The use of human pluripotent stem cells (hPSCs) in cell therapy is hindered by the tumorigenic risk from residual undifferentiated cells. Here we performed a high-throughput screen of over 52,000 small molecules and identified 15 pluripotent cell-specific inhibitors (PluriSIns), nine of which share a common structural moiety. The PluriSIns selectively eliminated hPSCs while sparing a large array of progenitor and differentiated cells. Cellular and molecular analyses demonstrated that the most selective compound, PluriSIn #1, induces ER stress, protein synthesis attenuation, and apoptosis in hPSCs. Close examination identified this molecule as an inhibitor of stearoyl-coA desaturase (SCD1), the key enzyme in oleic acid biosynthesis, revealing a unique role for lipid metabolism in hPSCs. PluriSIn #1 was also cytotoxic to mouse blastocysts, indicating that the dependence on oleate is inherent to the pluripotent state. Finally, application of PluriSIn #1 prevented teratoma formation from tumorigenic undifferentiated cells. These findings should increase the safety of hPSC-based treatments.

A hallmark feature of myosin-II is that it can spontaneously self-assemble into bipolar synthetic thick filaments (STFs) in low ionic strength buffers, thereby serving as a reconstituted in-vitro model for muscle thick filament. While these STFs have been extensively used for structural characterization, their functional evaluation has been limited.  In this report, we show that myosins in STFs mirror the more electrostatic and cooperative interactions that underlie the energy-sparing super-relaxed (SRX) state, which are not seen using shorter myosin sub-fragments, heavy meromyosin (HMM) and myosin subfragment-1 (S1). Using these STFs, we show several pathophysiological insults in hypertrophic cardiomyopathy, including the R403Q myosin mutation, phosphorylation of myosin light chains, and increased ADP:ATP ratio destabilize the SRX population. Furthermore, wild-type myosin containing STFs, but not S1, HMM, or STFs-containing R403Q myosin, recapitulated the ADP-induced destabilization of the SRX state. Studies involving a clinical-stage small molecule inhibitor, mavacamten, showed that it is not only more effective in increasing myosin SRX population in STFs than in S1 or HMM ,  but it also increases myosin SRX population equally well in STFs made of healthy and disease-causing R403Q myosin. Importantly, we also found that pathophysiological perturbations   such as elevated ADP concentration weakens the mavacamten’s ability to increase the myosin SRX population, suggesting that mavacamten-bound myosin heads are not permanently protected in the SRX state but can be recruited   into action. These findings collectively emphasize that STFs serve as a valuable tool to provide novel insights into the myosin SRX state in healthy, disease, and therapeutic conditions.

A small molecule inhibitor of NF-B-dependent cytokine expression was discovered that blocked tumor necrosis factor (TNF) ␣-induced IB␣ degradation in MM6 cells but not the degradation of ␤-catenin in Jurkat cells. Ro106-9920 blocked lipopolysaccharide (LPS)-dependent expression of TNF␣, interleukin-1␤, and interleukin-6 in fresh human peripheral blood mononuclear cells with IC 50 values below 1 M. Ro106-9920 also blocked TNF␣ production in a dose-dependent manner following oral administration in two acute models of inflammation (air pouch and LPS challenge). Ro106-9920 was observed to inhibit an ubiquitination activity that does not require ␤TRCP but associates with IB␣ and will ubiquitinate IB␣ S32E,S36E (IB␣ee) specifically at lysine 21 or 22. Ro106-9920 was identified in a cell-free system as a time-dependent inhibitor of IB␣ee ubiquitination with an IC 50 value of 2.3 ؎ 0.09 M. The ubiquitin E3 ligase activity is inhibited by cysteine-alkylating reagents, supported by E2UBCH7, and requires cIAP2 or a cIAP2-associated protein for activity. These activities are inconsistent with what has been reported for SCF ␤TRCP , the putative E3 for IB␣ ubiquitination. Ro106-9920 was observed to be selective for IB␣ee ubiquitination over the ubiquitin-activating enzyme (E1), E2UBCH7, nonspecific ubiquitination of cellular proteins, and 97 other molecular targets. We propose that Ro106-9920 selectively inhibits an uncharacterized but essential ubiquitination activity associated with LPSand TNF␣-induced IB␣ degradation and NF-B activation.

Osteoarthritis (OA) is a degenerative joint disease that is characterized by localized inflammatory and secondary proliferative changes. Suppressor of cytokine signaling 3 (SOCS3) is elevated during OA development. We investigated the effects of this protein on human chondrocyte survival in OA and the inflammatory response together with the mechanisms of these effects. Small interfering RNA (siRNA) was used to knock down the expression of SOCS3 in interleukin(IL)-1β-induced primary human osteoarthritic chondrocytes. We found that siRNA-mediated SOCS3 knock-down in human osteoarthritic chondrocytes increased production of IL-1β-induced prostaglandin E, cell growth, transcript level and nuclear translocation of cyclin D1. Silencing of SOCS3 resulted in altered expression of nuclear factor-kappa-B (NF-κB) and cyclooxygenase (COX2). Our findings indicate that enhanced SOCS3 could have contradictory influences on OA development. SOCS3 might protect damaged joints by its anti-inflammatory effect and by inhibition of over-augmented cartilage tissue repair, which could exhibit inhibitory properties for joint inflammation, abnormal chondrocyte clustering and osteophyte formation in OA. On the other hand, SOCS3 might reduce chondrocyte growth response, which would delay repair of subchondral cancellous bone damage in OA owing to its anti-proliferation effect. The anti-inflammation and growth inhibition effects exhibited by enhanced SOCS3 in OA appear to be related to its capacity to down-regulate expression levels of NF-κB and COX2.

Cell-cell and cell-substrate interactions alter the contractile behaviour of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cells are often cultured as a mono-layer on rigid tissue culture plastic (TCP) or glass, in contrast to the flexible extracellular matrix of myocardium. We examine the contractile behaviour of hiPSC-CMs on a range of substrates including standard TCP to determine the influence on the time course and synchronicity of cell contraction. HiPSC-CM (Ncardia Cor.4U or CDI iCell 2 ) were seeded on day À2 on either fibronectin-coated TCP or fibronectin-coated recombinant collagen-like polypeptide-based hydrogel (Fujifilm, Tilburg NL) with an estimated stiffness of 10kPa. Spontaneous contractility was monitored using video recordings made on days 0, 1, 3, 5 and 7 (D0-7). To assess the contractile behaviour, video frames (100fps) were subdivided into a 30x30 grid, and each grid square analysed using the MUSCLEMOTION algorithm (Sala et al., 2018). The % of single peaked-transients and the 10 th -90 th percentile difference (IP90) of contraction duration at 50% amplitude (CD50-IP90) and contraction start time (TStart-IP90) values were analysed. Values are reported as means5SD, and groups were compared to each other. Contraction synchronicity (IP90 TStart) and duration (IP90 CD50) in cultures on TCP and hydrogel were not significantly different. However, the percentage of grid squares with single-peaked transients was less in the TCP group (80.150.7% on D1 decreasing to 60.953.9% on D7) than on hydrogel (98.650.1% on D1 decreasing to 84.4513.1% on D7). The time course associated with cultures on TCP was unphysiological and indicates that the substrate is not satisfactory for hiPSC-CMs. In contrast, a single-peaked twitch contraction observed routinely on the collagen hydrogel is consistent with the normal contraction-relaxation cycle in individual cardiomyocytes. These results underline the importance of a suitable substrate for hiPSC-CM culture.