Abstract
This paper focuses on the development of renewable sources of isletreplacement tissue for the treatment of type I diabetes mellitus. Placental tissue-derived mesenchymal stem cells (MSCs) are a promising source for regenerative medicine due to their plasticity and easy availability. They have the potential to differentiate into insulin-producing cells. miR-375 is a micro RNA that is expressed in the pancreas and involved in islet development. Human placental decidua basalis MSCs (PDB-MSCs) were cultured from full-term human placenta. The immunophenotype of the isolated cells was checked for CD90, CD105, CD44, CD133 and CD34 markers. The MSCs (P3) were chemically transfected with hsa-miR-375. Total RNA was extracted 4 and 6 days after transfection. The expressions of insulin, NGN3, GLUT2, PAX4, PAX6, KIR6.2, NKX6.1, PDX1, and glucagon genes were evaluated using real-time qPCR. On day 6, we tested the potency of the clusters in response to the high glucose challenge and assessed the presence of insulin and NGN3 proteins via immunocytochemistry. Flow cytometry analysis confirmed that more than 90% of the cells were positive for CD90, CD105 and CD44 and negative for CD133 and CD34. Morphological changes were followed from day 2. Cell clusters formed during day 6. Insulin-producing clusters showed a deep red color with DTZ. The expression of pancreatic-specific transcription factors increased remarkably during the four days after transfection and significantly increased on day 7. The clusters were positive for insulin and NGN3 proteins, and C-peptide and insulin secretion increased in response to changes in the glucose concentration (2.8 mM and 16.7 mM). In conclusion, the MSCs could be programmed into functional insulin-producing cells by transfection of miR-375.
Highlights
The pancreatic islets have around a 70% beta cell content [1]
The expression of micro ribonucleic acid (miR)-375 is regulated by transcription factors that are important in the development and function of the pancreas, such as hepatocyte nuclear factor 6 (HNF6), neurogenin 3 (Ngn3), neuronal differentiation 1 (NEUROD1), and pancreatic and duodenal homeobox 1 (PDX-1) [26]
Joglekar et al revealed that the miRNA levels of miR-7, miR-9, miR-375 and miR-376 significantly increased during human pancreatic islet development [42]
Summary
The pancreatic islets have around a 70% beta cell content [1]. These cells secrete insulin, a hormone of great importance in regulating carbohydrate, fat and protein metabolism. Transplantation of functional pancreatic islet cells is a subject of considerable interest as an alternative to whole pancreas transplantation In this method, an infusion of fresh donor islets is followed by long-term non-steroidal immunosuppressive drug consumption. MicroRNAs [16, 17] play a direct role in pancreatic islet development [18], beta cell differentiation [19], insulin secretion [20], and the control of glucose homeostasis [21, 22]. The expression of miR-375 is regulated by transcription factors that are important in the development and function of the pancreas, such as hepatocyte nuclear factor 6 (HNF6), neurogenin 3 (Ngn3), neuronal differentiation 1 (NEUROD1), and pancreatic and duodenal homeobox 1 (PDX-1) [26]. The objective of this study was to transfect hPDMSCs with miR-375 in order to obtain effective insulin-producing clusters (IPCs) in a short period of time
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