Abstract
In-vitro expansion of functional adult human β-cells is an attractive approach for generating insulin-producing cells for transplantation. However, human islet cell expansion in culture results in loss of β-cell phenotype and epithelial-mesenchymal transition (EMT). This process activates expression of ZEB1 and ZEB2, two members of the zinc-finger homeobox family of E-cadherin repressors, which play key roles in EMT. Downregulation of ZEB1 using shRNA in expanded β-cell-derived (BCD) cells induced mesenchymal-epithelial transition (MET), β-cell gene expression, and proliferation attenuation. In addition, inhibition of ZEB1 expression potentiated redifferentiation induced by a combination of soluble factors, as judged by an improved response to glucose stimulation and a 3-fold increase in the fraction of C-peptide-positive cells to 60% of BCD cells. Furthermore, ZEB1 shRNA led to increased insulin secretion in cells transplanted in vivo. Our findings suggest that the effects of ZEB1 inhibition are mediated by attenuation of the miR-200c target genes SOX6 and SOX2. These findings, which were reproducible in cells derived from multiple human donors, emphasize the key role of ZEB1 in EMT in cultured BCD cells and support the value of ZEB1 inhibition for BCD cell redifferentiation and generation of functional human β-like cells for cell therapy of diabetes.
Highlights
Repressors, which activate epithelial-mesenchymal transition (EMT) by binding to E-box elements in the CDH1 promoter and suppressing its activity[22]
The levels of insulin transcripts were inversely proportional to the levels of ZEB1 transcripts, which were a function of the MOI of the ZEB1 shRNA virus (Fig. 2B)
ZEB proteins have been reported as downstream effectors of major EMT-inducing pathways, some of which already reported by our group to drive EMT during ex vivo expansion of BCD cells
Summary
Repressors, which activate EMT by binding to E-box elements in the CDH1 promoter and suppressing its activity[22]. ZEB1 promotes EMT by repressing expression of basement membrane components and cell polarity proteins. ZEB1 has been found to trigger a micro RNA (miR)-mediated double-negative feedback loop that stabilizes EMT. ZEB1 directly suppresses expression of the miR-200 family, and is one of the predominant targets of these miRs23–27. We show that ZEB1 expression is activated in expanded human islet cells. Inhibiting its expression by shRNA leads to BCD cell growth arrest, mesenchymal-epithelial transition (MET), and redifferentiation. ZEB1 inhibition synergizes with RC treatment, resulting in enhanced BCD cell redifferentiation. Our findings suggest that the ZEB1/ miR-200 feedback loop may mediate the effects of ZEB1 inhibition
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