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 .).

After the first report of induced pluripotent stem cells (iPSCs), considerable efforts have been made to develop more efficient methods for generating iPSCs without foreign gene insertions. Here we show that Sendai virus vector, an RNA virus vector that carries no risk of integrating into the host genome, is a practical solution for the efficient generation of safer iPSCs. We improved the Sendai virus vectors by introducing temperature-sensitive mutations so that the vectors could be easily removed at nonpermissive temperatures. Using these vectors enabled the efficient production of viral/factor-free iPSCs from both human fibroblasts and CD34+ cord blood cells. Temperature-shift treatment was more effective in eliminating remaining viral vector-related genes. The resulting iPSCs expressed human embryonic stem cell markers and exhibited pluripotency. We suggest that generation of transgenefree iPSCs from cord blood cells should be an important step in providing allogeneic iPSC-derived therapy in the future.regenerative medicine | nonintegrating RNA vector

Human induced pluripotent stem cells (hiPSCs) possess the capabilities of self-renewal and differentiation into multiple cell types, and they are free of the ethical problems associated with human embryonic stem cells (hESCs). These characteristics make hiPSCs a promising choice for future regenerative medicine research. There are significant obstacles, however, preventing the clinical use of hiPSCs. One of the most obvious safety issues is the presence of residual undifferentiated cells that have tumorigenic potential. To locate residual undifferentiated cells, in vivo teratoma formation assays have been performed with immunodeficient animals, which is both costly and time-consuming. Here, we examined three in vitro assay methods to detect undifferentiated cells (designated an in vitro tumorigenicity assay): soft agar colony formation assay, flow cytometry assay and quantitative real-time polymerase chain reaction assay (qRT-PCR). Although the soft agar colony formation assay was unable to detect hiPSCs even in the presence of a ROCK inhibitor that permits survival of dissociated hiPSCs/hESCs, the flow cytometry assay using anti-TRA-1-60 antibody detected 0.1% undifferentiated hiPSCs that were spiked in primary retinal pigment epithelial (RPE) cells. Moreover, qRT-PCR with a specific probe and primers was found to detect a trace amount of Lin28 mRNA, which is equivalent to that present in a mixture of a single hiPSC and 5.0×104 RPE cells. Our findings provide highly sensitive and quantitative in vitro assays essential for facilitating safety profiling of hiPSC-derived products for future regenerative medicine research.

Use of clinical-grade human induced pluripotent stem cell (iPSC) lines as a starting material for the generation of cellular therapeutics requires demonstration of comparability of lines derived from different individuals and in different facilities. This requires agreement on the critical quality attributes of such lines and the assays that should be used. Working from established recommendations and guidance from the International Stem Cell Banking Initiative for human embryonic stem cell banking, and concentrating on those issues more relevant to iPSCs, a series of consensus workshops has made initial recommendations on the minimum dataset required to consider an iPSC line of clinical grade, which are outlined in this report. Continued evolution of this field will likely lead to revision of these guidelines on a regular basis.

We investigated the intracellular signaling of OX40, a member of the tumor necrosis factor receptor family. Activation of NF-B in OX40-transfected HSB-2 cells was detected by electrophoretic mobility shift assay within 30 min after the binding of the ligand gp34. In vitro binding experiments showed that tumor necrosis factor receptor-associated factor (TRAF) 1, TRAF2, TRAF3, and TRAF5 but not TRAF4 associated with glutathione S-transferase-OX40 fusion protein. The cotransfection experiments using human embryo kidney cell derived HEK 293T cells showed that TRAF2, TRAF3, and TRAF5 associated with OX40 in vivo. Studies with OX40 deletion mutants demonstrated that the cytoplasmic portion consisting of amino acid sequence 256 -263 (GGSFRTPI) was required for the association with TRAFs and NF-B activation. The introduction of the dominant negative mutants of TRAF2 and TRAF5 into HSB-2-OX40 cells suppressed NF-B activation in a dose-dependent manner. In addition, the introduction of TRAF3 together with the dominant negative mutants of TRAF2 or TRAF5 further reduced NF-B activation. These results indicate that the NF-B activation resulting from OX40 stimulation is mediated by both TRAF2 and TRAF5, and is likely to be negatively modulated by TRAF3.Human OX40 is a 50-kDa cell surface glycoprotein expressed primarily on activated CD4 ϩ T cells and some human T cell leukemia virus type I (HTLV-I) 1 -infected T cell lines, but not on resting peripheral T cells, peripheral B cells, or thymocytes. OX40 was originally described as a cell surface antigen on the activated rat T cells (1). Molecular cloning of its cDNA (1-4) revealed that OX40 is a member of the nerve growth factor receptor/tumor necrosis factor receptor (NGF-R/TNF-R) superfamily which is now known to include low affinity nerve growth factor receptor (p75 NGF-R), tumor necrosis factor receptors (p50/55 TNF-R1 and p75/80 TNF-R2), lymphotoxin-␤ receptor, Fas antigen (CD95/APO-1), CD40, CD30, CD27, and 4-1 BB (5, 6). All the members of this superfamily share a characteristic repeating cysteine-rich motif in the extracellular domain, which is believed to be related to their ability to interact with the TNF-related ligands. The diverse cellular responses such as cell growth, differentiation, and programmed cell death (apoptosis) are triggered by the interaction between the members of the NGF-R/TNF-R superfamily and their ligands.The ligand for human OX40 was also cloned and identified as previously reported gp34, a cell surface protein expressed on HTLV-I-infected T cell lines and subsequently demonstrated to be induced by transactivator p40 tax of HTLV-I (7-9). As expected, the deduced amino acid sequence of gp34 revealed that it is a member of the TNF family. Gp34 has been reported to be expressed on some HTLV-I-infected cell lines such as Hut 102 and MT-2 (10), human umbilical vein endothelial cells (11), and stimulated B lymphoblastoid cell line MSAB (12).Since its first description, OX40 has been known to transmit costimulatory signals to T cells. Recent studies w...

Basic studies of human pluripotential stem cells have advanced rapidly and stem cell products are now seeing therapeutic applications. However, questions remain regarding the tumorigenic potential of such cells. Here, we report the tumorigenic potential of induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) for the treatment of wet-type, age-related macular degeneration (AMD). First, immunodeficient mouse strains (nude, SCID, NOD-SCID and NOG) were tested for HeLa cells’ tumor-forming capacity by transplanting various cell doses subcutaneously with or without Matrigel. The 50% Tumor Producing Dose (TPD50 value) is the minimal dose of transplanted cells that generated tumors in 50% of animals. For HeLa cells, the TPD50 was the lowest when cells were embedded in Matrigel and transplanted into NOG mice (TPD50 = 101.1, n = 75). The TPD50 for undifferentiated iPSCs transplanted subcutaneously to NOG mice in Matrigel was 102.12; (n = 30). Based on these experiments, 1×106 iPSC-derived RPE were transplanted subcutaneously with Matrigel, and no tumor was found during 15 months of monitoring (n = 65). Next, to model clinical application, we assessed the tumor-forming potential of HeLa cells and iPSC 201B7 cells following subretinal transplantation of nude rats. The TPD50 for iPSCs was 104.73 (n = 20) and for HeLa cells 101.32 (n = 37) respectively. Next, the tumorigenicity of iPSC-derived RPE was tested in the subretinal space of nude rats by transplanting 0.8–1.5×104 iPSC-derived RPE in a collagen-lined (1 mm×1 mm) sheet. No tumor was found with iPSC-derived RPE sheets during 6–12 months of monitoring (n = 26). Considering the number of rodents used, the monitoring period, the sensitivity of detecting tumors via subcutaneous and subretinal administration routes and the incidence of tumor formation from the iPSC-derived RPE, we conclude that the tumorigenic potential of the iPSC-derived RPE was negligible.

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The MLL-ELL chimeric gene is the product of the (11;19)(q23p13.1) translocation associated with de novo and therapy-related acute myeloid leukemias (AML). ELL is an RNA polymerase II elongation factor that interacts with the recently identified EAF1 (ELL associated factor 1) protein. EAF1 contains a limited region of homology with the transcriptional activation domains of three other genes fused to MLL in leukemias, AF4, LAF4, and AF5q31. Using an in vitro transformation assay of retrovirally transduced myeloid progenitors, we conducted a structure-function analysis of MLL-ELL. Whereas the elongation domain of ELL was dispensable, the EAF1 interaction domain of ELL was critical to the immortalizing properties of MLL-ELL in vitro. To confirm these results in vivo, we transplanted mice with bone marrow transduced with MLL fused to the minimal EAF1 interaction domain of ELL. These mice all developed AML, with a longer latency than mice transplanted with the wild-type MLL-ELL fusion. Based on these results, we generated a heterologous MLL-EAF1 fusion gene and analyzed its transforming potential. Strikingly, we found that MLL-EAF1 immortalized myeloid progenitors in the same manner as that of MLL-ELL. Furthermore, transplantation of bone marrow transduced with MLL-EAF1 induced AML with a shorter latency than mice transplanted with the MLL-ELL fusion. Taken together, these results indicate that the leukemic activity of MLL-ELL requires the EAF1 interaction domain of ELL, suggesting that the recruitment by MLL of a transactivation domain similar to that in EAF1 or the AF4/LAF4/AF5q31 family may be a critical common feature of multiple 11q23 translocations. In addition, these studies support a critical role for MLL partner genes and their protein-protein interactions in 11q23 leukemogenesis.11q23 translocations occur frequently in hematologic malignancies. The MLL gene spans the 11q23 chromosomal translocation breakpoint and contains significant homology with the Drosophila trithorax gene (9,26,29,32). More than 30 different recurring cytogenetic aberrations that affect the MLL gene have been described (5). The critical feature of these chromosomal rearrangements is the generation of a chimeric transcript consisting of 5Ј MLL and 3Ј sequences of the gene on the partner chromosome. At present, more than 20 MLL partner genes at 11q23 partner chromosomal breakpoints have been cloned (3, 5). The functions of most MLL partner genes are not yet known. Although no consistent homologies or motifs among the partner gene sequences have been identified that might explain how their fusion to MLL results in leukemia, certain groups of partner genes have similar features. These include ENL and AF9, which are serine-and proline-rich and share extensive amino acid homology (15,29). AF4, LAF4, and AF5q31 are also rich in serines and prolines and exhibit homology with ENL and AF9 (8,14,25). AF4, ENL, and AF9 contain transcriptional activation domains with similar properties in reporter gene assays (17,20).The (11;19)(q23;p13.1) transloca...