Gene expression profiling in equine polysaccharide storage myopathy revealed inflammation, glycogenesis inhibition, hypoxia and mitochondrial dysfunctions

<p>Abstract</p> <p>Background</p> <p>Several cases of myopathies have been observed in the horse Norman Cob breed. Muscle histology examinations revealed that some families suffer from a polysaccharide storage myopathy (PSSM). It is assumed that a gene expression signature related to PSSM should be observed at the transcriptional level because the glycogen storage disease could also be linked to other dysfunctions in gene regulation. Thus, the functional genomic approach could be conducted in order to provide new knowledge about the metabolic disorders related to PSSM. We propose exploring the PSSM muscle fiber metabolic disorders by measuring gene expression in relationship with the histological phenotype.</p> <p>Results</p> <p>Genotypying analysis of GYS1 mutation revealed 2 homozygous (AA) and 5 heterozygous (GA) PSSM horses. In the PSSM muscles, histological data revealed PAS positive amylase resistant abnormal polysaccharides, inflammation, necrosis, and lipomatosis and active regeneration of fibers. Ultrastructural evaluation revealed a decrease of mitochondrial number and structural disorders. Extensive accumulation of an abnormal polysaccharide displaced and partially replaced mitochondria and myofibrils. The severity of the disease was higher in the two homozygous PSSM horses.</p> <p>Gene expression analysis revealed 129 genes significantly modulated (p < 0.05). The following genes were up-regulated over 2 fold: IL18, CTSS, LUM, CD44, FN1, GST01. The most down-regulated genes were the following: mitochondrial tRNA, SLC2A2, PRKCα, VEGFα. Data mining analysis showed that protein synthesis, apoptosis, cellular movement, growth and proliferation were the main cellular functions significantly associated with the modulated genes (p < 0.05). Several up-regulated genes, especially IL18, revealed a severe muscular inflammation in PSSM muscles. The up-regulation of glycogen synthase kinase-3 (GSK3β) under its active form could be responsible for glycogen synthase (GYS1) inhibition and hypoxia-inducible factor (HIF1α) destabilization.</p> <p>Conclusion</p> <p>The main disorders observed in PSSM muscles could be related to mitochondrial dysfunctions, glycogenesis inhibition and the chronic hypoxia of the PSSM muscles.</p

Derivation of induced pluripotent stem cells from an infertile boar carrying a reciprocal translocation

International audienc

330 Derivation of porcine induced pluripotent stem cells from fibroblasts of a translocated azoospermic boar

International audienceChromosomal rearrangements have a crucial impact on the proper proceedings of meiosis and can lead by several mechanisms to the production of unbalanced gametes or to the complete arrest of gametes production. To assess the impact of these rearrangements in the early development of pig germ cells, we proposed to generate a library of stem cells from infertile boars that are carriers of chromosomal abnormalities as a new tool for the development of an in vitro differentiation system from pluripotent stem cells to germ cells. We report here the reprogramming of fibroblasts from an azoospermic boar carrying a reciprocal translocation t(Y:14) by integrative or nonintegrative viral overexpression of Oct4, Sox2, Klf4, and c-Myc. The iPS cell lines were characterised for pluripotency, cell cycle, and differentiation potential by conventional methods. Genomic stability was analysed by G-banding karyotype, comparative genomic hybridization, and FISH. The porcine iPS-like cell lines harbored characteristics of ground and naïve pluripotency when cultured in specific media. They expressed several pluripotency genes and harbored an ES-like cell cycle. Nevertheless, contrary to mouse and human iPS, they did not silence the integrated exogenes, leading to a poor differentiation potential. Moreover, cytogenetic analysis revealed a high genomic instability upon passaging, which suggests the development of a population with an increased selective advantage. We characterised the selected duplications and compared them to those previously described in other species. In contrast, the nonintegrative reprogrammation system gives us promising results regarding differentiation potential and genomic stability and will bring new insights into the molecular factors controlling and maintaining pluripotency in the pig species

Non integrative strategy decreases chromosome instability and improves endogenous pluripotency genes reactivation in porcine induced pluripotent-like stem cells

International audienceThe pig is an emerging animal model, complementary to rodents for basic research and for biomedical and agronomical purposes. However despite the progress made on mouse and rat models to produce genuine pluripotent cells, it remains impossible to produce porcine pluripotent cell lines with germline transmission. Reprogramming of pig somatic cells using conventional integrative strategies remains also unsatisfactory. In the present study, we compared the outcome of both integrative and nonintegrative reprogramming strategies on pluripotency and chromosome stability during pig somatic cell reprogramming. The porcine cell lines produced with integrative strategies express several pluripotency genes but they do not silence the integrated exogenes and present a high genomic instability upon passaging. In contrast, pig induced pluripotent-like stem cells produced with non-integrative reprogramming system (NI-iPSLCs) exhibit a normal karyotype after more than 12 months in culture and reactivate endogenous pluripotency markers. Despite the persistent expression of exogenous OCT4 and MYC, these cells can differentiate into derivatives expressing markers of the three embryonic germ layers and we propose that these NI-iPSLCs can be used as a model to bring new insights into the molecular factors controlling and maintaining pluripotency in the pig and other non-rodent mammalians