Modelling Duchenne muscular dystrophy in vitro with newly generated, blood cell-derived induced pluripotent stem cell line ORIONi003-A.

Here, we present newly derived in vitro model for modeling Duchenne muscular dystrophy. Our new cell line was derived by reprogramming of peripheral blood mononuclear cells (isolated from blood from pediatric patient) with Sendai virus encoding Yamanaka factors. Derived iPS cells are capable to differentiate in vitro into three germ layers as verified by immunocytochemistry. When differentiated in special medium, our iPSc formed spontaneously beating cardiomyocytes. As cardiomyopathy is the main clinical complication in patients with Duchenne muscular dystrophy, the cell line bearing the dystrophin gene mutation might be of interest to the research community.

In vitro modeling of amyotrophic lateral sclerosis with induced pluripotent stem cell technology-derived cell line ORIONi002-A.

We present here a new iPS cell line for modeling sporadic form of ALS. Cell line was generated by reprogramming skin fibroblasts isolated with explant culture technology from skin biopsy, donated by ALS patient. For reprogramming, polycistronic self-replicating RNA vector was used and derived iPS cells were characterized by immunocytochemistry and FACS (pluripotent factors expression), karyotyping, STR fingerprinting analysis and in vitro differentiation assay. New cell line showed normal (46, XY) karyotype and differentiated in vitro into cells from three germ layers. STR analysis proved the origin and originality of the cell line.

Establishment of PANDA - a new human pancreatic ductal adenocarcinoma cell line with 3D cell culture technology.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive type of malignancy with one of the worst prognoses amongst any type of cancer. Surgery is applicable only to the limited number of patients with locally resectable tumors and currently represents the only curative treatment option. Treatment with chemotherapy and radiotherapy can only extend patient survival. Despite advances in conventional therapies, the five-year survival of PDAC remained largely unchanged. New in vitro and in vivo models are therefore urgently needed to investigate this type of cancer. Here, we present the establishment and characterization of a novel pancreatic cancer cell line, isolated from a patient with PDAC. Cell line abbreviated as PANDA (PANncreatic Ductal Adenocarcinoma) was established with an optimized 3D culture protocol published previously by our group. The new cancer cell line "PANDA" represents a novel in vitro approach for PDAC cancer research and new therapy testing.

Generation of ORIONi001-A induced pluripotent stem cell line for in vitro modeling of sporadic form of amyotrophic lateral sclerosis.

We generated new in vitro model for sporadic form of amyotrophic lateral sclerosis by reprogramming isolated skin fibroblasts into iPSCs. Fibroblasts were reprogrammed with commercially available synthetic polycistronic, self-replicating RNA vector. As verified by FISH, an early passages of a new iPSC line showed mosaic karyotype (cells with normal and abnormal karyotype 46,XY,t(2;14)(q13;p12) were present), while late passages contained only cells with abnormal karyotype. New iPSCs differentiated into all three germ layers and formed a teratoma in nude mice. Our iPSC line represents a new model for therapy testing and drug development in the field of ALS research.

Fluoro Jade-B detection of dying cells in the SVZ and RMS of adult rats after bilateral olfactory bulbectomy.

A novel fluorochrome, Fluoro-Jade B, was used to detect dying precursor cells in the subventricular zone (SVZ) and rostral migratory stream (RMS) of adult rats after bilateral olfactory bulbectomy and in control intact rats. The animals in experimental group were left to survive 3 days and from 3 till 16 months after surgical procedure. 1. In the control animals, Fluoro-Jade B positive cells were visible in the SVZ and within the whole extent of the RMS. The number of Fluoro-Jade B positive cells increased in the elbow in comparison to the rest parts of the RMS. 2. In the experimental animals surviving either 3 days or from 3 till 16 months after bilateral olfactory bulbectomy, Fluoro-Jade B positive cells displayed the similar pattern of distribution as in the control animals. However, some quantitative differences in the labeled cells number along the rostral migratory pathway appeared. 3. The average number of degenerating cells within the control SVZ and RMS was 26.24+/- 0.686. In bulbectomized animals, regardless of survival time, an insignificant increase of Fluoro-Jade B positive cells number occurred. We can conclude that dying of precursor cells is a physiological process running within the SVZ/RMS in both control and experimental animals. Moreover, this physiological process is not influenced by survival period after bilateral olfactory bulbectomy. Our results demonstrate Fluoro-Jade B as a useful marker of dying cells.

Age-related changes of NADPH-diaphorase positivity in the rat rostral migratory stream.

Accumulating evidence confirms that nitric oxide (NO), a versatile diffusible signaling molecule, contributes to controling of adult neurogenesis. We have previously shown the timing of NADPH-diaphorase (NADPH-d) positivity within the rat rostral migratory stream (RMS) during the first postnatal month. The present study was designed to describe further age-related changes of NO presence in this neurogenic region. The presence of NO synthesizing cells in the RMS was shown by NADPH-d histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry. The phenotypic identity of nitrergic cells was examined by double labeling with GFAP and NeuN. Systematic qualitative and quantitative analysis of NADPH-d-positive cells was performed in the neonatal (P14), adult(5 months) and aging (20 months) rat RMS. 1. Nitrergic cells with different distribution pattern and morphological characteristics were present in the RMS at all ages examined. In neonatal animals, small, moderately stained NADPH-d-positive cells were identified in the RMS vertical arm and in the RMS elbow. In adult and aging rats a few labeled cells could be also detected in the RMS horizontal arm. NADPH-d-positive cells in adult and aging rats were characterized by long varicose processes and displayed dark labeling in comparison to the neonatal group. 2. Double immunolabeling has revealed that nNOS-immunoreactivity co-localized with that of NeuN. This indicates that nitrergic cells within the RMS are neurons. 3. Quantitative analysis showed that the number of NADPH-d-positive cells increases with advancing age. The presence of NO producing cells in the RMS of neonatal adult and aging rats indicates, that this proliferating and migratory area is under the influence of NO throughout the entire life of the animals.