Inhibition of ZEB1 expression induces redifferentiation of adult human ß cells expanded in vitro.

In-vitro expansion of functional adult human ß-cells is an attractive approach for generating insulin-producing cells for transplantation. However, human islet cell expansion in culture results in loss of ß-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 ß-cell-derived (BCD) cells induced mesenchymal-epithelial transition (MET), ß-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 ß-like cells for cell therapy of diabetes.

Redifferentiation of expanded human pancreatic ß-cell-derived cells by inhibition of the NOTCH pathway.

In vitro expansion of ß-cells from adult human pancreatic islets would overcome donor ß-cell shortage for cell replacement therapy for diabetes. Using a ß-cell-specific labeling system we have shown that ß-cell expansion is accompanied by dedifferentiation resembling epithelial-mesenchymal transition and loss of insulin expression. Epigenetic analyses indicate that key ß-cell genes maintain open chromatin structure in expanded ß-cell-derived (BCD) cells, although they are not transcribed. In the developing pancreas important cell-fate decisions are regulated by NOTCH receptors, which signal through the Hairy and Enhancer of Split 1 (HES1) transcription regulator. We have reported that BCD cell dedifferentiation and proliferation in vitro correlate with reactivation of the NOTCH pathway. Inhibition of HES1 expression using shRNA during culture initiation results in reduced ß-cell replication and dedifferentiation, suggesting that HES1 inhibition may also affect BCD cell redifferentiation following expansion. Here, we used HES1 shRNA to down-regulate HES1 expression in expanded human BCD cells, showing that HES1 inhibition is sufficient to induce BCD cell redifferentiation, as manifested by a significant increase in insulin expression. Combined treatment with HES1 shRNA, cell aggregation in serum-free medium, and a mixture of soluble factors further stimulated the redifferentiation of BCD cells. In vivo analyses demonstrated the ability of the redifferentiated cells to replace ß-cell function in hyperglycemic immunodeficient mice. These findings demonstrate the redifferentiation potential of ex vivo expanded BCD cells and the reproducible differentiating effect of HES1 inhibition in these cells.

HES-1 is involved in adaptation of adult human beta-cells to proliferation in vitro.

OBJECTIVE: In vitro expansion of beta-cells from adult human islets could solve the tissue shortage for cell replacement therapy of diabetes. Culture of human islet cells typically results in <16 cell doublings and loss of insulin expression. Using cell lineage tracing, we demonstrated that the expanded cell population included cells derived from beta-cells. Understanding the molecular mechanisms involved in beta-cell fate in vitro is crucial for optimizing expansion and redifferentiation of these cells. In the developing pancreas, important cell-fate decisions are regulated by NOTCH receptors, which signal through the hairy and enhancer of split (HES)-1 transcriptional regulator. Here, we investigated the role of the NOTCH signaling pathway in beta-cell dedifferentiation and proliferation in vitro. RESEARCH DESIGN AND METHODS: Isolated human islets were dissociated into single cells. beta-Cells were genetically labeled using a Cre-lox system delivered by lentiviruses. Cells were analyzed for changes in expression of components of the NOTCH pathway during the initial weeks in culture. HES-1 expression was inhibited by a small hairpin RNA (shRNA), and the effects on beta-cell phenotype were analyzed. RESULTS: Human beta-cell dedifferentiation and entrance into the cell cycle in vitro correlated with activation of the NOTCH pathway and downregulation of the cell cycle inhibitor p57. Inhibition of HES-1 expression using shRNA resulted in significantly reduced beta-cell replication and dedifferentiation. CONCLUSIONS: These findings demonstrate that the NOTCH pathway is involved in determining beta-cell fate in vitro and suggest possible molecular targets for induction of beta-cell redifferentiation following in vitro expansion.

Lineage tracing evidence for in vitro dedifferentiation but rare proliferation of mouse pancreatic beta-cells.

Understanding and manipulating pancreatic beta-cell proliferation is a major challenge for pancreas biology and diabetes therapy. Recent studies have raised the possibility that human beta-cells can undergo dedifferentiation and give rise to highly proliferative mesenchymal cells, which retain the potential to redifferentiate into beta-cells. To directly test whether cultured beta-cells dedifferentiate, we applied genetic lineage tracing in mice. Differentiated beta-cells were heritably labeled using the Cre-lox system, and their fate in culture was followed. We provide evidence that mouse beta-cells can undergo dedifferentiation in vitro into an insulin-, pdx1-, and glut2-negative state. However, dedifferentiated beta-cells only rarely proliferate under standard culture conditions and are eventually eliminated from cultures. Thus, the predominant mesenchymal cells seen in cultures of mouse islets are not of a beta-cell origin.

Expansion and redifferentiation of adult human pancreatic islet cells.

Beta-cell replacement represents the ultimate cure for type 1 diabetes, however it is limited by availability of organ donors. Adult human islets are difficult to propagate in culture, and efforts to expand them result in dedifferentiation. Here we describe conditions for expansion of adult human islet cells, as well as a way for their redifferentiation. Most cells in islets isolated from human pancreata were induced to replicate within the first week of culture in expansion medium. Cells were propagated for 16 population doublings, without a change in replication rate or noticeable cell mortality, representing an expansion of over 65,000-fold. Replication was accompanied by a decrease in expression of key beta-cell genes. Shift of the cells to differentiation medium containing betacellulin resulted in redifferentiation, as manifested by restoration of beta-cell gene expression and insulin content. These methods may allow transplantation of functional islet cells from single donors into multiple recipients.