TRAF2 recruitment via T61 in CD30 drives NFκB activation and enhances hESC survival and proliferation.

CD30 (TNFRSF8), a tumor necrosis factor receptor family protein, and CD30 variant (CD30v), a ligand-independent form encoding only the cytoplasmic signaling domain, are concurrently overexpressed in transformed human embryonic stem cells (hESCs) or hESCs cultured in the presence of ascorbate. CD30 and CD30v are believed to increase hESC survival and proliferation through NFκB activation, but how this occurs is largely unknown. Here we demonstrate that hESCs that endogenously express CD30v and hESCs that artificially overexpress CD30v exhibit increased ERK phosphorylation levels, activation of the canonical NFκB pathway, down-regulation of the noncanonical NFκB pathway, and reduced expression of the full-length CD30 protein. We further find that CD30v, surprisingly, resides predominantly in the nucleus of hESC. We demonstrate that alanine substitution of a single threonine residue at position 61 (T61) in CD30v abrogates CD30v-mediated NFκB activation, CD30v-mediated resistance to apoptosis, and CD30v-enhanced proliferation, as well as restores normal G2/M-checkpoint arrest upon H2O2 treatment while maintaining its unexpected subcellular distribution. Using an affinity purification strategy and LC-MS, we identified TRAF2 as the predominant protein that interacts with WT CD30v but not the T61A-mutant form in hESCs. The identification of Thr-61 as a critical residue for TRAF2 recruitment and canonical NFκB signaling by CD30v reveals the substantial contribution that this molecule makes to overall NFκB activity, cell cycle changes, and survival in hESCs.

Integration-free induced pluripotent stem cells model genetic and neural developmental features of down syndrome etiology.

Down syndrome (DS) is the most frequent cause of human congenital mental retardation. Cognitive deficits in DS result from perturbations of normal cellular processes both during development and in adult tissues, but the mechanisms underlying DS etiology remain poorly understood. To assess the ability of induced pluripotent stem cells (iPSCs) to model DS phenotypes, as a prototypical complex human disease, we generated bona fide DS and wild-type (WT) nonviral iPSCs by episomal reprogramming. DS iPSCs selectively overexpressed chromosome 21 genes, consistent with gene dosage, which was associated with deregulation of thousands of genes throughout the genome. DS and WT iPSCs were neurally converted at >95% efficiency and had remarkably similar lineage potency, differentiation kinetics, proliferation, and axon extension at early time points. However, at later time points DS cultures showed a twofold bias toward glial lineages. Moreover, DS neural cultures were up to two times more sensitive to oxidative stress-induced apoptosis, and this could be prevented by the antioxidant N-acetylcysteine. Our results reveal a striking complexity in the genetic alterations caused by trisomy 21 that are likely to underlie DS developmental phenotypes, and indicate a central role for defective early glial development in establishing developmental defects in DS brains. Furthermore, oxidative stress sensitivity is likely to contribute to the accelerated neurodegeneration seen in DS, and we provide proof of concept for screening corrective therapeutics using DS iPSCs and their derivatives. Nonviral DS iPSCs can therefore model features of complex human disease in vitro and provide a renewable and ethically unencumbered discovery platform.

Generation of a human embryonic stem cell line stably expressing high levels of the fluorescent protein mCherry.

AIM: The generation and characterization of a human embryonic stem cell (hESC) line stably expressing red fluorescent mCherry protein. METHODS: Lentiviral transduction of a ubiquitously-expressed human EF-1α promoter driven mCherry transgene was performed in MEL2 hESC. Red fluore-scence was assessed by immunofluorescence and flow cytometry. Pluripotency of stably transduced hESC was determined by immunofluorescent pluripotency marker expression, flow cytometry, teratoma assays and embryoid body-based differentiation followed by reverse transcriptase-polymerase chain reaction. Quantification of cell motility and survival was performed with time lapse microscopy. RESULTS: Constitutively fluorescently-labeled hESCs are useful tools for facile in vitro and in vivo tracking of survival, motility and cell spreading on various surfaces before and after differentiation. Here we describe the generation and characterization of a hESC line (MEL2) stably expressing red fluorescent protein, mCherry. This line was generated by random integration of a fluorescent protein-expressing cassette, driven by the ubiquitously-expressed human EF-1α promoter. Stably transfected MEL2-mCherry hESC were shown to express pluripotency markers in the nucleus (POU5F1/OCT4, NANOG and SOX2) and on the cell surface (SSEA4, TRA1-60 and TG30/CD9) and were shown to maintain a normal karyotype in long-term (for at least 35 passages) culture. MEL2-mCherry hESC further readily differentiated into representative cell types of the three germ layers in embryoid body and teratoma based assays and, importantly, maintained robust mCherry expression throughout differentiation. The cell line was next adapted to single-cell passaging, rendering it compatible with numerous bioengineering applications such as measurement of cell motility and cell spreading on various protein modified surfaces, quantification of cell attachment to nanoparticles and rapid estimation of cell survival. CONCLUSION: The MEL2-mCherry hESC line conforms to the criteria of bona fide pluripotent stem cells and maintains red fluorescence throughout differentiation, making it a useful tool for bioengineering and in vivo tracking experiments.