RESUMO
BACKGROUND: The present study aimed to evaluate whether bioengineered mouse islet cell sheets can be used for the treatment of diabetes mellitus. METHODS: Isolated mouse pancreatic islets were dispersed, and cells were plated on temperature-responsive culture plates coated with iMatrix-551. On day 3 of culture, the sheets were detached from the plates and used for further analysis or transplantation. The following parameters were assessed: (1) morphology, (2) expression of ß-cell-specific transcription factors and other islet-related proteins, (3) methylation level of the pancreatic duodenal homeobox-1 (Pdx-1) promoter, as determined by bisulfite sequencing, and (4) levels of serum glucose after transplantation of one or two islet cell sheets into the abdominal cavity of streptozotocin-induced diabetic severe combined immunodeficiency mice. RESULTS: From each mouse, we recovered approximately 233.3 ± 12.5 islets and 1.4 ± 0.1 × 105 cells after dispersion. We estimate that approximately 68.2% of the cells were lost during dispersion. The viability of recovered single cells was 91.3 ± 0.9%. The engineered islet cell sheets were stable, but the messenger RNA levels of various ß-cell-specific transcription factors were significantly lower than those of primary islets, whereas Pdx-1 promoter methylation and the expression of NeuroD, Pdx-1, and glucagon proteins were similar between sheets and islets. Moreover, transplantation of islet cell sheets did not revert serum hyperglycemia in any of the recipient mice. CONCLUSIONS: Engineering effective islet cell sheets require further research efforts, as the currently produced sheets remain functionally inferior compared with primary islets.
Assuntos
Diabetes Mellitus Experimental/terapia , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/metabolismo , Cultura Primária de Células/métodos , Engenharia Tecidual/métodos , Cavidade Abdominal/cirurgia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Glicemia , Sobrevivência Celular , Células Cultivadas , Metilação de DNA , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Glucagon/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Hiperglicemia/sangue , Hiperglicemia/terapia , Insulina , Camundongos , Camundongos SCID , Proteínas do Tecido Nervoso/metabolismo , Cultura Primária de Células/instrumentação , Regiões Promotoras Genéticas/genética , Estreptozocina/toxicidade , Transativadores/genética , Transativadores/metabolismo , Resultado do TratamentoRESUMO
Genetically modified pigs that express fluorescent proteins such as green and red fluorescent proteins have become indispensable biomedical research tools in recent years. Cell or tissue transplantation studies using fluorescent markers should be conducted, wherein the xeno-antigenicity of the fluorescent proteins does not affect engraftment or graft survival. Thus, we aimed to create a transgenic (Tg)-cloned pig that was immunologically tolerant to fluorescent protein antigens. In the present study, we generated a Tg-cloned pig harboring a derivative of Plum modified by a single amino acid substitution in the chromophore. The cells and tissues of this Tg-cloned pig expressing the modified Plum (mPlum) did not fluoresce. However, western blot and immunohistochemistry analyses clearly showed that the mPlum had the same antigenicity as Plum. Thus, we have obtained primary proof of principle for creating a cloned pig that is immunologically tolerant to fluorescent protein antigens.