Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder mainly affecting electric motor neurons. from the selective lack of motoneurons in the cerebral cortex, brainstem, and spinal-cord, resulting in atrophy of limb, axial, and respiratory muscle groups [1]. Mutations in superoxide dismutase-1 (SOD-1) take into account about 20% of familial ALS individuals [2], [3]. SOD1G93A mice can be a approved model for the ALS study broadly, which communicate mutant G93A of human being SOD-1 and develop medical symptoms just like those observed in ALS individuals [4]. Motoneurons from SOD1G93A mice could provide some provided info to review the system of ALS [5], [6]. A powerful way to obtain motoneurons carrying the genes responsible for this condition would help understand the causes of motoneuron death in ALS and develop new therapeutics for the disease. Recently, somatic cells can be reprogrammed to a pluripotent state through viral transduction of four transcription factors Oct4, Sox2, c-Myc, and Klf4 [7]C[9]. The induced pluripotent stem (iPS) cells were indistinguishable from ES cells in proliferative and developmental potential, and they can differentiate into derivatives of all germ layers. Several protocols have been developed to induce iPS cells to efficiently differentiate into neurons [10]C[14]. However, it remains unknown whether iPS cells with genetic deficiency possess neuronal differentiation potential similar to normal cells lines. In this study, we compared the neuronal differentiation potential between iPS cells derived from SOD1G93A mice and iPS cells derived from normal C57BL/6 mice and investigated whether SOD1 mutations could influence the neuronal differentiation, especially motoneuron generation from iPS cells. Results of the present study would provide evidence on the possibility of the efficient generation of motoneurons from iPS cells with SOD mutations. Results Generation and characterization of iPS cells from tail-tip fibroblasts Totally 6 iPS cell lines were generated by retroviral expression of mouse Oct4, Sox2, c-Myc, and Klf4 from B6SJL-TgN TTFs and C57BL/6 TTFs for characterization and comparison, in which 3 iPS cell lines were derived from 3 transgenic B6SJL-TgN mice (ALS-iPS) and 3 iPS cell line were derived from 3 C57BL/6 mice (C57-iPS) (Figs. 1A and 1C). To confirm that these iPS cells exhibit ES-like properties, we examined some ES cell markers that included alkaline phosphatase (AP) activity and ES cell-specific transcription factors Oct4 and SSEA-1. Results shown in Figs. 1B and 1D demonstrated that the iPS clones exhibited high AP activity. The selected iPS clones were Flurizan also shown to be positive for Oct4 and SSEA-1 (Figs. 2A and 2B). To assess the gene expression pattern of the iPS clones, we isolated RNA from iPS cells and the result indicated that the endogenous Oct4, Sox2, c-Myc, Klf4, and Nanog were expressed which confirmed activation of these loci. Results shown in Fig. 2C demonstrated that the transgenes of selected clones from both ALS-iPS-1 and C57-iPS-12 cells were silenced. Importantly, all analyzed iPS clones induced expression from the endogenous Oct4, Sox2, and Nanog loci, and none of these genes were expressed in the original TTF fibroblasts, further supporting of successful reprogramming. Karyotype analyses demonstrated that all analyzed ALS-iPS-1 clones (Fig. 2G) and C57-iPS-12 clones (data not shown) exhibited a Rabbit polyclonal to ZNF404 normal karyotype. Open in a separate home window Shape 1 Establishment of mouse iPS cell lines from SOD1G93A C57BL/6 and mice mice.(A) Phase comparison image demonstrates iPS cells from SOD1G93A mice (ALS-iPS-1) grew as colonies about mitomycin-treated Flurizan MEF feeder cells. (B) These clones exhibited high AP activity. (C) Stage contrast image demonstrates iPS cells from C57BL/6 mice grew as colonies on mitomycin-treated MEF feeder cells. (D) These clones exhibited high AP activity. Size pub: 500 m. Open up in another window Shape 2 Immunostaining demonstrates the founded Flurizan iPS cell range (ALS-iPS-1) was positive for Oct4 (A) and SSEA-1 (B). (C) The manifestation patterns of pluripotent genes in iPS clones, E14 cells, and fibroblasts. The full total outcomes exposed that examined iPS clones induced manifestation through the endogenous Oct4, Sox2, and Nanog loci, and non-e of the genes were indicated in the initial TTF fibroblasts. (DCF) Teratoma produced from ALS-iPS-1 cells included cells owned by all three germ levels, including endoderm-derived glandular (D), mesoderm-derived cartilage cells (E), and ectoderm-derived neural pipes (F). Karyotype analyses proven that ALS-iPS-1 clones demonstrated a standard karyotype (G). Size pub: 100 m inside a and B; 250 m in D, E, and F. To verify the.