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Outcomes have not improved for metastatic osteosarcoma for several decades. In part, this failure to develop better therapies stems from a lack of understanding of osteosarcoma biology, given the rarity of the disease and the high genetic heterogeneity at the time of diagnosis. We report here the successful establishment of a new human osteosarcoma cell line, COS-33, from a patient-derived xenograft and demonstrate retention of the biological features of the original tumor. We found high mTOR signaling activity in the cultured cells, which were sensitive to a small molecule inhibitor, rapamycin, a suppressor of the mTOR pathway. Suppressed mTOR signaling after treatment with rapamycin was confirmed by decreased phosphorylation of the S6 ribosomal protein. Increasing concentrations of rapamycin progressively inhibited cell proliferation in vitro. We observed significant inhibitory effects of the drug on cell migration, invasion, and colony formation in the cultured cells. Furthermore, we found that only a strong osteogenic inducer, bone morphogenetic protein-2, promoted the cells to differentiate into mature mineralizing osteoblasts, indicating that the COS-33 cell line may have impaired osteoblast differentiation. Grafted COS-33 cells exhibited features typical of osteosarcoma, such as production of osteoid and tumorigenicity in vivo. In addition, we revealed that the COS-33 cell line retained a complex karyotype, a homozygous deletion of the TP53 gene, and typical histological features from its original tumor. Our novel cellular model may provide a valuable platform for studying the etiology and molecular pathogenesis of osteosarcoma as well as for testing novel drugs for future genome-informed targeted therapy.

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The most highly studied endocytic pathway, clathrin-dependent endocytosis, mediates a wide range of fundamental processes including nutrient internalization, receptor recycling, and signal transduction. In order to model tissue specific and developmental aspects of this process, CRISPR/Cas9 genomic editing was utilized to fluorescently label the C-terminus of clathrin light chain A (CLTA) within the phenotypically normal, parental CRMi001-A human induced pluripotent stem cell line. Successfully edited cells were isolated by fluorescently activated cell sorting, remained karyotypically normal, and maintained their differentiation potential. This cell line facilitates imaging of endogenous clathrin trafficking within varied cell types.

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Although it has been speculated that proteasome dysfunction may contribute to the pathogenesis of Huntington's disease (HD), a devastating neurodegenerative disorder, how proteasome activity is regulated in HD affected stem cells and somatic cells remains largely unclear. To better understand the pathogenesis of HD, we analyzed proteasome activity and the expression of FOXO transcription factors in three wild-type (WT) and three HD induced-pluripotent stem cell (iPSC) lines. HD iPSCs exhibited elevated proteasome activity and higher levels of FOXO1 and FOXO4 proteins. Knockdown of FOXO4 but not FOXO1 expression decreased proteasome activity. Following neural differentiation, the HD-iPSC-derived neural progenitor cells (NPCs) demonstrated lower levels of proteasome activity and FOXO expressions than their WT counterparts. More importantly, overexpression of FOXO4 but not FOXO1 in HD NPCs dramatically enhanced proteasome activity. When HD NPCs were further differentiated into DARPP32-positive neurons, these HD neurons were more susceptible to death than WT neurons and formed Htt aggregates under the condition of oxidative stress. Similar to HD NPCs, HD-iPSC-derived neurons showed reduced proteasome activity and diminished FOXO4 expression compared to WT-iPSC-derived neurons. Furthermore, HD iPSCs had lower AKT activities than WT iPSCs, whereas the neurons derived from HD iPSC had higher AKT activities than their WT counterparts. Inhibiting AKT activity increased both FOXO4 level and proteasome activity, indicating a potential role of AKT in regulating FOXO levels. These data suggest that FOXOs modulate proteasome activity, and thus represents a potentially valuable therapeutic target for HD.

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Duplications of the long arm of chromosome 6 have been previously reported in a limited number of patients; however, most reported duplications encompass regions of chromosome 6 distal to band q21. Duplications restricted to the proximal portion of 6q are rare. We report an 8-year-old male with a 16.4 megabase (Mb) tandem duplication of chromosome 6q14.1q16.1 (chr6:78950191-95395865; hg19) who exhibited dysmorphic facial features, seizures, global developmental delay, intellectual disability, autism spectrum disorder, sensorineural hearing loss, and immune deficiency. This patient refines and potentially expands the current, poorly-characterized phenotype associated with duplication of this proximal 6q region. We recommend a low threshold for a hearing evaluation beyond newborn screening and for pursuing an immune work-up in patients with similar 6q duplications. © 2016 Wiley Periodicals, Inc.

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