Abstract
Background In-vitro expansion of functional beta cells from adult human islets is an attractive approach for generating an abundant source of cells for beta-cell replacement therapy of diabetes. Using genetic cell-lineage tracing we have recently shown that beta cells cultured from adult human islets undergo rapid dedifferentiation and proliferate for up to 16 population doublings. These cells have raised interest as potential candidates for redifferentiation into functional insulin-producing cells. Previous work has associated dedifferentiation of cultured epithelial cells with epithelial-mesenchymal transition (EMT), and suggested that EMT generates cells with stem cell properties. Here we investigated the occurrence of EMT in these cultures and assessed their stem cell potential.Methodology/Principal FindingsUsing cell-lineage tracing we provide direct evidence for occurrence of EMT in cells originating from beta cells in cultures of adult human islet cells. These cells express multiple mesenchymal markers, as well as markers associated with mesenchymal stem cells (MSC). However, we do not find evidence for the ability of such cells, nor of cells in these cultures derived from a non-beta-cell origin, to significantly differentiate into mesodermal cell types.Conclusions/SignificanceThese findings constitute the first demonstration based on genetic lineage-tracing of EMT in cultured adult primary human cells, and show that EMT does not induce multipotency in cells derived from human beta cells.
Highlights
In-vitro expansion of functional beta cells from the limited number of donated adult human pancreata is an attractive approach for generating an abundant source of cells for beta-cell replacement therapy of diabetes
This vector lacks the DsRed2 gene that was present in the previous version, following our finding that DsRed2 expression interfered with sorting of enhanced green fluorescent protein (eGFP)+ cells by FACS
Beta cells infected by both constructs are expected to express Cre protein, Cre will translocate into the nucleus only upon addition of tamoxifen, leading to removal of the floxed DNA fragment in the reporter construct and activation of eGFP expression
Summary
In-vitro expansion of functional beta cells from the limited number of donated adult human pancreata is an attractive approach for generating an abundant source of cells for beta-cell replacement therapy of diabetes. To monitor the fate of cultured human beta cells we developed a lineage-tracing approach based on a Cre-loxP-mediated DNA recombination system delivered by lentivirus vectors [8] Using this system .50% of the insulin-positive cells present in isolated human islets could be labeled with enhanced green fluorescent protein (eGFP). Using genetic cell-lineage tracing we have recently shown that beta cells cultured from adult human islets undergo rapid dedifferentiation and proliferate for up to 16 population doublings. These cells have raised interest as potential candidates for redifferentiation into functional insulinproducing cells. We investigated the occurrence of EMT in these cultures and assessed their stem cell potential
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