, and antimetastatic properties. The objective of this study was to evaluate the signaling pathways involved in melanoma cell death after treatment with DM-1 compared to the standard agent for melanoma treatment, DTIC. Cell death was evaluated by flow cytometry for annexin V and iodide propide, cleaved caspase 8, and TNF-R1 expression. Hoechst 33342 staining was evaluated by fluorescent microscopy; lipid peroxidation and cell viability (MTT) were evaluated by colorimetric assays. The antiproliferative effects of the drugs were evaluated by flow cytometry for cyclin D1 and Ki67 expression. Mice bearing B16F10 melanoma were treated with DTIC, DM-1, or both therapies. DM-1 induced significant apoptosis as indicated by the presence of cleaved caspase 8 and an increase in TNF-R1 expression in melanoma cells. Furthermore, DM-1 had antiproliferative effects in this the same cell line. DTIC caused cell death primarily by necrosis, and a smaller melanoma cell population underwent apoptosis. DTIC induced oxidative stress and several physiological changes in normal melanocytes, whereas DM-1 did not significantly affect the normal cells. DM-1 antitumor therapy in vivo showed tumor burden decrease with DM-1 monotherapy or in combination with DTIC, besides survival rate increase. Altogether, these data confirm DM-1 as a chemotherapeutic agent with effective tumor control properties and a lower incidence of side effects in normal cells compared to DTIC.

Relevance of death receptors in nervous system: role in the pathogenesis of neurodegenerative diseases and targets for therapy

L’apoptosi és un procés fisiològic que controla el nombre de cèl·lules en organismes superiors. L’apoptosi està estrictament regulada i s’ha vist que està implicada en la patogènesi d’algunes malalties del sistema nerviós. En aquest sentit, un excés de mort cel·lular contribueix a les malalties neurodegenerati- ves, mentre que, el seu dèficit és una de les raons del desenvolupament de tumors. El punt principal de regulació del procés apoptòtic és l’activació de les caspases, cisteïna-proteases que tenen especificitat pels residus aspàrtic. Les caspases es poden activar per dos mecanismes principals: (1) alliberament de citocrom C dels mitocondris alterats al citoplasma i (2) l’activació dels receptors de la membrana anomenats receptors de mort (DR, de l’anglès death receptor). Aquests receptors s’han caracteritzat extensament en el sistema immunitari, mentre que en el sistema nerviós les seves funcions són encara desconegudes. El present article se centra en el paper dels DR en la patogènesi de malalties neurodegeneratives i suggereix el seu potencial des del punt de vista terapèutic. També es descriuen diverses molècules intracel·lulars caracteritzades per la seva habilitat en la modulació dels DR. Entre elles, presentem dues noves proteïnes – lifeguard i FAIM – que s’expressen específicament al sistema nerviós.

Expression of somatostatin receptors in human melanoma cell lines: effect of two different somatostatin analogues, octreotide and SOM230, on cell proliferation

Somatostatin analogues (SAs) are potential anticancer agents. This study was designed to investigate the expression of somatostatin receptors (SSTRs) in melanoma cells and the effect of two SAs on cell proliferation and viability. Eighteen primary and metastatic human cutaneous melanoma cell lines were treated with octreotide and SOM230. Expression of SSTR1, SSTR2, SSTR3 and SSTR5 was assessed by real-time polymerase chain reaction. Proliferation, viability and cell death were assessed using standard assays. Inhibition was modelled by mixed-effect regression. Melanoma cells expressed one or more SSTR. Both SAs inhibited proliferation of most melanoma cell lines, but inhibition was less than 50%. Neither SA affected cell viability or induced cell death. The results suggest that melanoma cell lines express SSTRs. The SAs investigated, under the conditions used in this study, did not, however, significantly inhibit melanoma growth or induce cell death. Novel SAs, combination therapy with SAs and their anti-angiogenic properties should be further investigated.

Myelin-associated proteins block the migration of olfactory ensheathing cells: an in vitro study using single cell tracking and traction force microscopy

Newly generated olfactory receptor axons grow from the peripheral to the central nervous system aided by olfactory ensheathing cells (OECs). Thus, OEC transplantation has emerged as a promising therapy for spinal cord injuries and for other neural diseases. However, these cells do not present a uniform population, but, instead, a functionally heterogeneous population that exhibits a variety of responses including adhesion, repulsion and crossover during cell-cell and cell-matrix interactions. Some studies report that the migratory properties of OECs are compromised by inhibitory molecules and potentiated by chemical gradients. Here, we demonstrated that rodent OECs express all the components of the Nogo Receptor complex and that their migration is blocked by Myelin. Next, we used cell tracking and traction force microscopy to analyze OEC migration and its mechanical properties over Myelin. Our data relate the absence of traction force of OEC with lower migratory capacity, which correlates with changes in the F-Actin cytoskeleton and focal adhesion distribution. Lastly, OEC traction force and migratory capacity is enhanced after cell incubation with the Nogo Receptor inhibitor NEP1-40.

Potential of caveolae in the therapy of cardiovascular and neurological diseases.

Caveolae are membrane micro-domains enriched in cholesterol, sphingolipids and caveolins, which are transmembrane proteins with a hairpin-like structure. Caveolae participate in receptor-mediated trafficking of cell surface receptors and receptor-mediated signaling. Furthermore, caveolae participate in clathrin-independent endocytosis of membrane receptors. On the one hand, caveolins are involved in vascular and cardiac dysfunction. Also, neurological abnormalities in caveolin-1 knockout mice and a link between caveolin-1 gene haplotypes and neurodegenerative diseases have been reported. The aim of this article is to present the rationale for considering caveolae as potential targets in cardiovascular and neurological diseases.