This video is showing the in vitro induction of human dental pulp stem cells toward pancreatic lineages. Stem cells give an alternative treatment in many diseases including diabetes type I which cause biolaging of beta cells. Transplantation of insulin producing cells will be the new promising method for causing lifelong.
In this video two approaches for generating insulin producing cell, including the integrative and non integrative induction protocol are described. And in this part the integration protocol for inducing insulin producing cells from human dental pulp stem cell is explained. For the induction protocol is the for mixing the the natural pancreatic developmental process.
To get the natural and the functional insulin producing cells. For generating insulin producing cells from mesenchymal stem cells like dental pulp stem cells, multi step induction protocol is employed. Mesenchymal stem cells are used in the word definitive endoderm, pancreatic endoderm, pancreatic endocrine and then pancreatic beta-cell or insulin producing cells respectively.
To induce the cells, three step induction approach is used as a backbone protocol. The integrative protocol employed the over expiration of the essential pancreatic transcription factor, PDX-1, by human dental pulp stem cells. Then the PDX-1 over expressed stem cells will be further induced using three step differentiation protocol.
The pancreatic differentiation potential, by the integrative protocol, will be compared with the non integrative protocol as illustrated in the animation. This idea was performed under the approved protocol by the Human Research Ethic Committee, Faculty of Dentistry, Chulalongkorn University with patients'informed consent. This protocol started with over expiration of PDX-1.
Human dental pulp stem cells in parts of 2 to 5, with 70-80%confluence, are used for the transduction. Cells are trypsinized and counted. Then one million of cells are seated onto the transfection vessel and incubated overnight.
24 hours later freshly prepared antivirus carrying PDX-1 gene is added to the cultured vessel according to the desired multiplicity of infection. The transfected cells are then kept in the incubator for 24 hours. Then fresh cultured medium is changed and maintained for another 48 period.
Morphology of transfected cells is checked before using for further induction step. The human dental pulp stem cells with PDX-1 over expiration are then Tryptimized and suspended in the first pancreatic induction medium, so called medium A.In this step, low attachment cultured vessel is used. Morphology of PDX-1 over expressed human dental pulp stem cells on day 3 after Medium A induction is observed.
Cells with good morphology will be then used for further inductions step. Next step, the second induction medium, so called medium B is added to the cultured vessel. The morphology of PDX-1 over expressed human dental pulp stem cells on day two after medium induction is observed.
Cells with good morphology will be then used for further induction step. Next step, the third induction media, so called medium C is added to the cultured vessel. 5 days later cell morphology is observed.
Cell colonies are collected for further analysis. Including colony morphology and size, pancreatic gene marker expression and functional property regarding Glucose-stimulated C-peptide secretion or GSCS assay. For the non-integrative induction approach, three step induction protocol mentioned previously is employed.
One million of cells are re-suspended in the medium A and seated on to low attachment culture vessel. Then cultured for three days. After morphology are checking, series of induction medium, including medium B and C are subsequently added to the cultured vessel on day three and five, respectively.
At day 10, cell morphology is observed. Cell colonies are collected for further analysis, including colony morphology and size, pancreatic gene marker expression, and functional property regarding Glucose-stimulated C-peptide secretion or GSCS assay. The outcomes of both induction protocols are compared in both protocols.
Human dental pulp stem cells are able to form colony like structure since the very first day of the induction. The colonies appearance is round and dense. All of the colonies are floating in the culture vessels throughout the induction period.
The total colony count of the integrative induction protocol is a bit higher from that of non-integrative induction. The evolution under colony sized percentage shows a beneficial trend, from small to medium sized colony formation upon integrative induction, which is important to reduce the necrotic core inside the colony. Pancreatic gene marker analysis revealed that the integrative inductive protocol yields the colonies with high expiration of pancreatic markers, including MAP-A, GLUT-2, insulin, and GLP-1R, suggesting the differentiation toward the measured insulin producing cells.
To the function glucose C-peptide secretion is important. The results show that both induction protocol yields the colonies with good response to glucose challenge. From this study, both of pancreatic induction protocols are able to generate the insulin producing cells in vitro in term of pancreatic marker expiration and functional property, the protocols shows a better quality of insulin producing cell formation.
Suggesting the trends of human mesenchymal stem cells like human dental pulp stem cell application in stem cell based diabetes treatment in the near future.
