V-Myc immortalizes human neural stem cells in the absence of pluripotency-associated traits

© 2015 Pino-Barrio et al. A better understanding of the molecular mechanisms governing stem cell self-renewal will foster the use of different types of stem cells in disease modeling and cell therapy strategies. Immortalization, understood as the capacity for indefinite expansion, is needed for the generation of any cell line. In the case of v-myc immortalized multipotent human Neural Stem Cells (hNSCs), we hypothesized that v-myc immortalization could induce a more dedifferentiated state in v-myc hNSC lines. To test this, we investigated the expression of surface, biochemical and genetic markers of stemness and pluripotency in v-myc immortalized and control hNSCs (primary precursors, that is, neurospheres) and compared these two cell types to human Embryonic Stem Cells (hESCs) and fibroblasts. Using a Hierarchical Clustering method and a Principal Component Analysis (PCA), the v-myc hNSCs associated with their counterparts hNSCs (in the absence of v-myc) and displayed a differential expression pattern when compared to hESCs. Moreover, the expression analysis of pluripotency markers suggested no evidence supporting a reprogramming-like process despite the increment in telomerase expression. In conclusion, v-myc expression in hNSC lines ensures self-renewal through the activation of some genes involved in the maintenance of stem cell properties in multipotent cells but does not alter the expression of key pluripotency-associated genes.Spanish Ministry of Economy and Competitiveness (PLE2009–0101, SAF2010–17167); Comunidad Autónoma Madrid (S2011—BMD—2336); Instituto Salud Carlos III (RETICS TerCel, RD12/0019/0013) and European Union (Excell, NMP4—SL—2008–214706); (to PM): Instituto Salud Carlos III (RETICS TerCel, RD12/0019/0006; FISPeer Reviewe

V-Myc immortalizes human neural stem cells in the absence of pluripotency-associated traits

The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE63710A better understanding of the molecular mechanisms governing stem cell self-renewal will foster the use of different types of stem cells in disease modeling and cell therapy strategies. Immortalization, understood as the capacity for indefinite expansion, is needed for the generation of any cell line. In the case of v-myc immortalized multipotent human Neural Stem Cells (hNSCs), we hypothesized that v-myc immortalization could induce a more dedifferentiated state in v-myc hNSC lines. To test this, we investigated the expression of surface, biochemical and genetic markers of stemness and pluripotency in v-myc immortalized and control hNSCs (primary precursors, that is, neurospheres) and compared these two cell types to human Embryonic Stem Cells (hESCs) and fibroblasts. Using a Hierarchical Clustering method and a Principal Component Analysis (PCA), the v-myc hNSCs associated with their counterparts hNSCs (in the absence of v-myc) and displayed a differential expression pattern when compared to hESCs. Moreover, the expression analysis of pluripotency markers suggested no evidence supporting a reprogramming-like process despite the increment in telomerase expression. In conclusion, v-myc expression in hNSC lines ensures self-renewal through the activation of some genes involved in the maintenance of stem cell properties in multipotent cells but does not alter the expression of key pluripotency-associated genesThis work was supported by grants from (to AMS): Spanish Ministry of Economy and Competitiveness (PLE2009–0101, SAF2010–17167), Comunidad Autónoma Madrid (S2011—BMD— 2336), Instituto Salud Carlos III (RETICS TerCel, RD12/0019/0013) and European Union (Excell, NMP4—SL—2008–214706); (to PM): Instituto Salud Carlos III (RETICS TerCel, RD12/0019/0006; FIS P110/0449), ERANEt ISCIII—Fondos FEDER (PI12/ 03112) and the Spanish Association of Cancer Research (CIMEN2011). MJPB was funded by MINECO (PLE2009–0101) and Instituto Salud Carlos III (RETICS TerCel, RD06/0019/0023). This work was also supported by an institutional grant from Fundación Ramón Areces to the Center of Molecular Biology Severo Ochoa. PM lab is supported by: Instituto de Salud Carlos III (ISCIII; E-Rare-2 Call PI12/03112), Ministerio de Economía y Competitividad (MINECO; SAF2013-43065), Generalitat de Catalunya (SGR330) and Obra Social La Caixa-Fundaciò Josep Carrera

Clonal human fetal ventral mesencephalic dopaminergic neuron precursors for cell therapy research

A major challenge for further development of drug screening procedures, cell replacement therapies and developmental studies is the identification of expandable human stem cells able to generate the cell types needed. We have previously reported the generation of an immortalized polyclonal neural stem cell (NSC) line derived from the human fetal ventral mesencephalon (hVM1). This line has been biochemically, genetically, immunocytochemically and electrophysiologically characterized to document its usefulness as a model system for the generation of A9 dopaminergic neurons (DAn). Long-term in vivo transplantation studies in parkinsonian rats showed that the grafts do not mature evenly. We reasoned that diverse clones in the hVM1 line might have different abilities to differentiate. In the present study, we have analyzed 9 hVM1 clones selected on the basis of their TH generation potential and, based on the number of v-myc copies, v-myc down-regulation after in vitro differentiation, in vivo cell cycle exit, TH+ neuron generation and expression of a neuronal mature marker (hNSE), we selected two clones for further in vivo PD cell replacement studies. The conclusion is that homogeneity and clonality of characterized NSCs allow transplantation of cells with controlled properties, which should help in the design of long-term in vivo experimentsThis work was supported by grants from the Spanish Ministry of Economy and Competitiveness (formerly Science and Innovation; PLE2009-0101, SAF2010-17167), Comunidad Autónoma Madrid (S2011-BMD-2336), Instituto Salud Carlos III (RETICS TerCel, RD06/0010/0009) and European Union (Excell, NMP4-SL-2008-214706). This work was also supported by an institutional grant from Foundation Ramón Areces to the Center of Molecular Biology Severo Ocho

Plan de comunicación interna y externa de la Facultad de Ciencias Sociales. Programa de Actividades Complementarias y de Difusión Cultural de la Facultad II

Memoria ID-0258. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2014-2015

Targeted gene therapy in a mouse model of Fanconi anemia

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 27-10-2016Esta tesis tiene embargado el acceso al texto completo hasta el 2021-10-27Fanconi anemia (FA) is an inherited disease associated with bone marrow failure (BMF) and cancer predisposition. This disease is caused by mutations in any of the 20 FANC genes that belong to a DNA repair pathway known as FA/BRCA pathway. Gene therapy (GT) approaches with autologous hematopoietic stem and progenitor cells (HSPCs) may constitute an efficient treatment for FA patients. In fact, conventional non-targeted GT trials based on the use of lentiviral vectors are currently under development. Because the risk of insertional oncogenesis can not be completely ruled out in any of the GT trials that are at present undergoing with integrative vectors, gene-targeting approaches have been proposed as safer alternatives. These strategies are based on the use of artificial nucleases that generate double strand breaks at specific locations in the genome which can be repaired by the homologous recombination pathway of the cells. This pathway has been exploited to facilitate the integration, into specific sites of the cell genome, of donor templates harboring the gene of interest flanked by two homology arms. In this study we have conducted a gene-editing strategy aiming at the insertion of reporter and therapeutic donors into the mouse Mbs85 locus, an ortholog of the human AAVS1 safe harbor locus. In order to achieve our goal, TALE nucleases (TALEN) were nucleofected together with different donor templates carrying either the human therapeutic FANCA gene or the EGFP (Enhance Green Fluorescent Protein) reporter gene, both of which were under the regulation of a phosphoglycerate kinase (PGK) promoter. This strategy of gene editing was implemented by means of nucleofection of the TALEN and donor as DNA plasmids in Fanca-/- (FA-A) mouse embryonic fibroblasts (MEFs) and also in mouse HSPCs (mHSPCs) from WT and FA-A mice. We have first demonstrated the cleavage activity of designed TALEN in the Mbs85 locus of both FA-A MEFs and mHSPCs from WT and FA-A mice. Targeted integration (TI), as well as evidence of hFANCA expression were shown in gene-edited FA-A MEFs. Moreover, evidence of phenotypic correction was demonstrated in these cells by the reversion of the characteristic hypersensitivity to mitomycin C (MMC), and also by the reduction of the MMC-induced chromosomal aberrations. Gene-editing experiments in WT and FA-A mHSPCs also allowed us to demonstrate TI in these cell types. As it was observed in FA-A MEFs, evidence of phenotypic correction was also observed in FA-A hematopoietic colonies. In these experiments, we also observed a marked toxicity of plasmid DNA nucleofection that compromised the repopulating properties of gene-edited mHSPCs in irradiated recipients. Altogether, our results demonstrate for the first time the feasibility of implementing a therapeutic targeted integration strategy in a safe harbor locus of MEFs and mouse hematopoietic progenitors from a mouse model of Fanconi anemia. Additionally, our data suggest the necessity of limiting the toxicity associated to the nucleofection of mHSPCs with the TALEN and donor as plasmid DNA in order to improve the repopulating potential of geneedited HSCs.La anemia de Fanconi (AF) es una enfermedad hereditaria caracterizada por fallo de médula ósea y predisposición a cáncer. Esta enfermedad está causada por mutaciones en cualquiera de los 20 genes FANC descubiertos hasta la actualidad que participan en una vía de reparación del ADN conocida como ruta de AF/BRCA. Las aproximaciones de terapia génica (TG) con células madre hematopoyéticas (CMHs) autólogas podrían constituir un tratamiento eficaz para los pacientes con AF. De hecho, se están desarrollando ensayos de TG convencional no dirigida basados en el uso de vectores lentivirales. Puesto que el riesgo de oncogénesis insercional no puede descartarse por completo en ninguno de los ensayos de TG llevados a cabo en la actualidad con vectores integrativos, se han propuesto nuevas aproximaciones de TG dirigida como alternativas más seguras. Estas estrategias se basan en el uso de nucleasas artificiales que generan roturas de doble cadena en localizaciones específicas del genoma que pueden ser reparadas por la ruta de recombinación homóloga de las células. Esta ruta ha sido utilizada para facilitar la integración, en sitios específicos del genoma, de construcciones donadoras que contienen el gen de interés flanqueado por dos brazos de homología. En este estudio hemos llevado a cabo una estrategia de edición génica con el objetivo de integrar genes marcadores y terapéuticos en el locus Mbs85 de ratón, ortólogo al locus seguro AAVS1 humano. Con el fin de lograr nuestro objetivo, se nucleofectaron las nucleasas TALE (TALEN) junto a diferentes construcciones donadoras que contenían el gen terapéutico humano FANCA o el gen marcador EGFP, ambos bajo la regulación del promotor de la fosfoglicerato quinasa. Esta estrategia de edición génica se realizó por medio de nucleofección de las TALEN y del donador como ADN plasmídico en fibroblastos embrionarios de ratones con AF-A (Fanca-/-) y en células madre y progenitores hematopoyéticos (CMPHs) de ratones con AFA y ratones sanos (WT). Hemos demostrado por primera vez la actividad de corte de las TALEN diseñadas para cortar el locus Mbs85 de fibroblastos embrionarios AF-A y de CMPHs de ratones con AF-A o sanos (WT). En los clones editados genéticamente de fibroblastos embrionarios AF-A se demostró integración dirigida, así como expresión de hFANCA. Asimismo, se demostró evidencia de corrección fenotípica en estas células por la reversión de su característica hipersensibilidad a mitomicina C (MMC), y también por la disminución de aberraciones cromosómicas inducidas por MMC. Los experimentos de edición génica en CMPHs de ratones con AF-A o sanos (WT) nos permitieron demostrar integración dirigida en estos tipos celulares. Tal y como fue observado en los fibroblastos embrionarios AF-A, se demostraron también evidencias de corrección fenotípica en colonias hematopoyéticas AF-A. En estos experimentos, también observamos una toxicidad acusada debida a la nucleofección de ADN plasmídico que comprometió las propiedades de repoblación de las CMPHs editadas genéticamente en receptores irradiados. En conjunto, nuestros resultados demuestran por primera vez la posibilidad de llevar a cabo una estrategia terapéutica de integración dirigida en un locus seguro de fibroblastos embrionarios y progenitores hematopoyéticos de un modelo de ratón de anemia de Fanconi. Además, nuestros datos muestran la necesidad de limitar la toxicidad asociada a la nucleofección de CMPHs con las construcciones TALEN y los donadores en forma de ADN plasmídico para mejorar el potencial de repoblación de las CMHs editadas genéticamente.Para su ejecución, el trabajo de investigación realizado ha contado con la colaboración de los siguientes Programas de Investigación: Séptimo Programa Marco de la Comisión Europea (Proyecto PERSIST; Ref 222878). Ministerio de Economía y Competitividad (Proyecto SAF 2012-39834). Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III (RETICSRD06/ 0010/0015; RD12/0019/0023). Dirección General de Investigación de la Comunidad de Madrid (Proyecto CellCAM; Ref S2012/BMD-2420). Programa de transferencia de tecnología en el campo de la terapia génica de la Fundación Botín. Marí

Clonal Human Fetal Ventral Mesencephalic Dopaminergic Neuron Precursors for Cell Therapy Research

A major challenge for further development of drug screening procedures, cell replacement therapies and developmental studies is the identification of expandable human stem cells able to generate the cell types needed. We have previously reported the generation of an immortalized polyclonal neural stem cell (NSC) line derived from the human fetal ventral mesencephalon (hVM1). This line has been biochemically, genetically, immunocytochemically and electrophysiologically characterized to document its usefulness as a model system for the generation of A9 dopaminergic neurons (DAn). Long-term in vivo transplantation studies in parkinsonian rats showed that the grafts do not mature evenly. We reasoned that diverse clones in the hVM1 line might have different abilities to differentiate. In the present study, we have analyzed 9 hVM1 clones selected on the basis of their TH generation potential and, based on the number of v-myc copies, v-myc down-regulation after in vitro differentiation, in vivo cell cycle exit, TH+ neuron generation and expression of a neuronal mature marker (hNSE), we selected two clones for further in vivo PD cell replacement studies. The conclusion is that homogeneity and clonality of characterized NSCs allow transplantation of cells with controlled properties, which should help in the design of long-term in vivo experiments. © 2012 Ramos-Moreno et al.Spanish Ministry of Economy and Competitiveness; Comunidad Autónoma Madrid; Instituto Salud Carlos III; European Union (Excell, NMP4-SL-2008 214706); Foundation Ramon ArecesPeer Reviewe

Immunocytochemistry of stemness surface markers.

<p>Staining was performed 48 hours following the last passage. From left to right: staining displayed by hESCs (HS181 line, low and high magnification), hNPCs, <i>v-myc</i> immortalized lines (hNS1, hVM1, and hCTX) and fibroblasts (hFF-1). From top to bottom the markers studied were: SSEA1, SSEA4, TRA1–60 and TRA1–81. Hoechst 33258 nuclear staining is shown in blue. Scale bar represents 100 μm for hESCs in the left hand column (low magnification) and 25 μm for all the other microphotographs.</p

Gene expression pattern of the different cell lines, clustering and PCA analysis.

<p>A) The array data were normalized and a hierarchical clustering was run. On top of the heatmap, the dendrogram shows the clustering among the different samples. The classified list of the genes under study appears on the right side of the heatmap. The Color Bar Expression Levels show the differences in gene expression: black means no differences, green means a high expression, and red means low expression. Grey color mean No Expression (NE). B) Human Stem Cell Pluripotency Array (TaqMan) gene expression data from the different studied cell lines were subjected to a PCA. The graph presents the first, second and third components. For every cell line, the dots represent the value of each sample. To confirm the results of the PCA, a clustering analysis with the dataset of the components was done (right side of the panel). C) PCA analysis of <i>v-myc</i> immortalized cells and non-immortalized hNPCs and the clustering of the PCA data to confirm the similarity between <i>v-myc</i> immortalized and the hNPCs neurospheres according to the three components.</p

Immunocytochemistry of pluripotency markers.

<p>Staining was performed 48 hours following the last passage. From left to right: staining displayed by hESCs (HS181 line, low and high magnifications), hNPCs, <i>v-myc</i> immortalized lines (hNS1, hVM1, and hCTX) and fibroblasts (hFF-1). From top to bottom the markers studied were: NANOG, OCT3/4, SOX2 and AP. Hoechst 33258 nuclear staining is shown in blue. Scale bar represents 100 μm for hESCs in the left hand column (low magnification) and 25 μm for all the other microphotographs.</p

Expression of pluripotency markers and quantification of alkaline phosphatase activity.

<p>mRNA levels were quantified by RT-Q-PCR. NANOG (A), OCT3–4 (B), SOX2 (C) and hTERT (D) expression in the <i>v-myc</i> immortalized cell lines shows significant differences with the HS181 pluripotent cell line. Data are the average ± S.E.M (n = 4). * P<0.05; one-way ANOVA, followed by a <i>post-hoc</i> LSD and Bonferroni test or non parametric Kruskal-Wallis and median test followed by a <i>post-hoc multiple comparisons of mean ranks for all groups</i>. E) Quantification of alkaline phosphatase activity in the cell lines. Data are the average ± S.E.M (n = 3). * P<0.05; non parametric Kruskal-Wallis and median test followed by a <i>post-hoc multiple comparisons of mean ranks for all groups</i>.</p