RESUMO
Background/objective: The purpose of this study was to report the outcomes of a clinical trial conducted in Japan to assess the safety and effectiveness of third-generation autologous chondrocyte implantation (ACI) using IK-01 (CaReS™), which does not require flap coverage, in the treatment of patients with focal cartilage injury of the knee. Methods: This was an open label, exploratory clinical trial. Patients were enrolled between June 2012 and September 2016. The primary endpoint of the study was the International Knee Documentation Committee (IKDC) score at 52 weeks after implantation. The IKDC, Lysholm, and visual analog scale (VAS) scores were evaluated at the time of screening and at 4, 12, 24, 36, and 52 weeks after implantation. Improvements from the baseline scores were evaluated using the equation "(postoperative score) - (preoperative score)." Magnetic resonance imaging (MRI) was performed at 2, 12, 24, and 52 weeks after implantation, and MRI measurements were evaluated using T1 rho and T2 mapping. Results: Nine patients were enrolled in this study and were examined for safety. Product quality did not satisfy the specification in one patient, and bacterial joint infection occurred in one patient. As a result, seven patients were included in the outcome analyses. The mean IKDC score significantly improved from 36.4 preoperatively to 74.1% at 52 weeks after implantation (p < 0.0001). The mean Lysholm and VAS scores also significantly improved from 39.6 to 57.4 to 89.6 and 22.9, respectively, after surgery. In the MRI evaluation, the T1 rho and T2 values of the implanted area were similar to those of the surrounding cartilage at 52 weeks after implantation. Conclusions: Third generation ACI (IK-01) can be an effective treatment option for focal cartilage defects of the knee; however, surgeons must pay careful attention to the risk of postoperative joint infection.
RESUMO
Stevens-Johnson syndrome, ocular cicatricial pemphigoid, and severe thermal or chemical injury are considered severe ocular surface disorders (OSDs) because they affect the entire ocular surface, including corneal and conjunctival epithelial stem cells. In patients with severe OSDs, the long-term prognosis for limbal transplantation is poor, and the related corneal opacity and cicatrization lead to devastating visual impairment. To date, there is no standardized treatment to improve vision in cases with severe OSD. Investigating novel treatment methods for severe OSDs, our group began cultivated oral mucosal epithelial transplantation in 2002 and developed a limbal-supported rigid-type contact lens that can be applied as a nonsurgical treatment. When used in combination, these treatment methods make it possible to successfully restore vision in cases with severe OSDs.
Assuntos
Queimaduras Químicas/terapia , Lentes de Contato , Células Epiteliais/transplante , Queimaduras Oculares/induzido quimicamente , Mucosa Bucal/citologia , Penfigoide Mucomembranoso Benigno/terapia , Síndrome de Stevens-Johnson/terapia , Queimaduras Químicas/fisiopatologia , Células Cultivadas , Terapia Combinada , Doenças da Córnea/fisiopatologia , Doenças da Córnea/terapia , Epitélio Corneano/citologia , Epitélio Corneano/transplante , Queimaduras Oculares/fisiopatologia , Humanos , Penfigoide Mucomembranoso Benigno/fisiopatologia , Transplante de Células-Tronco , Síndrome de Stevens-Johnson/fisiopatologia , Transtornos da Visão/reabilitação , Acuidade Visual/fisiologiaRESUMO
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 .).
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Degeneração Macular/terapia , Epitélio Pigmentado da Retina/citologia , Idoso , Técnicas de Cultura de Células , Diferenciação Celular , Estudos de Viabilidade , Feminino , Fibroblastos , Humanos , Masculino , Epitélio Pigmentado da Retina/transplante , Transplante AutólogoRESUMO
Human Pluripotent Stem Cell (PSC)-derived cell therapy holds enormous promise because of the cells' "unlimited" proliferative capacity and the potential to differentiate into any type of cell. However, these features of PSC-derived cell products are associated with concerns regarding the generation of iatrogenic teratomas or tumors from residual immature or non-terminally differentiated cells in the final cell product. This concern has become a major hurdle to the introduction of this therapy into the clinic. Tumorigenicity testing is therefore a key preclinical safety test in PSC-derived cell therapy. Tumorigenicity testing becomes particularly important when autologous human induced Pluripotent Stem Cell (iPSC)-derived cell products with no immuno-barrier are considered for transplantation. There has been, however, no internationally recognized guideline for tumorigenicity testing of PSC-derived cell products for cell therapy. In this review, we outline the points to be considered in the design and execution of tumorigenicity tests, referring to the tests and laboratory work that we have conducted for an iPSC-derived retinal pigment epithelium (RPE) cell product prior to its clinical use.
RESUMO
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 (TPD50â=â10(1.1), nâ=â75). The TPD50 for undifferentiated iPSCs transplanted subcutaneously to NOG mice in Matrigel was 10(2.12); (nâ=â30). Based on these experiments, 1×10(6) iPSC-derived RPE were transplanted subcutaneously with Matrigel, and no tumor was found during 15 months of monitoring (nâ=â65). 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 10(4.73) (nâ=â20) and for HeLa cells 10(1.32) (nâ=â37) respectively. Next, the tumorigenicity of iPSC-derived RPE was tested in the subretinal space of nude rats by transplanting 0.8-1.5×10(4) 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 (nâ=â26). 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.
Assuntos
Carcinogênese , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/transplante , Degeneração Macular/patologia , Degeneração Macular/terapia , Epitélio Pigmentado da Retina/citologia , Transplante de Células-Tronco/efeitos adversos , Animais , Feminino , Células HeLa , Humanos , Camundongos , RatosRESUMO
We show that pigment epithelium-derived factor (PEDF), which is secreted from primary or iPSC-derived retinal pigment epithelium (RPE), dramatically inhibits the growth of iPSCs. PEDF is detected abundantly in culture supernatants of primary or iPSC-derived RPE. Apoptotic cell death is induced in iPSC when co-cultured with RPE, a process that is significantly blocked by addition of antibody against PEDF. Indeed, addition of recombinant PEDF to the iPSC cell culture induces apoptotic cell death in iPSCs, but the expression of pluripotency related-genes is maintained, suggesting that PEDF causes cell death, not differentiation, of iPSCs. To recapitulate this event in vivo, we examined tumor formation in NOG mice after subcutaneous injection of iPSCs with or without an iPSC-derived RPE sheet (2.5 × 10(5) RPE cells). We observed that the tumor forming potential of iPSCs was significantly suppressed by simultaneous transplantation with an iPSC-derived RPE sheet.
Assuntos
Apoptose/fisiologia , Proteínas do Olho/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/fisiologia , Fatores de Crescimento Neural/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Serpinas/metabolismo , cis-trans-Isomerases/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Camundongos , Camundongos Knockout , Células-Tronco Neoplásicas/efeitos dos fármacosRESUMO
Mesenchymal stem cells (MSCs) derived from human bone marrow have capability to differentiate into cells of mesenchymal lineage. The cells have already been applied in various clinical situations because of their expansion and differentiation capabilities. The cells lose their capabilities after several passages, however. With the aim of conferring higher capability on human bone marrow MSCs, we introduced the Sox2 or Nanog gene into the cells. Sox2 and Nanog are not only essential for pluripotency and self-renewal of embryonic stem cells, but also expressed in somatic stem cells that have superior expansion and differentiation potentials. We found that Sox2-expressing MSCs showed consistent proliferation and osteogenic capability in culture media containing basic fibroblast growth factor (bFGF) compared to control cells. Significantly, in the presence of bFGF in culture media, most of the Sox2-expressing cells were small, whereas the control cells were elongated in shape. We also found that Nanog-expressing cells even in the absence of bFGF had much higher capabilities for expansion and osteogenesis than control cells. These results demonstrate not only an effective way to maintain proliferation and differentiation potentials of MSCs but also an important implication about the function of bFGF for self-renewal of stem cells including MSCs.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Células-Tronco Mesenquimais/citologia , Medula Óssea , Técnicas de Cultura de Células , Proliferação de Células , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas HMGB/genética , Proteínas HMGB/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Humanos , Proteína Homeobox Nanog , Osteogênese , Fatores de Transcrição SOXB1 , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Transdução GenéticaRESUMO
Adult stem cells have been reported to exist in various tissues. The isolation of high-quality human stem cells that can be used for regeneration of fatal deseases from accessible resources is an important advance in stem cell research. In the present study, we identified a novel stem cell, which we named tooth germ progenitor cells (TGPCs), from discarded third molar, commonly called as wisdom teeth. We demonstrated the characterization and distinctiveness of the TGPCs, and found that TGPCs showed high proliferation activity and capability to differentiate in vitro into cells of three germ layers including osteoblasts, neural cells, and hepatocytes. TGPCs were examined by the transplantation into a carbon tetrachloride (CCl4)-treated liver injured rat to determine whether this novel cell source might be useful for cell-based therapy to treat liver diseases. The successful engraftment of the TGPCs was demonstrated by PKH26 fluorescence in the recipient's rat as to liver at 4 weeks after transplantation. The TGPCs prevented the progression of liver fibrosis in the liver of CCl4-treated rats and contributed to the restoration of liver function, as assessed by the measurement of hepatic serum markers aspartate aminotransferase and alanine aminotransferase. Furthermore, the liver functions, observed by the levels of serum bilirubin and albumin, appeared to be improved following transplantation of TGPCs. These findings suggest that multipotent TGPCs are one of the candidates for cell-based therapy to treat liver diseases and offer unprecedented opportunities for developing therapies in treating tissue repair and regeneration.
Assuntos
Cirrose Hepática Experimental/cirurgia , Transplante de Células-Tronco Mesenquimais , Dente Serotino/citologia , Células-Tronco Multipotentes/citologia , Germe de Dente/citologia , Animais , Tetracloreto de Carbono/toxicidade , Diferenciação Celular , Divisão Celular , Linhagem da Célula , Separação Celular/métodos , Células Cultivadas/citologia , Células Cultivadas/transplante , Sobrevivência de Enxerto , Humanos , Cirrose Hepática Experimental/sangue , Cirrose Hepática Experimental/prevenção & controle , Testes de Função Hepática , Regeneração Hepática , Células-Tronco Multipotentes/transplante , Neurônios/citologia , Osteócitos/citologia , Osteogênese , Ratos , Ratos Endogâmicos F344 , Transplante HeterólogoRESUMO
Mesenchymal stromal cells (MSCs) derived from human bone marrow are expected to be utilized for the purpose of tissue engineering, because of their extensive self-renewal or proliferation capability. The capability decreases after several passages, however. Basic fibroblast growth factor (bFGF) is commonly used for culture of various cells including bone marrow-derived MSCs. With the aim of conferring higher capability on human bone marrow MSCs, we introduced the bFGF gene into the passaged cells by retroviral system. The bFGF-expressing MSCs, even at 7 to 8 passages after the infection, showed consistent proliferation capability. The capability was not detected in control cells even in culture media containing the bFGF protein. Thus, we could not mimic the effect of forced expression of bFGF by exogenously adding the bFGF protein in culture media. Although we expressed the shortest isoform of bFGF, which was considered to be mostly cytosolic, we found the protein mostly in the nucleus. Our observations demonstrate not only an effective way to maintain proliferation potentials of MSCs, but also a possibility that there may be mechanistic and functional differences in the signal transduction events between endogenously expressed and exogenously added bFGF protein in MSCs.
Assuntos
Células da Medula Óssea/citologia , Fator 2 de Crescimento de Fibroblastos/genética , Mesoderma/citologia , Adulto , Idoso de 80 Anos ou mais , Linhagem Celular , Proliferação de Células , Criança , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Expressão Gênica , Humanos , Masculino , Retroviridae/genética , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , TransgenesRESUMO
The Rho family small guanosine triphosphatases (GTPases) play important roles in many cellular processes, especially in regulation of cytoskeletal organization. In this study, I examined the functions of Rac1 and Cdc42 for disc morphogenesis in Drosophila. I expressed either a constitutively active form or a dominant negative form of each protein during early morphogenesis of eye discs. Inactivation of Rac1 or Cdc42 resulted in small eye phenotypes. On the other hand, I found that activation of either Rac1 or Cdc42 induces ectopic antennae. In some cases, an almost complete antenna was observed instead of an eye, which was possibly transformation from an eye to an antenna. As a molecular evidence for the ectopic antennae, I found that the Distal-less protein, which is essential for the distalization process, was ectopically induced in the eye discs. I also found that the Decapentaplegic and Wingless proteins, which are upstream morphogenetic signaling proteins of the distalization process, could be ectopically induced by activation of Rac1 or Cdc42. My observations suggest novel functions of Rac1 and Cdc42 for disc morphogenesis.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/anatomia & histologia , Drosophila/crescimento & desenvolvimento , Olho/enzimologia , Olho/crescimento & desenvolvimento , Proteínas de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Homeodomínio/metabolismo , Morfogênese , Fenótipo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Proteína Wnt1 , Proteínas rac de Ligação ao GTP/genéticaRESUMO
Notch (N) signaling is an evolutionarily conserved mechanism that regulates many cell-fate decisions. deltex (dx) encodes an E3-ubiquitin ligase that binds to the intracellular domain of N and positively regulates N signaling. However, the precise mechanism of Dx action is unknown. Here, we found that Dx was required and sufficient to activate the expression of gene targets of the canonical Su(H)-dependent N signaling pathway. Although Dx required N and a cis-acting element that overlaps with the Su(H)-binding site, Dx activated a target enhancer of N signaling, the dorsoventral compartment boundary enhancer of vestigial (vgBE), in a manner that was independent of the Delta (Dl)/Serrate (Ser) ligands- or Su(H). Dx caused N to be moved from the apical cell surface into the late-endosome, where it accumulated stably and co-localized with Dx. Consistent with this, the dx gene was required for the presence of N in the endocytic vesicles. Finally, blocking the N transportation from the plasma membrane to the late-endosome by a dominant-negative form of Rab5 inhibited the Dx-mediated activation of N signaling, suggesting that the accumulation of N in the late-endosome was required for the Dx-mediated Su(H)-independent N signaling.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Animais , Membrana Celular/metabolismo , Polaridade Celular , Vesículas Citoplasmáticas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/genética , Microscopia Confocal , Transporte Proteico , Receptores Notch , Proteínas Repressoras/genética , Fatores de Tempo , Asas de Animais/metabolismoRESUMO
Eph receptor tyrosine kinases have been implicated in various developmental processes, including axonal guidance, angiogenesis and morphogenesis. In general, Eph receptors and their ligands, ephrins, are reciprocally compartmentalized during embryogenesis. However, they are expressed in an overlapping fashion in some developing neural and non-neural tissues. Results from the overexpression or mutant mice of ephrin ligands in the retino-tectal system suggest that ephrin-As regulate co-expressed EphA receptor activity, but little is known about the molecular mechanisms. Here we show that EphA receptors and co-expressed ephrin-A ligands interact directly in cis via their functional binding domains, and that this interaction does not seem to mediate intracellular signals, but has an inhibitory effect on the trans interaction.
Assuntos
Efrinas/metabolismo , Receptor EphA1/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Western Blotting/métodos , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Linhagem Celular , Galinhas , Embrião de Mamíferos , Embrião não Mamífero , Efrinas/química , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imuno-Histoquímica/métodos , Rim , Ligantes , Camundongos , Camundongos Mutantes , Modelos Biológicos , Fosforilação , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transfecção/métodosRESUMO
Rho family small GTPases are thought to be key molecules in the regulation of cytoskeletal organization, especially for actin filaments. In order to examine the functions of Rac1 and Cdc42 in axon guidance at the midline of the central nervous system in Drosophila embryos, we either activated or inactivated Rac1 and Cdc42 in all postmitotic neurons. We found that the phenotypes of Cdc42 activation and Rac1 inactivation were similar to those of roundabout mutants, in that many extra axons crossed the midline. We also found that Rac1 inactivation is dominant over Roundabout receptor activation. Our observations indicate that Rac1 and Cdc42 have distinct functions in downstream signalling events triggered by Roundabout receptors. In order to further examine the functional difference between Rac1 and Cdc42 in the growth cone morphogenesis, we used primary embryonic cultures to closely observe neurite formation. We showed that activation of Rac1 and Cdc42 has distinct effects on neurite formation, particularly on growth cone morphology and the actin filaments within. Both Rac1 and Cdc42 activation induced large growth cones and long filopodia, but Cdc42 did so more efficiently than Rac1. Only Rac1 activation, however, induced thick actin bundles in the filopodia. We also found a clear difference between Rac1 and Cdc42 in terms of the response to an inhibitor of actin polymerization. Our results suggest that Cdc42 is specifically involved in the regulation of actin filaments in growth cones, whereas Rac1 is involved in additional functions.