In order to apply human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to regenerative medicine, the cells should be produced under restricted conditions conforming to GMP guidelines. Since the conventional culture system has some issues that need to be addressed to achieve this goal, we developed a novel culture system. We found that recombinant laminin-511 E8 fragments are useful matrices for maintaining hESCs and hiPSCs when used in combination with a completely xeno-free (Xf) medium, StemFit™. Using this system, hESCs and hiPSCs can be easily and stably passaged by dissociating the cells into single cells for long periods, without any karyotype abnormalities. Human iPSCs could be generated under feeder-free (Ff) and Xf culture systems from human primary fibroblasts and blood cells, and they possessed differentiation abilities. These results indicate that hiPSCs can be generated and maintained under this novel Ff and Xf culture system.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have the potential to provide an infinite source of tissues for regenerative medicine. Although defined xeno-free media have been developed, culture conditions for reliable propagation of hESCs still require considerable improvement. Here we show that recombinant E8 fragments of laminin isoforms (LM-E8s), which are the minimum fragments conferring integrin-binding activity, promote greater adhesion of hESCs and hiPSCs than do Matrigel and intact laminin isoforms. Furthermore, LM-E8s sustain long-term self-renewal of hESCs and hiPSCs in defined xeno-free media with dissociated cell passaging. We successfully maintained three hESC and two hiPSC lines on LM-E8s in three defined media for 10 passages. hESCs maintained high level expression of pluripotency markers, had a normal karyotype after 30 passages and could differentiate into all three germ layers. This culture system allows robust proliferation of hESCs and hiPSCs for therapeutic applications.

Anti-p200 pemphigoid has been characterized by autoantibodies to an unidentified 200-kDa protein (p200) of the dermal؊epidermal junction. The objective of this study was to identify p200. We performed 2D gel electrophoresis of dermal extracts and immunoblotting with patients' sera, followed by MS analysis of a unique protein band. The protein band corresponded to laminin ␥1. Anti-laminin ␥1 mAb reacted with the anti-p200 immunoprecipitates by immunoblotting. Sera from 32 patients with anti-p200 pemphigoid showed 90% reactivity to the recombinant products of laminin ␥1. None of the healthy control sera reacted with laminin ␥1. By immunoblotting, reactivity of a patient's serum with p200 was competitively inhibited by adding anti-laminin ␥1 C-terminus mAb. Purified anti-p200 IgG also inhibited the reactivity of this mAb to dermal laminin ␥1. Most laminin ␥1-positive sera showed reactivity with recombinant laminin ␥1 C-terminal E8 fragment. Reactivity of patients' sera and purified IgG to dermal laminin ␥1 was higher than reactivity to blood vessel laminin ␥1 under reducing conditions. These results suggest that laminin ␥1 is the autoantigen for patients with anti-p200 pemphigoid. The autoantibodies may specifically recognize dermal laminin ␥1 with unique posttranslational modifications. The epitope is localized to the 246 C-terminal amino acids within the coiled-coil domain. The 9 C-terminal residues are known to be critically involved in laminin recognition by integrins.autoimmune disease ͉ basement membrane ͉ bullous pemphigoid ͉ proteomics