Abstract
In-vitro expansion of insulin-producing cells from adult human pancreatic islets could provide an abundant cell source for diabetes therapy. However, proliferation of β-cell-derived (BCD) cells is associated with loss of phenotype and epithelial-mesenchymal transition (EMT). Nevertheless, BCD cells maintain open chromatin structure at β-cell genes, suggesting that they could be readily redifferentiated. The transforming growth factor β (TGFβ) pathway has been implicated in EMT in a range of cell types. Here we show that human islet cell expansion in vitro involves upregulation of the TGFβ pathway. Blocking TGFβ pathway activation using short hairpin RNA (shRNA) against TGFβ Receptor 1 (TGFBR1, ALK5) transcripts inhibits BCD cell proliferation and dedifferentiation. Treatment of expanded BCD cells with ALK5 shRNA results in their redifferentiation, as judged by expression of β-cell genes and decreased cell proliferation. These effects, which are reproducible in cells from multiple human donors, are mediated, at least in part, by AKT-FOXO1 signaling. ALK5 inhibition synergizes with a soluble factor cocktail to promote BCD cell redifferentiation. The combined treatment may offer a therapeutically applicable way for generating an abundant source of functional insulin-producing cells following ex-vivo expansion.
Highlights
ALK5 inhibition synergized with Redifferentiation cocktail (RC) treatment in upregulation of transcripts encoding insulin, IAPP, and β-cell transcription factors (S5 Fig), as well as in inducing a 2.6-fold increase in the number of C-peptide-positive cells (S5C Fig), compared with cells treated with RC and NT short hairpin RNA (shRNA)
Our findings document the activation of the transforming growth factor β (TGFβ) pathway in expanded human BCD cells, and demonstrate that a 40% inhibition of ALK5 expression is sufficient for induction of BCD
Our findings suggest the involvement of AKT and its downstream effector forkhead box protein O1 (FOXO1) in mediating the effects of TGFβ pathway inhibition on redifferentiation and growth arrest of BCD
Summary
This study was conducted according to the principles expressed in the Declaration of Helsinki. Cells were cultured as previously described [1] in CMRL 1066 medium containing. PSG, was prepared and applied to cells as previously described [27]. Cells were infected at MOI 2:1 in CMRL 1066 medium containing 8 mg/ml polybrene overnight. Data analysis was performed with qBase software. Samples were blocked for 30 min at RT in blocking buffer (1% BSA, 10% fetal goat serum, and 0.2% saponin) and incubated overnight at 4°C, or 1 hour at RT, with primary antibodies (S2 Table) diluted in blocking buffer. TUNEL assay was performed using In Situ Cell Death Detection Kit (Roche), according to the manufacturer’s protocol. Data analysis was performed on CEL files using Partek Genomics Suite software (Partek). Clustering analysis was performed by Partek Genomics Suite software with Pearson’s dissimilarity correlation by average linkage methods. To account for multiple testing, the Bonferroni correction was applied
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