Objectives: To establish a straightforward single-cell passaging cultivation method that enables high-quality maintenance of human induced pluripotent stem cells without the appearance of karyotypic abnormalities or loss of pluripotency. Methods: Cells were kept in culture for over 50 passages, following a structured chronogram of passage and monitoring cell growth by population doubling time calculation and cell confluence. Standard procedures for human induced pluripotent stem cells monitoring as embryonic body formation, karyotyping and pluripotency markers expression were evaluated in order to assess the cellular state in long-term culture. Cells that underwent these tests were then subjected to differentiation into keratinocytes, cardiomyocytes and definitive endoderm to evaluate its differentiation capacity. Results: Human induced pluripotent stem cells clones maintained its pluripotent capability as well as chromosomal integrity and were able to generate derivatives from the three germ layers at high passages by embryoid body formation and high-efficient direct differentiation into keratinocytes, cardiomyocytes and definitive endoderm. Conclusions: Our findings support the routine of human induced pluripotent stem cells single-cell passaging as a reliable procedure even after long-term cultivation, providing healthy human induced pluripotent stem cells to be used in drug discovery, toxicity, and disease modeling as well as for therapeutic approaches.

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Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype of kidney cancer. This carcinoma is histologically characterized by the presence of clear and abundant cytoplasm. In the present study, we sought to identify genes differentially expressed in ccRCC and build a molecular profile of this cancer. We selected genes described in the literature related to cellular differentiation and proliferation. We analyzed the gene and protein expression by quantitative PCR (qPCR) and immunohistochemistry, respectively, and examined possible epigenetic mechanisms that regulate their expression in ccRCC samples and cell lines. Occludin (OCLN) and growth arrest-specific 1 (GAS1) genes were underexpressed in ccRCC, and we report that miR-122 and miR-34a, respectively, may regulate their expression in this cancer. Furthermore, we showed by qPCR and immunohistochemistry that solute carrier family 2 member 1 (SLC2A1) was significantly overexpressed in ccRCC. The set of genes identified in the present study furthers our understanding of the molecular basis and development of ccRCC.

In spite of the great advance in human induced pluripotent stem cells knowledge, several nonconsensual protocols to cultivate this peculiar cell type can be found in the literature. Laboratories and companies worldwide have been trying to provide equivalent results regarding long-term cultivation of hiPSCs and their derivatives, so it is mandatory the establishment of reproducible pipelines for cell generation, cultivation, differentiation, etc. Here, we validated a straightforward single-cell passaging cultivation method that enabled high-quality maintenance of human induced pluripotent stem cells (hiPSCs) over 50 passages without the appearance of karyotypic abnormalities or loss of pluripotency. Further, the hiPSC clones were able to generate derivatives from the three germ layers at high passages by embryoid body formation and high-efficient direct differentiation into keratinocytes, cardiomyocytes and definitive endoderm (DE). Thus, our findings support the routine of hiPSCs single-cell passaging as a reliable procedure even after long-term cultivation, providing healthy PSCs to be used in high-standard cellular modeling research and therapeutic approaches.

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Astrocytoma is the most common and aggressive tumor of the central nervous system. Genetic and environmental factors, bacterial infection, and several other factors are known to be involved in gliomagenesis, although the complete underlying molecular mechanism is not fully understood. Tumorigenesis is a multistep process involving initiation, promotion, and progression. We present a human model of malignant astrocyte transformation established by subjecting primary astrocytes from healthy adults to four sequential cycles of forced anchorage impediment (deadhesion). After limiting dilution of the surviving cells obtained after the fourth deadhesion/readhesion cycle, three clones were randomly selected, and exhibited malignant characteristics, including increased proliferation rate and capacity for colony formation, migration, and anchorage-independent growth in soft agar. Functional assay results for these clonal cells, including response to temozolomide, were comparable to U87MG—a human glioblastoma-derived cell lineage—reinforcing malignant cell transformation. RNA-Seq analysis by next-generation sequencing of the transformed clones relative to the primary astrocytes revealed upregulation of genes involved in the PI3K/AKT and Wnt/β-catenin signaling pathways, in addition to upregulation of genes related to epithelial–mesenchymal transition, and downregulation of genes related to aerobic respiration. These findings, at a molecular level, corroborate the change in cell behavior towards mesenchymal-like cell dedifferentiation. This linear progressive model of malignant human astrocyte transformation is unique in that neither genetic manipulation nor treatment with carcinogens are used, representing a promising tool for testing combined therapeutic strategies for glioblastoma patients, and furthering knowledge of astrocytoma transformation and progression.

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