Biochemistry Department - Chemistry Institute
Universidade de São Paulo
Embryonic stem cell differentiation into insulin-producing cells (IPCS): functional characterization of the Purkinje Cell Protein 4 (PCP4) gene in this process
Patricia Kossugue1, Camila Leal-Lopes1,2, Renato Astorino1, Fernando Lojudice1, Mari Sogayar1,2.
1NUCEL/NETCEM-Cell and Molecular Therapy Center, School of Medicine, University of São Paulo, São Paulo, Brazil; 2Biochemistry Department, Chemistry Institute, University of São Paulo, São Paulo, Brazil
Alternative cellular sources for type 1 Diabetes mellitus treatment have been previously investigated, the most promising of which seems to be the insulin producing cells (IPCs), obtained by stem cells differentiation. Some reports show that murine embryonic stem cells (mESCs) are able to form islet-like clusters, however, their insulin production is insufficient to render diabetic mice normoglycemic. This work aims at developing an adequate protocol for generation of IPCs by searching for new genes which may be involved in the pancreatic organogenesis process.
Early on during mESCs differentiation into IPCs, we observed the presence of progenitor cells, which were able to express pancreatic β-cell markers. At the end of the differentiation process, the islet-like clusters positively stained for the insulin-specific dithizone, and were able to express proinsulin, PDX1, GLUT2, PAX4 and other β-cell markers (immunofluorscence). These clusters were microencapsulated in a novel biopolymer (Bioprotect®) generated and deposited as a patent by our group and subjected them to in vivo maturation by introducing them into normal animals. A dramatic increase in expression was observed for the above mentioned genes, indicating complete maturation of the differentiated cells. Moreover, when these encapsulated cell clusters were transplanted into diabetized mice, the animals achieved and maintained normoglycemia during 60 days. In addition, at day 42 after transplant, the glucose tolerance test (OGTT) response displayed by these animals was more similar to that of normal mice than to the control diabetized not treated mice (p<0.05).
In parallel, we compared undifferentiated mESCs to IPCs using a microarray platform and selected some of the up-regulated genes for functional analysis. . One of these genes is that coding for the Purkinje cell protein 4 (Pcp4), which is 1,000 times more expressed in the differentiated cell IPC clusters than in undifferentiated mESC cells. We adopted a functional genomics approach to investigate the role played by the Pcp4 gene in β-cells and in β-cell differentiation, by overexpressing and knocking down this gene in MIN-6 insulinoma and mESC cells. Overexpression of Pcp4 in MIN-6 did not interfere with the expression of the genes analyzed. On the other hand, Pcp4 knock-down caused increased cell growth rates, with increased doubling time and decreased cell viability (apoptosis induction assay) . In addition, over-expression of Pcp4 in mESCs subjected to differentiation into IPCs apparently increase the expression of genes related to β-cell differentiation, such as the Isl1 gene (p<0.01), which is important for islet-like aggregates formation.
In conclusion, we developed a new protocol for ESCs differentiation into IPCs, which generates cell clusters that are able to revert diabetes in diabetized mice, and we also describe here, for the first time, that the Pcp4 gene is expressed in pancreatic β-cells, being possibly related to β-cell maturation and maintenance of their viability.
BNDES, CAPES, CNPq, FAPESP, FINEP, MCTI, MS-DECIT.
11:00 - 12:30
|Cellular Encapsulation||BioSafe: a novel biomaterial for pancreatic islets encapsulation and type 1 diabetes mellitus therapy||Room 111-112|
15:30 - 17:00
|Islet Cell Transplantation - Miscellaneous||Embryonic stem cell differentiation into insulin-producing cells (IPCS): functional characterization of the Purkinje Cell Protein 4 (PCP4) gene in this process||Room 111-112|