High frequency targeted mutagenesis using engineered endonucleases and DNA-end processing enzymes.

Targeting DNA double-strand breaks is a powerful strategy for gene inactivation applications. Without the use of a repair plasmid, targeted mutagenesis can be achieved through Non-Homologous End joining (NHEJ) pathways. However, many of the DNA breaks produced by engineered nucleases may be subject to precise re-ligation without loss of genetic information and thus are likely to be unproductive. In this study, we combined engineered endonucleases and DNA-end processing enzymes to increase the efficiency of targeted mutagenesis, providing a robust and efficient method to (i) greatly improve targeted mutagenesis frequency up to 30-fold, and; (ii) control the nature of mutagenic events using meganucleases in conjunction with DNA-end processing enzymes in human primary cells.

The first reported generation of several induced pluripotent stem cell lines from homozygous and heterozygous Huntington's disease patients demonstrates mutation related enhanced lysosomal activity.

Neuronal disorders, like Huntington's disease (HD), are difficult to study, due to limited cell accessibility, late onset manifestations, and low availability of material. The establishment of an in vitro model that recapitulates features of the disease may help understanding the cellular and molecular events that trigger disease manifestations. Here, we describe the generation and characterization of a series of induced pluripotent stem (iPS) cells derived from patients with HD, including two rare homozygous genotypes and one heterozygous genotype. We used lentiviral technology to transfer key genes for inducing reprogramming. To confirm pluripotency and differentiation of iPS cells, we used PCR amplification and immunocytochemistry to measure the expression of marker genes in embryoid bodies and neurons. We also analyzed teratomas that formed in iPS cell-injected mice. We found that the length of the pathological CAG repeat did not increase during reprogramming, after long term growth in vitro, and after differentiation into neurons. In addition, we observed no differences between normal and mutant genotypes in reprogramming, growth rate, caspase activation or neuronal differentiation. However, we observed a significant increase in lysosomal activity in HD-iPS cells compared to control iPS cells, both during self-renewal and in iPS-derived neurons. In conclusion, we have established stable HD-iPS cell lines that can be used for investigating disease mechanisms that underlie HD. The CAG stability and lysosomal activity represent novel observations in HD-iPS cells. In the future, these cells may provide the basis for a powerful platform for drug screening and target identification in HD.

Induced pluripotent stem cell lines from Huntington's disease mice undergo neuronal differentiation while showing alterations in the lysosomal pathway.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an excessive expansion of a CAG trinucleotide repeat in the gene encoding the protein huntingtin, resulting in an elongated stretch of glutamines near the N-terminus of the protein. Here we report the derivation of a collection of 11 induced pluripotent stem (iPS) cell lines generated through somatic reprogramming of fibroblasts obtained from the R6/2 transgenic HD mouse line. We show that CAG expansion has no effect on reprogramming efficiency, cell proliferation rate, brain-derived neurotrophic factor level, or neurogenic potential. However, genes involved in the cholesterol biosynthesis pathway, which is altered in HD, are also affected in HD-iPS cell lines. Furthermore, we found a lysosomal gene upregulation and an increase in lysosome number in HD-iPS cell lines. These observations suggest that iPS cells from HD mice replicate some but not all of the molecular phenotypes typically observed in the disease; additionally, they do not manifest increased cell death propensity either under self-renewal or differentiated conditions. More studies will be necessary to transform a revolutionary technology into a powerful platform for drug screening approaches.

Comparison of gene expression pattern in SP cell populations from four tissues to define common "stemness functions".

The goal of our study was to identify a subset of genes commonly expressed in Side Populations (SP), isolated by Hoechst staining followed by flow cytometry, from adult mouse bone marrow, male adult germinal cells, muscle primary culture, and mesenchymal cells. These SP cells have been proposed to be a "stem-like" population and are used here as a "model" that may reveal mechanisms which would be relevant for a better understanding of stem cell properties. Transcriptional profiles for SP and the more differentiated non-SP cells isolated from the four tissues were compared by hybridization on microarray using a common external reference. Among the 503 genes differentially expressed, which discriminate SP and non-SP cells in all the tissues, the genes upregulated in SP cells are implicated in the quiescent status of the cells, the maintenance of their pluripotency and the capacity to undergo asymmetric division. These genes may be responsible for the decision for self-renewal of these cells, whereas the repression of lineage-affiliated genes in SP cells could be responsible for their undifferentiated state. These genes, acting in concert, may be the key players that mediate the mechanisms that control stem cell functions, and our results suggest that we have identified common "stemness functions" of these "stem-like" cells.

Global transcriptional characterization of SP and MP cells from the myogenic C2C12 cell line: effect of FGF6.

With the use of Hoechst staining techniques, we have previously shown that the C2C12 myogenic cell line contains a side population (SP) that is largely increased in the presence of fibroblast growth factor 6 (FGF6). Here, we compared transcriptional profiles from SP and main population (MP) cells from either C2C12 or FGF6-expressing C2C12. Expression profiles of SPs show that these cells are less differentiated than MPs and display some similarities to stem cells. Moreover, principal component analysis made it possible to distinguish specific contributions of either FGF6 or differentiation effects on gene expression profiles. This demonstrated that FGF6-expanded SPs were similar to parental C2C12-derived SPs. Conversely, FGF6-treated MPs differed from parental MPs and were more related to SP cells. These results show that FGF6 pushed committed myogenic cells toward a more immature phenotype resulting in the accumulation of cells with a SP phenotype. We propose that FGF6 conditioning could provide a way to expand the pool of immature cells by myoblast dedifferentiation.