Human embryonic stem cells (hESCs) have two properties of interest for the development of cell therapies: self-renewal and the potential to differentiate into all major lineages of somatic cells in the human body. Widespread clinical application of hESC-derived cells will require culture methods that are low-cost, robust, scalable and use chemically defined raw materials. Here we describe synthetic peptide-acrylate surfaces (PAS) that support self-renewal of hESCs in chemically defined, xeno-free medium. H1 and H7 hESCs were successfully maintained on PAS for over ten passages. Cell morphology and phenotypic marker expression were similar for cells cultured on PAS or Matrigel. Cells on PAS retained normal karyotype and pluripotency and were able to differentiate to functional cardiomyocytes on PAS. Finally, PAS were scaled up to large culture-vessel formats. Synthetic, xeno-free, scalable surfaces that support the self-renewal and differentiation of hESCs will be useful for both research purposes and development of cell therapies.

KLF8 (Krüppel-like factor 8) is a member of the Krüppel transcription factor family that binds CACCC elements in DNA and activates or represses their target genes in a context-dependent manner. Here we present sumoylation as a novel mechanism that regulates KLF8 post-translationally. We found that KLF8 can be covalently modified by small ubiqitin-like modifier (SUMO)-1, SUMO-2, and SUMO-3 in vivo. We showed that KLF8 interacted with the PIAS family of SUMO E3 ligases PIAS1, PIASy, and PIASx␣ but not with E2 SUMO-conjugating enzyme Ubc9. Furthermore, we demonstrated that the E2 and E3 ligases enhanced the sumoylation of KLF8. In addition, site-directed mutagenesis identified lysine 67 as the major sumoylation site on KLF8. Lysine 67 to arginine mutation strongly enhanced activity of KLF8 as a repressor or activator to its physiological target promoters and as an inducer of the G 1 cell cycle progression. Taken together, our results demonstrated that sumoylation of KLF8 negatively regulates its transcriptional activity and cellular functions.Post-translational modifications of proteins, such as phosphorylation, acetylation, methylation, ubiquitination, and sumoylation, play crucial roles in many cellular processes due to their ability to cause rapid changes in the conformation and functions of preexisting proteins. Small ubiqitin-like modifier (SUMO) 2 -1, -2, and -3 are ubiquitin-like proteins that can be covalently attached to a large number of proteins through the formation of isopeptide bonds between the C terminus of mature SUMOs and the -amino group of a lysine in the acceptor proteins (1, 2). The conjugated forms of SUMO-2 and SUMO-3 differ from one another only by three N-terminal residues. They form a distinct subfamily known as SUMO-2/3 and are 50% identical in sequence to SUMO-1 (2, 3). Sumoylation of proteins proceeds via a multi-enzymatic pathway that shares similarity with the ubiquitin-conjugation system but uses a SUMO-specific enzymatic machinery (4).Like ubiquitin, SUMOs are expressed in inactive precursors that have to be processed by SUMO-specific proteases to expose a C-terminal double glycine motif that is required for SUMO conjugation. The processed form of SUMO is specifically activated in an ATP-dependent manner by an E1-activating enzyme consisting of an SAE1 (AOS1)-SAE2 (UBA2) heterodimer. Activated SUMO is transferred to Ubc9, the E2-conjugating enzyme, and is subsequently attached to the -amino group of a specific lysine in the target protein (4, 5). In Saccharomyces cerevisiae, sumoylation involves an E3 ligase (e.g. Siz1 and Siz2) for ligation of SUMO to its substrates. In contrast, E3 enzyme is not required for the addition of SUMO to its target proteins in mammals, although specific E3 ligases can promote the conjugation of SUMO from the E2 to its target protein (6 -8). To date, three types of SUMO E3 ligases, the nucleoporin RanBP2 (4, 9), the PIAS proteins (10), and the polycomb group protein Pc2 (11), have been described. The PIAS proteins were initially described as protein inh...

Human induced pluripotent stem cells have the potential to become an unlimited cell source for cell replacement therapy. The realization of this potential, however, depends on the availability of culture methods that are robust, scalable, and use chemically defined materials. Despite significant advances in hiPSC technologies, the expansion of hiPSCs relies upon the use of animal-derived extracellular matrix extracts, such as Matrigel, which raises safety concerns over the use of these products. In this work, we investigated the feasibility of expanding and differentiating hiPSCs on a chemically defined, xeno-free synthetic peptide substrate, i.e. Corning Synthemax® Surface. We demonstrated that the Synthemax Surface supports the attachment, spreading, and proliferation of hiPSCs, as well as hiPSCs’ lineage-specific differentiation. hiPSCs colonies grown on Synthemax Surfaces exhibit less spread and more compact morphology compared to cells grown on Matrigel™. The cytoskeleton characterization of hiPSCs grown on the Synthemax Surface revealed formation of denser actin filaments in the cell-cell interface. The down-regulation of vinculin and up-regulation of zyxin expression were also observed in hiPSCs grown on the Synthemax Surface. Further examination of cell-ECM interaction revealed that hiPSCs grown on the Synthemax Surface primarily utilize αvβ5 integrins to mediate attachment to the substrate, whereas multiple integrins are involved in cell attachment to Matrigel. Finally, hiPSCs can be maintained undifferentiated on the Synthemax Surface for more than ten passages. These studies provide a novel approach for expansion of hiPSCs using synthetic peptide engineered surface as a substrate to avoid a potential risk of contamination and lot-to-lot variability with animal derived materials.

FIP200 (focal adhesion kinase [FAK] family interacting protein of 200 kD) is a newly identified protein that binds to the kinase domain of FAK and inhibits its kinase activity and associated cellular functions. Here, we identify an interaction between FIP200 and the TSC1–TSC2 complex through FIP200 binding to TSC1. We found that association of FIP200 with the TSC1–TSC2 complex correlated with its ability to increase cell size and up-regulate S6 kinase phosphorylation but was not involved in the regulation of cell cycle progression. Conversely, knockdown of endogenous FIP200 by RNA interference reduced S6 kinase phosphorylation and cell size, which required TSC1 but was independent of FAK. Furthermore, overexpression of FIP200 reduced TSC1–TSC2 complex formation, although knockdown of endogenous FIP200 by RNA interference did not affect TSC1–TSC2 complex formation. Lastly, we showed that FIP200 is important in nutrient stimulation-induced, but not energy- or serum-induced, S6 kinase activation. Together, these results suggest a cellular function of FIP200 in the regulation of cell size by interaction with the TSC1–TSC2 complex.

FIP200 is a novel protein inhibitor for focal adhesion kinase (FAK), which binds to FAK directly and inhibits its kinase activity and associated cellular functions, such as cell adhesion, spreading, and motility in fibroblasts. Here we show that FIP200 inhibits G 1 -S phase progression, proliferation, and clonogenic survival in human breast cancer cells. Consistent with the G 1 arrest induced by FIP200, we found that FIP200 increased p21 and decreased cyclin D1 protein levels in breast cancer cells. In addition, FIP200 significantly induced p21 promoter activity in MCF-7 cells and this response was abolished upon deletion of p53 binding sites within p21 promoter. Furthermore, we found that FIP200 could interact with exogenous and endogenous p53 protein and significantly increase its half-life compared with the control cells. We also found that the NH 2 -terminal 154 residues of FIP200 were sufficient to mediate p53 interaction and G 1 arrest in cells. The increase in p53 half-life correlated with the increased phosphorylation at Ser 15 and decreased proteasomal degradation via ubiquitin and Hdm2-independent mechanism. Stabilization of p53 by FIP200 could be partially reversed by NQO1 inhibitor, dicoumarol. In contrast to p53, FIP200 decreased cyclin D1 protein half-life by promoting proteasome-dependent degradation of cyclin D1. In summary, our results suggest that FIP200 increases p21 protein levels via stabilization of its upstream regulator p53 and decreases cyclin D1 protein by promoting its degradation. Both effects are critical for FIP200-induced G 1 arrest and may contribute to the putative antitumor activities of FIP200 in breast cancer. (Cancer Res 2005; 65(15): 6676-84)