We assessed the feasibility of transplanting a sheet of retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs) in a patient with neovascular age-related macular degeneration. The iPSCs were generated from skin fibroblasts obtained from two patients with advanced neovascular age-related macular degeneration and were differentiated into RPE cells. The RPE cells and the iPSCs from which they were derived were subject to extensive testing. A surgery that included the removal of the neovascular membrane and transplantation of the autologous iPSC-derived RPE cell sheet under the retina was performed in one of the patients. At 1 year after surgery, the transplanted sheet remained intact, best corrected visual acuity had not improved or worsened, and cystoid macular edema was present. (Funded by Highway Program for Realization of Regenerative Medicine and others; University Hospital Medical Information Network Clinical Trials Registry [UMIN-CTR] number, UMIN000011929 .).

bTo develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wildtype strain required approximately 1 g of biotin per liter for normal growth, the bioY disruptant (⌬bioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ⌬bioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ⌬bioY strain. By selectively using the resulting two strains (⌬bioB and ⌬bioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 g to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 g per liter).

SummaryThe first-in-human trial of induced pluripotent stem cell (iPSC)-based autologous transplantation was successfully performed on a female patient with age-related macular degeneration. Here we delineated the base-resolution methylome of the iPSC-derived retinal pigment epithelium (iRPE) used in this trial. The methylome of iRPE closely resembled that of native RPE (nRPE), although partially methylated domains (PMDs) emerged in iRPE but not nRPE. Most differentially methylated regions between iRPE and nRPE appeared to originate from (de)methylation errors during differentiation, whereas errors at reprogramming resulted in aberrant genomic imprinting and X chromosome reactivation. Moreover, non-CpG methylation was prominent in nRPE but not iRPE. Intriguingly, xenotransplantation to mouse remodeled the iRPE methylome to demethylate a subset of suppressed genes and accumulate non-CpG methylation, but failed to resolve PMDs and hypermethylated CpG islands. Although the impacts of these alterations remain elusive, our findings should provide a useful guide for methylome analyses of other iPSC-derived cells.

The migration of Caenorhabditis elegans gonadal distal tip cells (DTCs) offers an excellent model to study the migration of epithelial tubes in organogenesis. mig-18 mutants cause meandering or wandering migration of DTCs during gonad formation, which is very similar to that observed in animals with mutations in mig-17, which encodes a secreted metalloprotease of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family. MIG-18 is a novel secreted protein that is conserved only among nematode species. The mig-17(null) and mig-18 double mutants exhibited phenotypes similar to those in mig-17(null) single mutants. In addition, the mutations in fbl-1/fibulin-1 and let-2/collagen IV that suppress mig-17 mutations also suppressed the mig-18 mutation, suggesting that mig-18 and mig-17 function in a common genetic pathway. The Venus-MIG-18 fusion protein was secreted from muscle cells and localized to the gonadal basement membrane, a tissue distribution reminiscent of that observed for MIG-17. Overexpression of MIG-18 in mig-17 mutants and vice versa partially rescued the relevant DTC migration defects, suggesting that MIG-18 and MIG-17 act cooperatively rather than sequentially. We propose that MIG-18 may be a cofactor of MIG-17/ADAMTS that functions in the regulation of the gonadal basement membrane to achieve proper direction of DTC migration during gonadogenesis.T HE ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family of the secreted zinc metalloproteases has important roles in development. Nineteen ADAMTS genes have been identified in the human genome, and mutations in many result in hereditary diseases that are related to disorders of the extracellular matrix (Apte 2009). The functions of ADAMTS-5, -9, and -20 are required for digit formation, and ADAMTS-9 and -20 are needed for closure of the palate in mice (McCulloch et al. 2009;Enomoto et al. 2010). ADAMTS-5 and -15 act in myoblast fusion (Stupka et al. 2013). However, the precise roles of ADAMTS proteases in development still remain elusive.Among five ADAMTS genes in Caenorhabditis elegans, gon-1 and mig-17 play essential roles in the development of the somatic gonad (Blelloch and Kimble 1999;Nishiwaki et al. 2000). GON-1 is required for active migration of gonadal distal tip cells (DTCs), whereas MIG-17 acts in the directional control of DTC migration. Genetic suppressor analyses of mig-17 mutants identified dominant gain-of-function (gf) mutations in two genes that encode basement membrane proteins, FBL-1C/ fibulin-1C and LET-2/a2 subunit of collagen IV (Kubota et al. 2004(Kubota et al. , 2008. The suppressor fbl-1(gf) mutations result in substitutions of evolutionarily conserved amino acids within the second EGF-like motif of FBL-1C. FBL-1C is recruited to the gonadal basement membrane by MIG-17 activity, where it is likely to be required for directional control of DTC migration (Kubota et al. 2004). The suppression by fbl-1(gf) mutations depends on NID-1/nidogen, a basement membrane protein et al. ...

No abstract