SUMMARY Human pluripotent stem cells (hPSCs) are potential sources of cells for modeling disease and development, drug discovery, and regenerative medicine. However, it is important to identify factors that may impact the utility of hPSCs for these applications. In an unbiased analysis of 205 hPSC and 130 somatic samples, we identified hPSC-specific epigenetic and transcriptional aberrations in genes subject to X chromosome inactivation (XCI) and genomic imprinting, which were not corrected during directed differentiation. We also found that specific tissue types were distinguished by unique patterns of DNA hypomethylation, which were recapitulated by DNA demethylation during in vitro directed differentiation. Our results suggest that verification of baseline epigenetic status is critical for hPSC-based disease models in which the observed phenotype depends on proper XCI or imprinting, and that tissue-specific DNA methylation patterns can be accurately modeled during directed differentiation of hPSCs, even in the presence of variations in XCI or imprinting.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system mediated by CD4+ T cells reactive with myelin basic protein (MBP). Rats were rendered resistant to the induction of EAE by vaccination with synthetic peptides corresponding to idiotypic determinants of the beta chain VDJ region and J alpha regions of the T cell receptor (TCR) that are conserved among encephalitogenic T cells. These findings demonstrate the utility of TCR peptide vaccination for modulating the activity of autoreactive T cells and represent a general therapeutic approach for T cell-mediated pathogenesis.

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Objective. Notch signaling is implicated in the repression of mesenchymal stem cell (MSC) chondrogenic differentiation. The purpose of this study was to examine the mechanism of this repression and how it is modulated to permit chondrogenesis.Methods. Notch intracellular domain (NICD) protein levels were monitored via Western blotting throughout chondrogenic differentiation of human MSCs in pellet cultures. Overexpression of Notch signaling components and their effect on chondrogenesis was achieved by transfecting plasmids coding for NICD, HES-1, and HERP-2/HEY-1. COL2A1 and AGGRECAN expression was monitored via quantitative polymerase chain reaction analysis. Chromatin immunoprecipitation (ChIP) was used to test whether HES-1 and HEY-1 bind putative N-box domains in intron 1 of COL2A1.Results. High levels of NICD proteins were reduced during chondrogenesis of human MSCs, and this was mediated by transforming growth factor ␤3 (TGF␤3). COL2A1 gene expression was repressed following overexpression of NICD (2-fold) and HES-1 (3-fold) and was markedly repressed by overexpression of HEY-1 (80-fold). HEY-1 repressed AGGRECAN expression 10-fold, while NICD and HES-1 had no effect. We identified 2 putative N-box domains adjacent to, and part of, the SOX9 enhancer binding site located in intron 1 of COL2A1. ChIP studies showed that endogenous HES-1 and HEY-1 bound to these sites.Transducin-like enhancer, the HES-1 corepressor protein, was displaced during chondrogenic differentiation and following TGF␤3 treatment.Conclusion. These results reveal novel mechanisms by which Notch signaling represses gene expression. Notch signaling proteins act on the SOX9 binding site in the COL2A1 enhancer and prevent SOX9-mediated transcriptional activation of COL2A1 and, thus, chondrogenic differentiation.

Antiphospholipid antibodies, including anti-

Antiphospholipid antibodies (aPL), including antibodies detected in anti-cardiolipin (aCL) enzyme-linked immunosorbent assays and in lupus anticoagulant (LA) tests, are strongly associated with recurrent thrombosis and recurrent fetal loss, i.e. the antiphospholipid syndrome (APS). Although recent studies suggest that most APS-associated aCL are directed against the phospholipid (PL)-binding plasma protein beta2-glycoprotein 1 (beta2GP1), the precise nature of aCL binding specificities remains controversial. To address the issue of aCL specificity we generated five new monoclonal IgG aCL from two patients with APS. Characterization of these five aCL, as well as two previously published IgG aCL, revealed three patterns of reactivity: (1) four antibodies reacted strongly with human beta2GP1-cardiolipin (CL) complexes and weakly with human beta2GP1 alone; (2) two antibodies recognized bovine beta2GP1, but not human beta2GP1; (3) one antibody reacted with complexes of human beta2GP1 and CL, but not with human beta2GP1 alone. Only one monoclonal displayed weak LA activity. These patient-derived IgG monoclonal antibodies, and additional ones to be generated, may help define varying species of antibodies detected in aCL assays and identify the specific antibodies that may be pathogenic.

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SummaryAlthough much has been learned about the molecular basis of immunoglobulin M (IgM) rheumatoid factors (KFs) in healthy individuals and in patients with mixed cryoglobulinemia and rheumatoid arthritis, little is known about the genetic origins of the potentially pathogenic IgG RFs in the inflamed rheumatoid synovia of patients. Recently, we generated from unmanipulated synovium B cells several hybridomas that secreted self-associating IgG KFs. To delineate the genetic origins of such potentially pathogenic RFs, we adapted the anchored polymerase chain reaction to rapidly clone and characterize the expressed Ig V genes for the L1 and the D1 IgG RFs. Then, we identified the germline counterparts of the expressed L1 IgG KF V genes. The results showed that the L1 heavy chain was encoded by a Vh gene that is expressed preferentially during early ontogenic development, and that is probably located within 240 kb upstream of the Jh locus. The overlap between this KF Vh gene and the restricted fetal antibody repertoire is reminiscent of the natural antibody-associated Vh genes, and suggests that at least part of the "potential pathogenic" IgG RFs in rheumatoid synovium may derive from the "physiological" natural antibody repertoire in a normal immune system. Indeed, the corresponding germline Vh gene for L1 encodes the heavy chain of an IgM KF found in a 19-wk-old fetal spleen. Furthermore, the comparisons of the expressed ILF V genes and their germline counterparts reveal that the L1 heavy and light chain variable regions had, respectively, 16 and 7 somatic mutations, which resulted in eight and four amino acid changes. Strikingly, all eight mutations in the complementarity determining regions of the V gene-encoded regions were replacement changes, while only 6 of 11 mutations in the framework regions caused amino acid changes. Combined with Ll's high binding affinity toward the Fc fragment, these results suggest strongly that the L1 IgG KF must have been driven by the Fc antigen.