Virtually all researchers on pancreatic and islet cell biology are familiar with the occasional sight of a single islet cell or small islets seemingly sprouting from the ducts of a section of adult pancreas. The incidence of such observations is amplified under a number of experimental or pathological conditions. For a long time, the obvious conclusion has been that islets might indeed be formed in or near the ducts, migrating at a later point to the acinar surroundings. BrdU labeling studies led to Bonner-Weir and colleagues to hypothesize that pancreatic regeneration in the partially pancreatectomized rat occurs through two pathways, namely: the self-replication of existing endocrine and exocrine cells; and the proliferation and differentiation of the ductal epithelium into new pancreatic lobules consisting of islets, acinar, and ductal tissue in the same proportions normally found in the organ. Pdx1 messenger RNA (mRNA) was detected in pancreatic ducts at a level of approximately 10% that of islets a few days after partial pancreatectomy. When human pancreatic tissue partially depleted of islets (leftovers of clinical islet preparations) was cultured in conditions favorable for ductal tissue expansion, abundant cells coexpressing ductal (CK-19) and beta cell markers (chiefly insulin and Pdx1) were identified. Adult mouse and human ductal cells transduced with adenoviruses expressing Pdx1, Ngn3, Pax4, and NeuroD strongly up-regulated the expression of the insulin gene – with the latter yielding the highest degree of induction.
The “ductal origin” hypothesis suffered a strong setback in 2004, when Dor and co-workers, using a pulse–chase strategy in a mouse transgenic model (see next section), established that adult islet regeneration occurs through self-replication rather than differentiation from non-insulin-producing pancreatic progenitors (see below). However, a very recent report using a similar lineage-tracing experimental design (in which transgenic mice with the ductal-specific carbonic anhydrase II promoter driving Cre recombinase are mated with floxed beta-galactosidase reporter mice) suggests that ductal cells do indeed give rise to new islets and acini both during normal islet turnover and after injury (ductal ligation). This would be in line with our recent finding that the expression of both Pdx1 and insulin was activated in the ductal epithelium of transplanted human pancreata upon recurrence of autoimmunity. However, these cells still retained a hybrid ductal–beta cell phenotype and might just represent an attempt at compensating for the loss of beta cell mass, possibly stimulated by hyperglycemia and chronic inflammation. In a recent study, Hao et al. explored the ability of non-endocrine epithelial cells from the adult pancreas to give rise to endocrine cells. The pancreas is mostly made of two cell types, namely mesenchymal and epithelial. The latter include ductal, acinar, and islet endocrine cell types. Among the former are pancreatic fibroblasts, endothelial cells, vascular smooth muscle cells, and stellate cells. Mesenchymal cells, in general, tend to take over the culture when pancreatic tissues are plated in conditions that favor adherence. However, treatment with the drug G418 is effective at getting rid of mesenchymal cells. The above investigators cultured the byproduct of islet isolation procedures, which were largely devoid of both endocrine (due to the mechanical separation of the islets) and mesenchymal cells (due to G418 treatment). When co-transplanted with fetal islet-like clusters in recipient immunodeficient mice, some of these CK-19-positive “non-endocrine pancreatic cells” differentiated into insulin-, glucagon-, and (more rarely) somatostatin-producing cells. Both the origin (ductal or acinar?) of the cells with this potential and the nature ( bona fide beta cells, or insulin-positive cellular byproducts?) of the differentiated progeny remain to be ascertained.
Do New Beta Cells Arise from the Islet?
A number of studies have pinpointed the origin of new beta cells to stem cells contained within islets. Thus, using the STZ model of regeneration, Fernandes et al. identified a population of somatostatin + /Pdx1 + cells inside the damaged islets. Follow-up of these cells led to the observation that they ended up turning into insulin-positive cells. These putative precursors were similarly observed in nonobese diabetic mice, where beta cell destruction is mediated by an autoimmune response. Similar findings were later reported by Guz et al., who documented islet regeneration in STZ-treated mice that received supportive insulin administration. Beta cell neogenesis was detected during the first week after the restoration of normoglycemia, and two putative beta cell progenitors were identified (Glut2 + and Ins + /somatostatin + ).
These results appear to be in contradiction with those of Dor et al., who also identified the islet as the source of new islets, but through a completely different mechanism .
Do New Islets Arise from the Bone Marrow?
The migration of transplanted bone marrow cells to many different tissues (particularly in response to insults or pathological conditions) is a phenomenon commonly observed both in animalsand humans. This apparent “transdifferentiation” potential of bone marrow cells led to the early hypothesis that they could be the basis of a universal self-repair mechanism – even if it is not normally active under physiological conditions . However, this idea suffered an important setback in 2002 with the publication of two studies showing that multipotent cells can fuse with differentiated ones, therefore adopting their phenotype. This was the case in a experimental setting where wild-type bone marrow transplantation rescued the liver of FAH −/− mice, which are a model of fatal hereditary type I tyrosinemia. Further investigation on the mechanisms behind the rescue revealed that donor bone marrow cells had migrated to the defective liver and fused with resident cells. The ensuing cells were indistinguishable from the local hepatocytes, but the complementation with the wild-type gene of the fused bone marrow cell resulted in a stronger hybrid with a selective proliferative advantage over the non-fused cells. These “corrected” cells eventually took over the liver, restoring function. The burden of the proof was now on those researchers claiming that bone marrow-derived cells could indeed differentiate into the target derivatives. Thus, Ianus et al. transplanted bone marrow cells from male transgenic INS2-EGFP mice into irradiated wild-type female recipients. Up to 3% of the cells within each islet exhibited EGFP expression 4–6 weeks after the procedure, most of them expressing insulin and Pdx1. This could be explained either by direct transdifferentiation of bone marrow cells into beta cells (which would activate the insulin promoter and therefore the reporter) or fusion to resident cells resulting in reprogramming of the donor ones. To rule out the latter, the authors transplanted the bone marrow of male INS2-Cre mice into ROSA-stoplox-EGFP female recipients. The rationale behind this approach was that any cell fusion event would be manifested by the Cre-mediated removal of the stop codon preventing EGFP expression.
Abundant cells containing the Y chromosome were found in the pancreas of the recipient, but none of them was fluorescent. Since forced in vitro fusion of these two types of genetically modified cells did indeed result in EGFP expression, it was concluded that bone marrow cells can contribute to the endocrine pancreas in a fusion-independent fashion. However, two reports published shortly thereafter found little or no evidence of bone marrow transdifferentiation into pancreatic beta cells. Using again a GFP-labeled donor population, the authors of the first study observed fluorescent cells in the islets of the recipient animals, but none of them co-expressed insulin, either in healthy or in STZ-treated animals. The second group extended these studies to another model of pancreatic regeneration (partial pancreatectomy). Despite substantial contribution of the donor cells to blood, lymphatic, and interstitial cells in the pancreas, they could find only two cells positive for GFP in a screening of more than 100,000 beta cells – which turned out to be in control animals. They concluded, therefore, that the bone marrow does not significantly contribute to the endocrine component of the pancreas. A third study confirmed these findings but provided additional evidence that bone marrow-derived endothelial progenitor cells were recruited to the pancreas in response to islet injury, which could be theoretically exploited to improve vascularization and/or endogenous regeneration of injured beta cells.
If bone marrow (BM) cells contributed to islet regeneration, BM derived from GFPpositive donor mice could be tracked upon transplantation into wild-type animals and found in the recipient’s islets. However, this approach does not account for cell fusion.
An alternative approach to rule out cell fusion is the transplantation of BM cells from Ins-Cre mice into recipients in which GFP will not be expressed unless there is a Cre-mediated excision of a stop codon. Cells with a Y chromosome that express insulin within the islets would provide evidence of BM-mediated regeneration. If cell fusion occurred, GFP-positive cells would be detected. In the absence of GFP fluorescence, it could be concluded that the observation is not due to cell fusion.
Tags: Beta cell neogenesis, bone marrow cells, cell fusion, ductal origin hypothesis, ductal-specific carbonic anhydrase, NeuroD, New Beta Cells Arise from the Islet, Ngn3, Pax4, Pdx1, Pdx1 messenger RNA, proliferation and differentiation of the ductal epithelium, single islet cell