Computational comparative analysis identifies potential stemness-related markers for mesenchymal stromal/stem cells.

Mesenchymal stromal/stem cells (MSCs) are multipotent cells that reside in multiple tissues are capable of self-renewal and differentiation into various cell types. These properties make them promising candidates for regenerative therapies. MSC identification is critical in yielding pure populations for successful therapeutic applications; however, the criteria for MSC identification proposed by the International Society for Cellular Therapy (ISCT) are inconsistent across different tissue sources. This study aimed to identify potential markers to be used together with the ISCT criteria to provide a more accurate means of MSC identification. Thus, we carried out a computational comparative analysis of the gene expression in human and mouse MSCs derived from multiple tissues to identify the differentially expressed genes that are shared between the two species. We show that six members of the proteasome degradation system are similarly expressed across MSCs derived from bone marrow, adipose tissue, amnion, and umbilical cord. Additionally, with the help of predictive models, we found that the expression profile of these genes correctly validated the identity of the MSCs across all the tissue sources tested. Moreover, using genetic interaction networks, we showed a possible link between these genes and antioxidant enzymes in the MSC antioxidant defense system, thereby pointing to their potential role in prolonging the life span of MSCs. According to our findings, members of the proteasome degradation system may serve as stemness-related markers.

LC3A Silencing Hinders Aggresome Vimentin Cage Clearance in Primary Choroid Plexus Carcinoma.

Aggresomes are transient microtubule-dependent inclusion bodies that sequester misfolded proteins and are ultimately removed by autophagy. Here we report the generation of a choroid plexus carcinoma cell line; Children's Cancer Hospital Egypt (CCHE)-45, which is characterized by the constitutive formation of aggresomes. When examining the autophagy pathway as the main route for aggresomes clearance, CCHE-45 cells displayed increased autophagy flux mediated by MAP1LC3B. MAP1LC3A-Variant1 gene expression was silenced by promoter methylation. Restoring MAP1LC3A-Variant1 expression resulted in the formation of MAP1LC3A positive autophagosmes and the disruption of the aggresomes' vimentin cage independent of MAP1LC3B positive autophagosomes. Our data supports the notion that basal quality control autophagy and vimentin cage clearance in CCHE-45 are mediated by MAP1LC3A. Hence we propose that absence of MAP1LC3A disrupts the autophagic pathway and leads to the failure of aggresome vimentin cage degradation. Consequently, this could represent a targetable pathway in autophagy-dependent cancers.

Enrichment, Propagation, and Characterization of Mouse Testis-Derived Mesenchymal Stromal Cells.

The therapeutic potential of multipotent stromal cells (MSCs) largely depends on the isolation and expansion methods used. In this study, we propose a laminin-based technique to select and enrich for MSCs isolated from the mouse testis. Primary cell cultures were prepared from juvenile mouse testes and the capacity to generate colony forming units together with population doubling time (PDT) during expansion were determined. The identity of MSCs was assayed using reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry for the active expression of cell surface markers, such as CD44, CD73, and CD29; absence of the CD45 hematopoietic cell marker; and in vitro differentiation of the cells into osteoblasts and adipocytes. Testis-derived MSCs (tMSCs) displayed self-renewal properties and in the early passages, exhibited high proliferation patterns with an average PDT of 44.1 hours. The lack of Vasa expression implied that the tMSCs were not of germ cell origin. The RT-PCR data, which were confirmed by immunophenotyping, revealed high expression of CD44 and the absence of CD45 expression in tMSCs. The strong Alizarin Red stain in tMSCs that were stimulated into making bone cells was indicative of the presence of calcium-producing cells (osteoblasts). Likewise, the adipogenic potential of tMSCs was demonstrated based on Oil Red O staining of lipid vacuoles in differentiated cells. Loss of fibroblast-like morphology in late passage cells along with the increase in PDT and the decrease in the mRNA levels of CD73 and CD29 suggested that the tMSCs developmental program is reformed at this stage.

Incidence of kiaa1549-braf fusion gene in Egyptian pediatric low grade glioma.

BACKGROUND: Low grade gliomas are the most common brain tumor in children. Tandem duplication involving the KIAA1549 and the BRAF kinase genes results in a gene fusion that has been recently characterized in a subset of low grade glioma While there is no clear evidence that the KIAA1549-BRAF gene fusion has an effect on prognosis, it is an attractive target for therapy development and as a diagnostic tool. METHODS: In the current study we examine the prevalence of KIAA1549-BRAF gene fusion in pediatric patients diagnosed with low grade glioma in the Egyptian population and its relationship to clinical and histological subtypes. Sixty patients between the ages of 1 to 18 years were analyzed for the presence of KIAA1549-BRAF fusion gene products using reverse transcription-PCR and sequencing. The clinicopathologic tumor characteristics were then analyzed in relation to the different fusion genes. RESULTS: KIAA1549-BRAF fusion genes were detected in 56.6% of patients. They were primarily associated with pilocytic astrocytoma (74.2%) and pilomyxoid astrocytoma (60%). Translocation 15-9 was the most common, representing (55.8%) of all positive samples followed by 16-9 (26.4%) and 16-11 (8.8%). Pilocytic astrocytomas presented primarily with 15-9 (32.2%), 16-9 (25.8%) and 16-11 (6.4%) while pilomyxoid astrocytomas presented with 15-9 (46.6%), 16-9 (6.6%) and 16-11 (6.6%) translocations. CONCLUSION: Gene fusion is found to be significantly increased in cerebellar pilocytic astrocytoma tumors. Furthermore, 15-9 was found to have a higher incidence among our cohort compared to previous studies. While most of the gene fusion positive pilomyxoid astrocytomas were 15-9, we find the association none significant.