It is now appreciated that both type 1 and type 2 diabetes are diseases of β-cell insufficiency. As such, there is a good deal of interest in mechanisms of β-cell regeneration that hold some therapeutic potential (1). This could apply to increasing numbers of remaining endogenous β-cells in vivo, or in vitro expansion of β-cells derived from isolated human islets, to be subsequently used for β-cell replacement therapy. Regeneration of β-cells may occur by several means, including replication of existing β-cells, generation of new β-cells from pancreatic ductal precursor cells (by a relatively undefined mechanism known as neogenesis), and perhaps transdifferentiation of pancreatic exocrine cells into endocrine β-cells. It has also been suggested that some pancreatic cell types, including β-cells, can undergo a reversible epithelial-mesenchymal transition (EMT) to enable a marked increase in cell numbers (2,3). However, in this issue of Diabetes, Chase et al. (4), by use of lineage-tracing, demonstrate that EMT is unlikely to be a major source of β-cell regeneration.
What is EMT? Epithelial cells have a consistent and polarized morphology that are found in co-adherent organized multicellular assemblies, often in single-cell layers. In contrast, mesenchymal cells are of irregular shape, often with a spindle-like form characteristic of fibroblasts. They are less adherent and relatively unstructured. Mesenchymal cells often exhibit greater rates of cell division and have the capacity for migration into, and through, tissues. A remarkable phenomenon was described in the early 1980s (5) where an epithelial cell can phenotypically morph into a mesenchymal cell, called a “transdifferentiation” event. This process of EMT is now recognized as a basic mechanism for tissue morphogenesis in the developing embryo, being required for many functions such as gastrulation, kidney formation, and neural crest migration. Much recent attention has focused on the role of EMT in the pathology of cancer invasiveness and metastasis, as well as in fibrosis (6). While a number of molecular components/markers have been described, EMT and its complex control mechanisms is a rapidly growing research field, but still in its infancy (7). For example, the reverse process, mesenchymal-epithelial transition, while clearly involved in embryogenesis and likely in cancer metastasis, remains poorly characterized.
Based on morphological appearance, protein marker immunostaining, and RT-PCR analysis, which gives a temporal snapshot of cellular changes during culture of isolated islets, two groups (2,3) have suggested that a β-cell can transdifferentiate into a mesenchymal-like cell with an enhanced growth rate and then redifferentiate back into a β-like “insulin immunopositive” cell via the process of EMT. Chase et al. (4) tested those conclusions using an elegant technique of conditional genetic lineage tracing. They bred mice strains that expressed the cre recombinase either in any cell that had ever expressed insulin (i.e., β-cells [ins2cre]) or in any cell that had ever expressed pdx1 (i.e., all pancreatic duct, exocrine and endocrine cells, or their known precursors [Pdx1cre]). Both strains also contained a cre-sensitive “switchable” reporter system (Z/EG) that was transcriptionally active in all cell types and encoded two different marker gene products. If cre is not expressed in a cell, the first reporter βgeo is expressed, and its termination signal blocks translation of the second reporter, green fluorescent protein (GFP). If cre is expressed, βgeo and its termination signal are permanently excised from the genome of that cell, and its progeny will now always express GFP irrespective of how future cells of that lineage may otherwise change. The prediction is simple: All β-cells in an in2cre-Z/EG mouse islet and any cell derived from a β-cell will be GFP+. After culture of these islets, almost no mesenchymal-like cells expressed GFP. Furthermore, all pancreatic epithelial cells in a pdx1cre-Z/EG mouse will express GFP (since they all are derived from pdx1-expressing progenitors), and any subsequent progeny should also be GFP+. Again, the mesenchymal-like cells did not express GFP. Thus, in either case, the “insulin-expressing cells” arising in islet-like aggregates cannot have been produced by EMT from mesenchymal-like cells. Moreover, the pdx1cre-Z/EG mouse strategy also questions the idea of pancreatic exocrine to endocrine cell transdifferentiation in the process of β-cell regeneration.
Indeed, Bensley, examining regeneration of the endocrine pancreas nearly 100 years ago at the University of Chicago, came to a similar conclusion, stating “I am accordingly of the opinion that the normal regulation of islet content in the pancreas is by interstitial growth of preexisting islets, and by the formation of new islets from the duct epithelium, and not at all by the formation of new islets out of acini” (8). Sometimes modern technology acts as an excellent and convincing confirmation of ideas established quite a while ago.
Nonetheless, the observation remains that “insulin immunopositive cells” can emerge from in vitro manipulations designed to increase β-cell numbers (2,3,9). Indeed, though a very rare occurrence, Chase et al. (4) presented this finding in their study too, but it seems unlikely, at least in mice, that this occurs via a reversible EMT route. Although this is a “first observation,” it needs to be substantiated (especially in human islet cells); however, the bar will be raised high for future studies to be convincing. The difficulties and pitfalls of using immunofluorescence (10) and RT-PCR gene expression analysis (prone to amplification artifacts) need to be realized to eliminate technical artifacts for assessing insulin-positive cells. Alternative approaches to evaluate β-cell phenotype and function need to be applied in order to credibly demonstrate that insulin-positive cells derived from attempts to promote β-cell regeneration indeed do have the potential for a differentiated β-cell’s capabilities.
Finally, some might dismissively claim that Chase et al.’s findings (4) are essentially negative. Maybe so, but this is an example of how a negative study could have excellent scientific value to the diabetes research community by guiding toward other paths of β-cell regeneration that now hold more promise for eventual therapeutic applications. Time will tell whether this holds true, but, for the moment, it seems that EMT participation in mechanisms of β-cell expansion has been preempted.
See accompanying Rapid Publication on pg. 3.