Stage no. 1/2022
Name stage 1: Obtaining primary cultures of hepatocytes (1-2 cultures) (partially). Analysis of the role of specific miRNAs in the transdifferentiation process of liver to pancreas (partially) Optimization of work protocols animal study – in vivo (partially)
Abstract: During the first stage, the isolation of liver cells (2 donors) was initiated and optimized in order to create the master cell bank (P0-P4). Gene and protein expression analyzes were established for a representative panel of biomarkers (vimentin, N and E-Cadherin, CK-18, Snail) to be included for the two types of cells, hepatocytic and transdifferentiated, pancreatic β type. The miRNA expression manipulation experiments will be initially performed for miR-424-5p and miR-26a before the transdifferentiation process and will be carried out according to an experimental design that involves inhibition and overexpression in a two-dimensional, adherent culture system, of the stocks generated within miR-Diab from diabetic patients, comparing their expression with stocks from non-diabetic reference patients. At the same time, we started preparing batches of animals to further analyze the effectiveness of the transdifferentiation process by subcutaneous implantation (Sub-Q) in immunodeficient SCID-Beige mice.
Stage no. 2/2023
Name stage 2: Obtaining primary cultures of hepatocytes (3-5 patients). Analysis of the role of selected miRNAs in the transdifferentiation process of liver to pancreas Animal study in vivo – follow-up 3-4 months (2 models) (partially) Dissemination.
Abstract: During stage 2, the activity of including new patients in the study was continued, respectively of liver cell isolation, and the bank of master cells (P0-P4) was created for 4 donors in order to carry out the proposed experiments. The transcriptional factors PDX1, NeuroD1, MAFA and FOXA2, the miRs mimic and inhibitory (miR-424-3p and miR-424-5p) were cloned in the pAdTrack-CMV expression vector. Also, the corresponding adenoviruses were constructed for each construct, by packaging and amplification in Ad-HEK-293 cells. Using chromatography, the amplified adenoviruses were purified and then titrated to determine the number of infectious viral particles to be used in the next step to transdifferentiate liver cells into insulin-secreting pancreatic β-cells. The miRNA expression manipulation experiments were initially performed for miR-424-5p and miR-26a using the RNAiMax transfection reagent before the transdifferentiation process with 24, 48 and 96 hours (n=3). At the same time, a study was completed to establish the importance of the VEGF-A factor in vivo on SCID-Beige mice (n=75) and we started preparing the batches for the 2 proposed models to further analyze the effectiveness of the transdifferentiation process through subcutaneous implantation (Sub-Q) of untransdifferentiated (NT), transdifferentiated (TD), miR-overexpressed TD (TDmiR+), and miR-inhibited TD (TDmiR-) cells together with cells co-expressing VEGF-A (n=128).