AbstractThe replacement of β-cells through the transplantation of isolated pancreatic islets is a promising treatment for type 1 diabetes. However, the success of islet transplantation has been limited by progressive decline in islet function and viability during isolation and post transplantation. Mesenchymal stromal cells (MSCs) are a multipotent and multifunctional adult progenitor cell which have been extensively shown to improve islet function and viability in vitro. Understanding how MSCs facilitate this, and under what conditions, may allow us to improve clinical islet transplantation protocols in the treatment of type 1 diabetes.
We initially explored how MSCs could protect islet function and viability under stress conditions. Mouse islets were cultured with low or high concentrations of mixed cytokines (IL-1β, TNF-α, IFN-γ) or incubated in hypoxic conditions (1% O2) before culture with mouse bone marrow-derived MSCs. Islet function was assessed by static incubation and insulin secretion measured by radioimmunoassay. Islet viability was assessed by flow cytometry. Co-cultured islets exposed to acute hypoxia, or a high concentration of pro-inflammatory cytokines had significantly greater cell viability than control islets. Similarly, islets exposed to low and high concentrations of cytokines had significantly improved glucose stimulated insulin secretion (GSIS). These results demonstrate that MSCs are protective against key transplantation stressors and may increase their response to more damaged islets.
We then assessed gene expression changes in co-cultured islets in a broad and exploratory manner by employing scRNA-Seq to describe the transcriptomes of 3 biological replicates of islets co-cultured with MSCs for 72hrs. Data were analysed in R Studio with Seurat v3. 8 distinct cell populations that have previously been described in islet single cell studies were identified: β, α, δ, PP, endothelial, MSCs, immune and Schwann. Differential gene expression analysis between control β-cells and co-cultured β-cells (1.3-fold change threshold) revealed 26 significantly differentially expressed genes (DEGs) common between all replicates. These DEGs have a wide range of downstream functional roles including cell death, stress and trafficking. qPCR analysis demonstrated a strong positive correlation (r = 0.82) between qPCR and scRNA-Seq fold-change in gene expression, confirming RNA-Seq results in a different mouse model.
We chose four candidate genes, which had all been upregulated, to investigate in knockdown studies. Upregulation of three of these genes (Lgals3bp, Mt1, Zdhhc2) was confirmed at the protein level by western blotting. In order to efficiently deliver knockdown reagents to all cells in the islet we optimised the use of pseudoislets which are broken down to single cell before being seeded into agarose based microwells to reform. Lipid mediated siRNA transfection highly efficient 97% gene KD in pseudoislets, however they were not responsive to glucose, so we opted to use partially dissociated islets seeded into microwells which prevented clumping upon reformation. Lipid mediated siRNA transfection of partially dissociated islets achieved 45-57% protein knockdown (KD) of candidate genes. KD of extracellular matrix protein Lgals3bp lead to an 12.5% decrease in islet cell viability suggesting that signalling through LGALS3BP maintains islet viability.
To explore how MSCs might affect β-cell biological processes in a broader sense we performed differential expression analysis with a lower fold change threshold of 1.1 (10% change). This returned 322 DEGs between control and co-cultured β-cells that were common to all replicates. Gene set enrichment analysis revealed enrichment of Gene Ontology biological processes associated with pancreatic development and differentiation. This prompted further sub-clustering of β-cells into mature and immature-like populations based on expression of key β-cell maturity markers. There was significant downregulation of key maturity markers - Ins2, Mafa, Slc2a2 and Nkx6.1 - in mature co-cultured β-cells compared to mature controls. However, there are no changes in the expression of β-cell disallowed genes such as Aldh13, Ldha, and Sox9. This suggests that MSCs are inducing loss of the mature β -cell phenotype/ a mild dedifferentiation which we predict may be protective to islets. To investigate this, we aimed to induce dedifferentiation in β-cells through several methods: FGF2 treatment, inhibition of Foxo1, and siRNA KD of Ins2, Mafa, Slc2a2 and Nkx6.1. Inhibition of Foxo1 led to significant downregulation of Ins2, Mafa, and Ucn3 and upregulation of Neurog3 indicating a successful dedifferentiation but this did not rescue islet function and viability under stress conditions. siRNA KD of Ins2, Mafa, and Slc2a2 was successful but KD of Nkx6.1 was not. Under normal conditions and stress conditions we did not see an effect on islet secretory function or islet cell viability with either Mafa KD or Mafa/Ins2/Slc2a2 KD. We were therefore unable to confirm if MSC mediated dedifferentiation is protective to islets.
|Date of Award||1 Sept 2022|
|Supervisor||Peter Jones (Supervisor), Timothy Pullen (Supervisor) & Chloe Rackham (Supervisor)|