Liver transdifferentiation • Pdx1 • VP16 • Reprogramming • Hepatocytes

Expression of Pdx1 in the foregut (e8) is one of the earlier molecular events that mark the specification of the pancreas as a separate organ (see the chapter “Pancreatic Development”). The role of Pdx1 as a “master regulator” of pancreatic development has led many investigators to test whether its ectopic expression would induce pancreatic differentiation by itself. This strategy has yielded somewhat modest results in most cellular substrates examined which suggests that Pdx1 expression is necessary, but not sufficient, to initiate pancreatic development. The conclusion from the observation that the initial evagination of the pancreatic epithelium occurs even in the absence of Pdx1.A possible exception to this rule, however, is observed when the target tissue is liver.In fact, there is a wealth of studies indicating that the liver and pancreas are especially susceptible to interconversion. Many invertebrates have a single organ that comprises both hepatic and pancreatic functions, which suggests that the separation of these two organs is a relatively late evolutionary event. In vertebrates, fibroblast growth factor (FGF) signals from the cardiac mesoderm have been shown to play an essential role for the ventral endoderm to differentiate into early hepatic cells and it has been demonstrated that both organs originate from common endodermal progenitors in the early foregut.According to the model described by Deutsch et al., 662 cardiac FGF will have inductive and blocking effects on liver and pancreas specification, respectively.

In general, hepatocytes and beta cells share not only many developmental features but also similar molecular machinery for glucose sensing and secretion. Many studies confirm that interconversion of liver and pancreas occurs under a variety of experimental conditions, including copper depletion in rats treatment with dexamethasone or diethylnitrosamine and certain tumoral processes.

FGF signalling from the cardiac mesoderm will induce liver specification proximally ( dotted region ), but will have a blocking effect on the distal portion of the ventral foregut, which will become pancreas ( stripes ) (Adapted from Deutsch et al.

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.

Keywords Foregut epithelium • Pancreatic buds • Pdx1 • Ptf1a • Ngn3 • Secondary transition

For obvious reasons, most of our knowledge on pancreatic development comes from the mouse model. Indeed, despite a few minor differences that will be pointed out throughout this chapter, the most important molecular players are highly conserved between mouse and human. Research conducted over the last decade has outlined a basic “roadmap” of the major molecular events that shape mouse beta cell development from the early blastocyst. Critical developmental milestones are: generation of definitive endoderm/gut epithelium; pancreatic differentiation; endocrine specification; and beta cell differentiation. We will now describe what is known about this process, emphasizing the role of the genes that act as master regulators of the transition between each stage and the next.

Fertilized egg
Morula
Blastocyst Inner Cell Mass (ICM)
Primitive ectoderm
Definitive endoderm
Primitive gut tube
Posterior foregut
Pancreatic endoderm
Endocrine precursor - Exocrine precursor
Islet endocrine cells – Acinar tissue

Generation of Endoderm/Gut Epithelium

Primitive endoderm and epiblast are, respectively, the outer and inner layers of the inner cell mass (ICM) immediately before gastrulation . The primitive endoderm will become part of the yolk sac, without contribution to the embryo proper. In contrast, the definitive endoderm is formed during gastrulation when epiblast cells leave the ICM through the primitive streak. There is an intermediate stage in definitive endoderm formation, called mesendoderm . Although visceral and definitive endoderm are similar, mesendoderm-specific genes such as goosecoid ( Gsc ) and Brachyury ( Bry ) do not appear during visceral endoderm differentiation, and therefore can be used to identify true definitive endoderm. The anterior part of the definitive endoderm will evolve into the foregut, from which pancreas, liver, and lungs will eventually bud out. The posterior definitive endoderm, on the other hand, becomes the midgut and hindgut, which will differentiate into large and small intestine. Nodal, a member of the transforming growth factor (TGF)- b family, is the main signaling molecule responsible for the initial patterning of the primitive gut epithelium. The gradients of Nodal are finely tuned, as shown in experiments where significant reductions in its expression resulted in preferential formation of mesoderm at the expense of endoderm. Additional studies imply that the regulation of Nodal gradients is a dynamic process that involves not only the secretion of the protein, but also the activity of specific repressors such as Drap1 .

Many genes have been associated with the formation of true endoderm, including Foxa1–2 , Mixl 1, Eomes , GATA4–6 , and several members of the Sox family . Although there is a potential redundancy with other Sox genes, Sox 17 is essential for embryonic cells to become endoderm in mouse. At least in Xenopus , Sox 17 also appears to be sufficient to induce endodermal fates.

Pancreatic Differentiation

The interaction between the gut endoderm and the surrounding mesoderm is primarily mediated by Sonic Hedgehog ( Shh ) signaling. Shh is highly expressed throughout the gut epithelium, but is down-regulated in a Ptf1a(p48) / Pdx1 + region that will later become the pancreas at e8. Both Shh repression and activation of Ptf1a and Pdx1 are defining events of pancreatic specification. Chemical inhibition of Shh by the steroid alkaloid cyclopamine enhances pancreatic differentiation, as Pdx1 expression is no longer restricted throughout the posterior foregut. Conversely, ectopic expression of Shh under the control of the Pdx1 promoter induces intestinal fates (including smooth muscle and interstitial cells of Cajal) instead of pancreatic fates .

A theoretical model for the molecular interplay leading to the development of definitive endoderm from mesendoderm. Nodal signaling is essential for the specification of mesendodermal progenitors. Definitive endoderm formation requires the concerted activity of Mixl1, b -catenin, and Tcf2 (HNF-1 b ). Mesoderm specification, in contrast, is influenced by Fgfr1, Tbx6, Brachyury, and Wnt3a. Different requirements for Foxa2, Sox17, and Nodal are found throughout the gut endoderm.

The pancreas is specified from a region of the embryonic foregut where Shh expression has been excluded due to active signaling from the notochord and surrounding mesenchyme. This region will express the pancreatic and duodenal homeobox 1 (Pdx1), as well as Ptf1 . A transversal cut of the foregut at this point would give a pattern similar to that depicted to the right , bottom : top and bottom Pdx1 + /Shh − areas, which will form the dorsal and the ventral pancreatic buds upon evagination, and a middle , Pdx1 − /Shh + region with pro-intestinal cells.

During regular pancreatic development, an area is defined in the posterior foregut in which Pdx1 expression occurs at the expense of Shh. This patterns the early pancreas as two Shh-excluded regions that will bud out dorsally and ventrally. Shh + areas, in contrast, will adopt an intestinal fate. Ectopic expression of Shh under the expression of the Pdx1 promoter will extend the latter phenotype in every direction, preventing appropriate pancreatic specification.

Pdx1

The pancreatic and duodenal homeobox 1 gene is also known as insulin promoter factor 1 (Ipf1) or islet/duodenum homeobox 1 (IDX1). In the adult mouse, it is selectively expressed in islet beta cells, where it binds to and regulates the insulin promoter. Pdx1 is first expressed in the region of the foregut endoderm that will later become the pancreas and the duodenum ( ~ e8.5 or 10-somite stage, see main text). Up to ~ e10, it is uniformly expressed in the dorsal and pancreatic buds. Pdx1 is subsequently down-regulated in the entire organ, to reappear again in arising beta cells from e11 onward. Lack of Pdx1 expression results in selective agenesis of the pancreas, both in knockout mice and in humans with a single-nucleotide mutation. However, it was also shown that the earlier events of pancreatic morphogenesis take place even in the absence of functional Pdx1, which suggests that Pdx1 acts in concert with other factors. In addition to its well-studied role during pancreatic development, expression of Pdx1 is essential for the maintenance of the phenotype in adult beta cells, as evidenced by conditional knockout experiments. Heterozygous Pdx1 +/− mice exhibit an age-dependent worsening of glucose tolerance, reduced glucose-stimulated insulin release, and higher susceptibility to apoptosis. The impaired glucose response of Psammomys obesus , a model of type 2 diabetes, was also associated to Pdx1 deficiency. Because of its critical role in orchestrating the early events of pancreatic development, as well as in the acquisition of beta cell properties, Pdx1 has been extensively used as a tool for the differentiation of stem cells.

In the mouse, the areas defined by expression of Pdx1 and repression of Shh will start to branch out dorsally and ventrally. This initial separation between the dorsal and the ventral pancreas will persist until later in development, when the two primordia will fuse. The influence of blood vessels in the overall development of the pancreatic primordia is well established. Thus, while removal of the dorsal aorta in frog embryos abrogated insulin expression, transgenic mice where the posterior foregut was ectopically vascularized developed hyperplasic islets and elevated insulin expression. It is in this context that endothelial cell signaling has been identified as a major morphogenetic agent in pancreatic specification.

Ptf1 a (p48) Ptf1 a is the a -subunit of the pancreas-specific transcription factor 1 (Ptf1), a basic helix-loop-helix (Bhlh) protein first described as a DNA-binding element regulating the expression of a -amylase 2, elastase 2, and trypsin in the acinar pancreas. p48 knockouts have a complete absence of exocrine pancreatic tissue, suggesting that the gene is a key regulator of acinar tissue development. This role was confirmed by the finding that endocrine cells (relocated to the spleen) were not affected by the abrogation of p48 expression. Later studies, however, found an additional role for p48 in the initiation of pancreatic development, because its expression is observed in the Shhexcluded area of the foregut endoderm around e8.5. The expression patterning at this stage of p48, but not that of Pdx1, is thought to be partially mediated by aortal endothelial signaling. In Xenopus , the combination of both Pdx1 and p48 expression was sufficient to induce ectopic pancreatic formation, but the initiation of mouse pancreatic development might require additional genes, such as Hlxb9 .

HNF-6 (OC-1)

Hepatocyte nuclear factor (HNF-6), also termed Onecut (OC)-1, is a member of the OC family of transcription factors, generally characterized by a single cut domain and a homeodomain distinct from that of other homeoproteins, including those of the cut subfamily. During embryonic development, it is highly expressed in the developing central nervous system (CNS) and from e9.5 in the foregut–midgut junction and liver primordium. Pancreatic expression is detectable throughout the epithelium from e10.5 onward, although it seems to be excluded from the islets at e18. Pancreatic growth and endocrine cell differentiation were severely impaired in Hnf-6 knockout mice, with an almost total abrogation of Ngn3 expression. The same authors demonstrated that Ngn3 is indeed a downstream target of Hnf-6. Interestingly, however, islets were able to “regrow” after birth. This is consistent with the view that adult islet regeneration occurs typically through Ngn3-independent processes, with only one known experimental exception (in which reactivation of the embryonic developmental program was observed after partial duct ligation; see the chapter “Pancreatic Regeneration”). Notwithstanding this, the newly generated beta cells were defective in Glut-2 and these animals remained diabetic.Additional studies demonstrated not only that Hnf-6 expression precedes that of Pdx1 in the foregut endoderm, but also that the expression of the latter is delayed in Hnf6 −/− embryos; and (2) Hnf-6 binds to the Pdx1 promoter and stimulates its activity.

TCF2 (HNF 1beta)

Transcription factor 2 (Tcf2), also called hepatocyte nuclear factor (HNF) 1beta is a POU homeobox transcription factor that has been associated with a variant of maturity-onset diabetes of the young (MODY). Other mutations of the gene result in pancreatic atrophy and hypoplasia in humans. The gene is highly expressed from e8.5 in the entire foregut–midgut region and in the pancreatic primordia by e9.5, where it colocalizes with Ptf1 a and Pdx1.

Although Tcf2 −/− knockout mice display early embryonic lethality due to defective formation of the visceral endoderm, tetraploid rescue with Tcf2 −/− embryonic stem (ES) cells results in embryos that can proceed throughout development. In these embryos, the formation of the dorsal, but not the ventral pancreatic bud could be observed. However, this bud was hypoplastic throughout development and disappeared around e13.5. This phenotype is similar to that of Ptf1 a knockouts, albeit more severe; indeed, a Tcf2-binding site was identified in the Ptf1 a promoter, which would be consistent with a role of the former in the regulation of the latter. 92 Pdx1 expression, however, was still detectable at e9.5 in Tcf2 −/− embryos, suggesting that the latter is not absolutely essential for the initiation of the pancreatic program. Experimental evidence indicates that both Hnf6 and Tcf2 are indispensable for Ngn3 expression.

While branching and the progression of differentiation are arrested in Pdx1-null embryos (lack of Pdx1 results in pancreatic agenesis ), the initial evagination of the pancreatic buds, and even the appearance of scattered insulin- and glucagonpositive cells, does still occur in the absence of Pdx1 . Recent evidence suggests that the expression of Ptf1 a , previously thought to be exclusively a marker of exocrine progenitor cells, may actually precede that of Pdx1 . Additional experimental evidence (e.g., simultaneous ectopic expression of both Pdx1 and Ptf1a induces stable conversion of posterior endoderm into pancreas ) seems to confirm that the concerted action of both is necessary for the initiation of the pancreatic program.

Hlxb9

Human homeobox gene 9 ( Hlxb9 ), also known as its encoded protein, HB9, is expressed in fully differentiated beta cells and from very early on (eight somite stage, ~ e8) in the notochord and the ventral and dorsal pancreatic endoderm. Pdx1 , in contrast, is expressed only in the ventral pancreatic endoderm at this stage of development. The observation that Hlxb9 expression precedes that of Pdx1 (at least in the dorsal anlagen) suggests an active role of this gene in shaping the early events of pancreatic specification. Hlxb9 knockouts show a selective agenesis of the dorsal pancreas. Although the ventral lobe still develops, its islets are smaller and beta cells within them less numerous, with evident reduction in beta cell-specific factors such as Nkx6.1 and Glut2.

Ectopic expression of Hlxb9 beyond e8 in Pdx1 – Hlxb9 transgenic mice led to severe impairment in pancreatic development, with decreased endocrine and exocrine differentiation and a partial adoption of intestinal fates.