RESUMO
Xenotransplantation of pig tissues has great potential to overcome the shortage of organ donors. One approach to address the vigorous immune rejection associated with xenotransplants is the use of embryonic precursor tissue, which induces and utilizes host vasculature upon its growth and development. Recently, we showed in mice that embryonic pig pancreatic tissue from embryonic day 42 (E42) exhibits optimal properties as a beta cell replacement therapy. We now demonstrate the proof of concept in 2 diabetic Cynomolgus monkeys, followed for 393 and 280 days, respectively. A marked reduction of exogenous insulin requirement was noted by the fourth month after transplantation, reaching complete independence from exogenous insulin during the fifth month after transplantation, with full physiological control of blood glucose levels. The porcine origin of insulin was documented by a radioimmunoassay specific for porcine C-peptide. Furthermore, the growing tissue was found to be predominantly vascularized with host blood vessels, thereby evading hyperacute or acute rejection, which could potentially be mediated by preexisting anti-pig antibodies. Durable graft protection was achieved, and most of the late complications could be attributed to the immunosuppressive protocol. While fine tuning of immune suppression, tissue dose, and implantation techniques are still required, our results demonstrate that porcine E-42 embryonic pancreatic tissue can normalize blood glucose levels in primates. Its long-term proliferative capacity, its revascularization by host endothelium, and its reduced immunogenicity, strongly suggest that this approach could offer an attractive replacement therapy for diabetes.
Assuntos
Diabetes Mellitus Experimental/cirurgia , Pâncreas/embriologia , Pâncreas/cirurgia , Suínos/embriologia , Suínos/cirurgia , Transplante Heterólogo , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Rejeição de Enxerto/imunologia , Macaca fascicularis , Masculino , Pâncreas/irrigação sanguínea , Pâncreas/imunologia , Transplante de Pâncreas , Estreptozocina/farmacologia , Transplante Heterólogo/imunologiaRESUMO
Cell therapy as an alternative to orthotopic liver transplantation represents a major challenge, since negligible proliferation of isolated hepatocytes occurs after transplantation because of the stringent homeostatic control displayed by the host liver. Thus, different modalities of liver injury as part of the pretransplant conditioning are a prerequisite for this approach. The major objective of the present study was to test whether xenotransplantation of pig fetal liver fragments, in which potential cell-cell and cell-stroma interactions are spared, might afford more robust growth and proliferation compared with isolated pig fetal hepatoblasts. After transplantation into SCID mice, fetal liver tissue fragments exhibited marked growth and proliferation, in the setting of a quiescent host liver, compared with isolated fetal hepatoblasts harvested at the same gestational age (embryonic day 28). The proliferative advantage of fetal pig liver fragments was clearly demonstrated by immunohistochemical and morphometric assays and was observed not only after implantation into the liver but also into extrahepatic sites, such as the spleen and the subrenal capsule. The presence of all types of nonparenchymal liver cells that is crucial for normal liver development and regeneration was demonstrated in the implants. Preservation of the three-dimensional structure in pig fetal liver fragments enables autonomous proliferation of transplanted hepatic cells in the setting of a quiescent host liver, without any requirement for liver injury in the pretransplant conditioning. The marked proliferation and functional maturation exhibited by the pig fetal liver fragments suggests that it could afford a preferable source for transplantation.
Assuntos
Hepatócitos/citologia , Homeostase , Transplante de Fígado , Fígado/citologia , Fígado/embriologia , Animais , Diferenciação Celular , Proliferação de Células , Separação Celular , Células Epiteliais/citologia , Regulação da Expressão Gênica , Fígado/irrigação sanguínea , Mesoderma/citologia , Camundongos , Camundongos SCID , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células-Tronco/citologia , Suínos , Transplante HeterólogoRESUMO
BACKGROUND: Transplantation of embryonic pig pancreatic tissue as a source of insulin has been suggested for the cure of diabetes. However, previous limited clinical trials failed in their attempts to treat diabetic patients by transplantation of advanced gestational age porcine embryonic pancreas. In the present study we examined growth potential, functionality, and immunogenicity of pig embryonic pancreatic tissue harvested at different gestational ages. METHODS AND FINDINGS: Implantation of embryonic pig pancreatic tissues of different gestational ages in SCID mice reveals that embryonic day 42 (E42) pig pancreas can enable a massive growth of pig islets for prolonged periods and restore normoglycemia in diabetic mice. Furthermore, both direct and indirect T cell rejection responses to the xenogeneic tissue demonstrated that E42 tissue, in comparison to E56 or later embryonic tissues, exhibits markedly reduced immunogenicity. Finally, fully immunocompetent diabetic mice grafted with the E42 pig pancreatic tissue and treated with an immunosuppression protocol comprising CTLA4-Ig and anti-CD40 ligand (anti-CD40L) attained normal blood glucose levels, eliminating the need for insulin. CONCLUSIONS: These results emphasize the importance of selecting embryonic tissue of the correct gestational age for optimal growth and function and for reduced immunogenicity, and provide a proof of principle for the therapeutic potential of E42 embryonic pig pancreatic tissue transplantation in diabetes.
Assuntos
Diabetes Mellitus Experimental/cirurgia , Diabetes Mellitus Tipo 1/cirurgia , Transplante de Pâncreas , Transplante Heterólogo , Abatacepte , Tirosina Quinase da Agamaglobulinemia , Aloxano , Animais , Glicemia/análise , Ligante de CD40/antagonistas & inibidores , Feminino , Idade Gestacional , Rejeição de Enxerto/prevenção & controle , Humanos , Imunocompetência , Imunoconjugados/uso terapêutico , Imunossupressores/uso terapêutico , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/ultraestrutura , Rim , Leucócitos Mononucleares/transplante , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Endogâmicos NOD , Camundongos Mutantes , Camundongos Nus , Camundongos SCID , Pâncreas/embriologia , Transplante de Pâncreas/imunologia , Pâncreas Exócrino/ultraestrutura , Gravidez , Proteínas Tirosina Quinases/deficiência , Sus scrofa/embriologia , Transplante Heterólogo/imunologia , Transplante Heterotópico/imunologiaRESUMO
Very little is known about the mechanisms that contribute to organ size differences between species. In the present study, we used a mouse model of embryonic pig tissue implantation to define the role of host Factor VIII in controlling the final size attained by the implant. We show here that pig embryonic spleen, pancreas, and liver all grow to an increased size in mice that are deficient in the Factor VIII clotting cascade. Similar results were obtained using the transplantation model after treatment with the low molecular weight heparin derivative Clexane which markedly enhanced transplant size. Likewise, enhanced size was found upon treatment with the direct thrombin inhibitor Dabigatran, suggesting that organ size regulation might be mediated by thrombin, downstream of Factor VIII. Considering that thrombin was shown to mediate various functions unrelated to blood clotting, either directly by cleavage of protease-activated receptors (PARs) or indirectly by cleaving osteopontin (OPN) on stroma cells, the role of PAR1 and PAR4 antagonists as well as treatment with cleaved form of OPN (tcOPN) were tested. While the former was not found to have an impact on overgrowth of embryonic pig spleen implants, marked reduction of size was noted upon treatment with the (tcOPN). Collectively, our surprising set of observations suggests that factors of the coagulation cascade have a novel role in organ size control.
Assuntos
Coagulação Sanguínea/fisiologia , Embrião de Mamíferos/fisiologia , Fator VIII/metabolismo , Implantes Experimentais , Animais , Coagulação Sanguínea/efeitos dos fármacos , Transferência Embrionária , Embrião de Mamíferos/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Fígado/efeitos dos fármacos , Fígado/embriologia , Camundongos , Camundongos Knockout , Camundongos SCID , Modelos Biológicos , Neovascularização Fisiológica/efeitos dos fármacos , Tamanho do Órgão , Especificidade de Órgãos/efeitos dos fármacos , Osteopontina/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/embriologia , Receptores Ativados por Proteinase/metabolismo , Baço/irrigação sanguínea , Baço/efeitos dos fármacos , Baço/embriologia , Coloração e Rotulagem , Sus scrofa , Trombina/farmacologiaRESUMO
Previous clinical attempts to correct genetic deficiencies such as hemophilia or Gaucher disease by transplantation of allogeneic spleen were associated with aggressive graft versus host disease, mediated by mature T cells derived from the donor spleen. We show that a fetal pig spleen harvested at the embryonic day 42 stage, before the appearance of T cells, exhibited optimal growth potential upon transplantation into SCID mice, and the growing tissue expressed factor VIII. Transplantation of embryonic day 42 spleen tissue into hemophilic SCID mice led to complete alleviation of hemophilia within 2-3 months after transplant, as demonstrated by tail bleeding and by assays for factor VIII blood levels. These results provide a proof of principle to the concept that transplantation of a fetal spleen, obtained from a developmental stage before the appearance of T cells, could provide a novel treatment modality for genetic deficiencies of an enzyme or a factor that can be replaced by the growing spleen tissue.
Assuntos
Hemofilia A/terapia , Baço/transplante , Transplante de Tecidos/efeitos adversos , Animais , Biomarcadores , Doença , Embrião de Mamíferos/cirurgia , Fator VIII/metabolismo , Sobrevivência de Enxerto/imunologia , Hemofilia A/imunologia , Hemofilia A/metabolismo , Hemofilia A/patologia , Camundongos , Baço/imunologia , Baço/patologia , Suínos , Transplante HeterólogoRESUMO
Pig embryonic tissues represent an attractive option for organ transplantation. However, the achievement of optimal organogenesis after transplantation, namely, maximal organ growth and function without teratoma development, represents a major challenge. In this study, we determined distinct gestational time windows for the growth of pig embryonic liver, pancreas, and lung precursors. Transplantation of embryonic-tissue precursors at various gestational ages [from E (embryonic day) 21 to E100] revealed a unique pattern of growth and differentiation for each embryonic organ. Maximal liver growth and function were achieved at the earliest teratoma-free gestational age (E28), whereas the growth and functional potential of the pancreas gradually increased toward E42 and E56 followed by a marked decline in insulin-secreting capacity at E80 and E100. Development of mature lung tissue containing essential respiratory system elements was observed at a relatively late gestational age (E56). These findings, showing distinct, optimal gestational time windows for transplantation of embryonic pig liver, pancreas, and lung, might explain, in part, the disappointing results in previous transplantation trials and could help enhance the chances for successful implementation of embryonic pig tissue in the treatment of a wide spectrum of human diseases.