RESUMO
Spontaneous reversion of disease-causing mutations has been observed in some genetic disorders. In our clinical observations of severe generalized recessive dystrophic epidermolysis bullosa (RDEB), a currently incurable blistering genodermatosis caused by loss-of-function mutations in COL7A1 that results in a deficit of type VII collagen (C7), we have observed patches of healthy-appearing skin on some individuals. When biopsied, this skin revealed somatic mosaicism resulting in the self-correction of C7 deficiency. We believe this source of cells could represent an opportunity for translational 'natural' gene therapy. We show that revertant RDEB keratinocytes expressing functional C7 can be reprogrammed into induced pluripotent stem cells (iPSCs) and that self-corrected RDEB iPSCs can be induced to differentiate into either epidermal or hematopoietic cell populations. Our results give proof-of-principle that an inexhaustible supply of functional patient-specific revertant cells can be obtained--potentially relevant to local wound therapy and systemic hematopoietic cell transplantation. This technology may also avoid some of the major limitations of other cell therapy strategies, e.g., immune rejection and insertional mutagenesis, which are associated with viral- and nonviral-mediated gene therapy. We believe this approach should be the starting point for autologous cellular therapies using 'natural' gene therapy in RDEB and other diseases.
Assuntos
Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Pluripotentes Induzidas/fisiologia , Mosaicismo , Diferenciação Celular/fisiologia , Criança , Epidermólise Bolhosa Distrófica/patologia , Epigênese Genética/fisiologia , Genes Recessivos , Humanos , Queratinócitos/fisiologia , Masculino , Pele/patologiaAssuntos
Epidermólise Bolhosa Juncional/terapia , Terapia Genética/métodos , Queratinócitos/fisiologia , Células-Tronco Pluripotentes/fisiologia , Moléculas de Adesão Celular/genética , Epidermólise Bolhosa Juncional/genética , Epidermólise Bolhosa Juncional/patologia , Humanos , Lactente , Queratinócitos/citologia , Células-Tronco Pluripotentes/citologia , Transdução Genética/métodos , CalininaRESUMO
Mucopolysaccharidosis type I (MPS IH; Hurler syndrome) is a congenital deficiency of α-L-iduronidase, leading to lysosomal storage of glycosaminoglycans that is ultimately fatal following an insidious onset after birth. Hematopoietic cell transplantation (HCT) is a life-saving measure in MPS IH. However, because a suitable hematopoietic donor is not found for everyone, because HCT is associated with significant morbidity and mortality, and because there is no known benefit of immune reaction between the host and the donor cells in MPS IH, gene-corrected autologous stem cells may be the ideal graft for HCT. Thus, we generated induced pluripotent stem cells from 2 patients with MPS IH (MPS-iPS cells). We found that α-L-iduronidase was not required for stem cell renewal, and that MPS-iPS cells showed lysosomal storage characteristic of MPS IH and could be differentiated to both hematopoietic and nonhematopoietic cells. The specific epigenetic profile associated with de-differentiation of MPS IH fibroblasts into MPS-iPS cells was maintained when MPS-iPS cells are gene-corrected with virally delivered α-L-iduronidase. These data underscore the potential of MPS-iPS cells to generate autologous hematopoietic grafts devoid of immunologic complications of allogeneic transplantation, as well as generating nonhematopoietic cells with the potential to treat anatomical sites not fully corrected with HCT.
Assuntos
Diferenciação Celular , Sistema Hematopoético/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Pré-Escolar , Metilação de DNA , Células HEK293 , Sistema Hematopoético/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Iduronidase/genética , Iduronidase/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Lactente , Queratinócitos/citologia , Queratinócitos/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Mucopolissacaridose I/genética , Mucopolissacaridose I/metabolismo , Mucopolissacaridose I/patologia , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Células Estromais/citologia , Células Estromais/metabolismo , TransfecçãoRESUMO
Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in the COL7A1 gene-encoding type VII collagen (Col7), the major component of anchoring fibrils at the dermal-epidermal junction. Individuals with RDEB develop painful blisters and mucosal erosions, and currently, there are no effective forms of therapy. Nevertheless, some advances in patient therapy are being made, and cell-based therapies with mesenchymal and hematopoietic cells have shown promise in early clinical trials. To establish a foundation for personalized, gene-corrected, patient-specific cell transfer, we generated induced pluripotent stem (iPS) cells from three subjects with RDEB (RDEB iPS cells). We found that Col7 was not required for stem cell renewal and that RDEB iPS cells could be differentiated into both hematopoietic and nonhematopoietic lineages. The specific epigenetic profile associated with de-differentiation of RDEB fibroblasts and keratinocytes into RDEB iPS cells was similar to that observed in wild-type (WT) iPS cells. Importantly, human WT and RDEB iPS cells differentiated in vivo into structures resembling the skin. Gene-corrected RDEB iPS cells expressed Col7. These data identify the potential of RDEB iPS cells to generate autologous hematopoietic grafts and skin cells with the inherent capacity to treat skin and mucosal erosions that typify this genodermatosis.
Assuntos
Epidermólise Bolhosa Distrófica , Genes Recessivos , Transplante de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Mesenquimais , Células-Tronco Pluripotentes/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/patologia , Epidermólise Bolhosa Distrófica/terapia , Epigênese Genética/fisiologia , Fibroblastos/citologia , Humanos , Técnicas In Vitro , Queratinócitos/citologia , Medicina de PrecisãoRESUMO
OBJECTIVE: To investigate whether syngeneic BM-derived DCs generated in vitro and fused with syngeneic C1498 tumor cells (murine AML line) could induce a better antitumor protective effect compared to similarly generated DCs pulsed with C1498 lysate with or without co-injection of a class B CpG oligodeoxynucleotide (CpG 7909) in vivo. METHODS: DCs were pulsed with C1498 lysate prior to intravenous administration 14 and 7 days prior to tumor challenge. Separate cohorts received DCs electrically fused to irradiated C1498 cells. Cohorts were administered DCs that were lysate-pulsed or fused with tumor cells on days 14 and 7 prior to tumor injection. Some cohorts were co-injected with CpG 7909 at the time of DC administration. RESULTS: All DC vaccines significantly improved survival (p < 0.01) vs nonvaccinated controls. There was no difference in the antitumor protective response between mice that received pulsed vs fused DCs (47% vs 45% survival). Both DC vaccines generated a fivefold increase in splenic tumor-reactive cytotoxic T-lymphocyte precursor cells and significantly (p < 0.05) higher mean frequencies of IFN-gamma-producing splenocytes compared to controls. CpG 7909 improved the survival of mice receiving the fused DCs (p < 0.05) but not the pulsed DCs. Surviving mice were rechallenged and found to be resistant to lethal tumor injection. CONCLUSIONS: DC vaccine strategies may be effective in generating anti-AML responses. No advantage was observed between lysate-pulsed and tumor cell-fused DCs. CpGs may provide an adjuvant effect depending on the type of DC vaccine administered.
Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/uso terapêutico , Células Dendríticas/transplante , Leucemia Mieloide Aguda/terapia , Vacinação , Adjuvantes Imunológicos/farmacologia , Animais , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Sistema Livre de Células/imunologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Feminino , Interferon gama/imunologia , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Camundongos , Transplante de Neoplasias/imunologia , Transplante de Neoplasias/métodos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/terapia , Oligodesoxirribonucleotídeos/imunologia , Oligodesoxirribonucleotídeos/farmacologia , Baço/imunologia , Baço/patologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
We have developed a mouse system by which to track the migration and homing of cells in a setting of bone marrow transplantation (BMT)-induced graft-versus-host disease (GVHD) after systemic infusion using enhanced green fluorescence protein (eGFP) transgenic (Tg) cells and a simple application of a fluorescence stereomicroscope outfitted with a color charge-coupled device (CCD) camera. Whole body images of anesthetized mice taken at various time points after cell infusion revealed the early migration of allogeneic cells to peripheral lymphoid organs, with later infiltration of GVHD target organs. Localization of eGFP Tg cells could be seen through the skin of shaved mice, and internal organs were easily discernible. After allogeneic or syngeneic eGFP Tg cell infusion, representative mice were dissected to better visualize deeper internal organs and tissues. Infusion of different cell populations revealed distinct homing patterns, and this method also provided a simple way to identify the critical time points for expansion of the transplanted cells in various organs. This simple application of the fluorescence stereomicroscope will be valuable for GVHD and graft-versus-tumor studies in which visualization of cellular migration, expansion, and cell-cell interactions will be more informative when analyzed by such an intravital method.