Differentiation of Human-Induced Pluripotent Stem Cells Into Insulin-Producing Clusters by MicroRNA-7.

OBJECTIVES: Diabetes results from inadequate insulin production from pancreatic ß-cells. Islet cell replacement is an effective approach for diabetes treatment; however, it is not sufficient for all diabetic patients. Thus, finding a new source with effective maturation of ß-cells is the major goal of many studies. MicroRNAs are a class of small noncoding ribonucleic acid that regulate gene expression through posttranscriptional mechanisms. MicroRNA-7 has high expression level during pancreatic islet development in humans, thereby playing a critical role in pancreatic ß-cell function. We study aimed to develop a protocol to differentiate human-induced pluripotent stem cells efficiently into isletlike cell clusters in vitro by using microRNA-7. MATERIALS AND METHODS: Human-induced pluripotent stem cell colonies were transfected with hsa-microRNA-7 by using siPORT NeoFX transfection agent. Total ribonucleic acid was extracted 24 and 48 hours after transfection. The expression of transcription factors which were important during pancreases development was also performed. On the third day, the potency of the clusters was assessed in response to high glucose levels. Diphenylthiocarbazone was used to identify the existence of the ß-cells. The presence of insulin and Neurogenin-3 proteins was investigated by immunocytochemistry. RESULTS: Morphologic changes were observed on the first day after chemical transfection, and cell clusters were formed on the third day. The expression of pancreatic specific transcription factors was increased on the first day and significantly increased on the second day. The isletlike cell clusters were positive for insulin and Neurogenin-3 proteins in immunocytochemistry. The clusters were stained with Diphenylthiocarbazone and secreted insulin in a glucose challenge test. CONCLUSIONS: MicroRNA-7 transcription factor network is important in pancreatic endocrine differentiation. Chemical transfection with microRNA-7 can differentiate human induced pluripotent stem cells into functional isletlike cell clusters in a short time.

Differentiation of human-induced pluripotent stem cells into insulin-producing clusters.

OBJECTIVES: In diabetes mellitus type 1, beta cells are mostly destroyed; while in diabetes mellitus type 2, beta cells are reduced by 40% to 60%. We hope that soon, stem cells can be used in diabetes therapy via pancreatic beta cell replacement. Induced pluripotent stem cells are a kind of stem cell taken from an adult somatic cell by "stimulating" certain genes. These induced pluripotent stem cells may be a promising source of cell therapy. This study sought to produce isletlike clusters of insulin-producing cells taken from induced pluripotent stem cells. MATERIALS AND METHODS: A human-induced pluripotent stem cell line was induced into isletlike clusters via a 4-step protocol, by adding insulin, transferrin, and selenium (ITS), N2, B27, fibroblast growth factor, and nicotinamide. During differentiation, expression of pancreatic ß-cell genes was evaluated by reverse transcriptase-polymerase chain reaction; the morphologic changes of induced pluripotent stem cells toward isletlike clusters were observed by a light microscope. Dithizone staining was used to stain these isletlike clusters. Insulin produced by these clusters was evaluated by radio immunosorbent assay, and the secretion capacity was analyzed with a glucose challenge test. RESULTS: Differentiation was evaluated by analyzing the morphology, dithizone staining, real-time quantitative polymerase chain reaction, and immunocytochemistry. Gene expression of insulin, glucagon, PDX1, NGN3, PAX4, PAX6, NKX6.1, KIR6.2, and GLUT2 were documented by analyzing real-time quantitative polymerase chain reaction. Dithizone-stained cellular clusters were observed after 23 days. The isletlike clusters significantly produced insulin. The isletlike clusters could increase insulin secretion after a glucose challenge test. CONCLUSIONS: This work provides a model for studying the differentiation of human-induced pluripotent stem cells to insulin-producing cells.

Isolation and characterization of Human Mesenchymal Stromal Cells Derived from Placental Decidua Basalis; Umbilical cord Wharton's Jelly and Amniotic Membrane.

OBJECTIVE: Mesenchymal stromal cells (MSCs) are considered as an excellent source in regenerative medicine, but availability and ethical problems limited their routine use. Therefore, another available source with easy procedure and exempt from ethical debate is important. The purpose of this study is to isolate and characterize the MSCs from human placenta. The stromal cells were isolated from Placental Decidua Basalis (PDB-MSC), Umbilical cord Wharton's Jelly (WJ-MSC) and Amniotic Membrane (AM-MSC). METHODS: Full term human placentas (n=4), from cesarean section delivery were collected. Small fragments from different parts were cultures as explants. The immunophenotyping, mesodermal differentiation, growth kinetics and stemness gene expression was studied. RESULTS: The cultivated cells from three sources expressed CD44, CD105, and CD90. Gene expression of NANOG and OCT4 confirmed the undifferentiated state. The doubling-times for WJ-MSCs, PLC-MSCs and AM-MSCs, respectively, were 21±8h, 28±9h and 25±9h at passage three and 30±5h, 45±7h and 45±7h at passage tenth. The proliferative potential of WJ-MSCs tended to be higher than the other two sources. CONCLUSION: The fetal derives stromal cells; especially the early passages of WJ-MSCs are available supplies for large scale production of MSC for using in clinical studies or research projects.

miR-375 induces human decidua basalis-derived stromal cells to become insulin-producing cells.

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.

Differentiation of human induced pluripotent stem cells into insulin-like cell clusters with miR-186 and miR-375 by using chemical transfection.

Diabetes mellitus is characterized by either the inability to produce insulin or insensitivity to insulin secreted by the body. Islet cell replacement is an effective approach for diabetes treatment; however, it is not sufficient for all the diabetic patients. MicroRNAs (miRNAs) are a class of small noncoding RNAs that play an important role in mediating a broad and expanding range of biological activities, such as pancreas development. The present study aimed to develop a protocol to efficiently differentiate human induced pluripotent stem (iPS) cells into islet-like cell clusters (ILCs) in vitro by using miR-186 and miR-375. The human iPS colonies were transfected with hsa-miR-186 and hsa-miR-375 by using siPORT™ NeoFX™ Transfection Agent, and the differentiation was compared to controls. Total RNA was extracted 24 and 48 h after transfection. The gene expressions of insulin, NGN3, GLUT2, PAX4, PAX6, KIR6.2, NKX6.1, PDX1, Glucagon, and OCT4 were then evaluated through real-time qPCR. On the third day, the potency of the clusters was assessed in response to high glucose levels. Dithizone (DTZ) was used to identify the existence of the ß-cells. Besides, the presence of insulin and NGN3 proteins was investigated by immunocytochemistry. Morphological changes were observed on the first day after the chemical transfection, and cell clusters were formed on the third day. The expression of pancreatic specific transcription factors was increased on the first day and significantly increased on the second day. The ILCs were positive for insulin and NGN3 proteins in the immunocytochemistry. Besides, the clusters were stained with DTZ and secreted insulin in glucose challenge test. Overexpression of miR-186 and miR-375 can be an alternative strategy for producing ILCs from the iPS cells in a short time. This work provides a new approach by using patient-specific iPSCs for ß-cell replacement therapy in diabetic patients.