Gene transfer of neurogenin3 and betacellulin reverses hyperglycemia and restores glucose-stimulated insulin secretion in insulin-deficient diabetic mice
Diabetic mice with stable hyperglycemia for at least a week were administered a single IV injection of HDAd-Ngn3, which resulted in a rapid reversal of hyperglycemia within a week and the significantly reduced blood glucose was sustained for about two months, beyond which hyperglycemia recurred (). Betacellulin (Btc), an islet growth factor, was added to HDAd-Ngn3 to amplify the Ngn3 effects (Kojima et al., 2003
). HDAd-Ngn3 combined with HDAd-Btc led to a rapid reversal of hyperglycemia within a week, which, in contrast to Ngn3 alone, was sustained for at least 6 months (). Empty vector and Btc alone had no effect on the hyperglycemia (). Ngn3-Btc-treated mice also gained weight () and displayed little hepatotoxicity, as evidenced by normal serum transaminases ().
Ngn3-Btc reverses diabetes in insulin-deficient mice
Fasting plasma insulin () was normalized in a sustained fashion by Ngn3-Btc gene transfer at a level comparable to nondiabetic controls. In contrast, empty vector-treated diabetic mice remained hyperglycemic with barely detectable plasma insulin levels. Insulin tolerance test indicated that a difference in insulin sensitivity did not account for the euglycemia (Supplemental Figure 1
An IP glucose tolerance test (GTT) showed that Ngn3-Btc-treated mice had a normal glucose disposal, indistinguishable from that of nondiabetic controls (), with the GTT-stimulated plasma insulin excursions also being similar between the two groups (). In contrast, empty vector-treated diabetic mice were profoundly glucose intolerant with no change in plasma insulin in response to the glucose load. Thus, Ngn3-Btc treatment restored glucose-stimulated insulin secretion (GSIS) in vivo. We also excluded endogenous islet regeneration as a significant source of the insulin in these animals ().
It is important for β-cells to sense low blood glucose and shut off insulin secretion to forestall severe hypoglycemia. Blood glucose levels during a 72 hour fast in Ngn3-Btc treated diabetic and in nondiabetic control mice () were indistinguishable between the two groups, indicating that insulin secretion in the Ngn3-Btc treated diabetic mice was appropriately turned off with fasting. Similar results were obtained in nondiabetic mice, with intact pancreatic islets, treated with Ngn3-Btc (Supplemental Figure 2A
Ngn3 and betacellulin treatment induces insulin transcript and protein in the liver
mRNA were expressed, by q-PCR, in the liver of Ngn3-Btc-treated mice, both in the early and late time points, and also in the liver of Ngn3-only treated mice in the absence of Btc co-treatment (). Determination of the total insulin content of the liver by acid-ethanol extraction revealed that insulin was readily detectable after Ngn3-Btc, its level reaching a plateau at 12-16 weeks after treatment, at which time the insulin content/mg total protein was ~ 20 % that of the nondiabetic pancreas (, top panel). We estimated the total liver insulin content 12 weeks after treatment to be similar to that of normal pancreas (, middle panel) since the liver is 4-7 times the size of the pancreas, and also proportionate to the total body weight, similar to that seen with nondiabetic pancreas (, bottom panel). Similar results were obtained for total liver C-peptide content (Supplemental Figure 3
). The presence of insulin mRNA and C-peptide in the Ngn3-Btc liver confirmed that the insulin measured in the liver of the treated mice was actually synthesized in the liver as opposed to that taken up from the plasma. Thus, Ngn3-Btc treatment restores a full complement of insulin to the liver comparable to that in normal pancreas.
Ngn3-Btc induces regulated insulin production from the liver
Ngn3 and betacellulin treatment induces glucose-stimulated insulin secretion (GSIS) from the liver
To determine if the liver is the source of the GSIS, we performed an in situ
liver perfusion experiment () wherein we cannulated the portal vein and infused solutions containing different concentrations of glucose and measured insulin and glucose in the liver effusate (). In contrast to empty vector treatment, Ngn3-Btc treatment led to GSIS as evidenced by a stepwise increase in insulin secretion in response to a stepwise increase in glucose concentration in the infusate from 2.8 mM to 11.1 mM and 25 mM (, bottom panel). Though this is lower than that seen with pancreas perfusion or islet perifusion studies, this magnitude of response is similar to that observed in perfusion studies on livers containing transplanted islets (Lau et al., 2007
; Mattsson et al., 2004
). Furthermore, the Ngn3-Btc-treated liver responded to a 0 mM glucose infusion by rapidly decreasing its insulin production, indicating that hepatic insulin production is tightly controlled by the glucose concentration of the infusate. Addition of glibenclamide (10 μM, abbreviated SU for sulfonylurea in , middle panel), a secretagogue belonging to the sulfonylurea class of drugs used to treat diabetic patients, to the infusate markedly stimulated insulin production only from the liver of Ngn3-Btc-treated mice (, bottom panel).
We also performed an in vitro
GSIS assay to complement the in situ
study. We repeated the gene delivery experiment in transgenic mip-GFP mice, which express eGFP driven by the mouse insulin promoter (Hara et al., 2003
). The same Ngn3-Btc regimen also reversed diabetes in STZ-diabetic mip-GFP mice. GFP-positive but not GFP-negative cells from the liver of these mice, isolated by flow cytometry, displayed a significant increase in insulin secretion when the concentration of glucose in the medium was increased from 2.8 to 25mM (). Incubation of untreated hepatocytes failed to display GSIS into the medium.
Ngn3 treatment induces an islet-like transcription profile in the liver
We asked if Ngn3 treatment induced the complete set of islet hormones in the liver, in addition to insulin. By q-PCR, both Ngn3-Btc-treated and Ngn3-only-treated, but not Btc- or empty vector-treated, liver produced readily detectable levels of transcripts of the other major islet hormones, viz., glucagon (Gcg), pancreatic polypeptide (PP), and somatostatin (Sst) ().
This suggests that Ngn3 is capable of activating a cascade of transcription factors that collectively specify the different islet cell lineages, a prediction that is corroborated by direct quantification of the transcripts for multiple transcription factors, including Pdx1, Neurod1, Isl1, Pax4, Pax6, Nkx2.2 and Nkx6.1 by q-PCR in both the Ngn3-Btc- and Ngn3-only treated liver (). All these factors are expressed during fetal endocrine pancreas development, and all of them except Pax4 (Sosa-Pineda et al., 1997
) are known to be expressed in the adult pancreas as well. Interestingly, unlike normal adult pancreatic islets, Ngn3-Btc- and Ngn3-only treated liver continued to express Pax4. We note that induced expression of Ngn3 in concert with HNF-1α can directly activate Pax4 expression (Smith et al., 2003
) and that Btc itself has been shown to induce Pax4 (Brun et al., 2008
). Pdx1, a pancreas-defining transcription factor upstream of Ngn3, was also found to be expressed with Ngn3-Btc and Ngn3-only treatment. This is not unexpected as Pdx1 is normally expressed in mature β-cells and is required for normal insulin secretion (Holland et al., 2005
). Interestingly, ectopic Pdx1 expression by gene transfer (Kojima et al., 2003
; Ber et al., 2003
) or in transgenic mice (Miyatsuka et al., 2003
) was shown to lead to pancreatic exocrine enzyme expression in the liver, but the appearance of Pdx1 in the Ngn3-induced neo-islets did not lead to exocrine enzyme expression (Supplemental Figure 4
Other β-cell specific transcripts essential for normal β-cell function, such as sulfonylurea receptor-1 (Abcc8
), Kir6.2 (Kcnj11
) and the prohormone convertases 1/3 (Pcsk1
) and 2 (Pcsk2
) were expressed in the Ngn3-Btc- and Ngn3-only treated liver (, Supplemental Table 1
). Therefore, Ngn3, with or without Btc, induces in the liver the developmental cascade of transcription factors that are normally seen in developing islets, along with other molecules characteristic of mature pancreatic islets.
Ngn3 induces two waves of insulin production – First wave occurs in parenchymal hepatocytes
Ngn3-Btc gene transfer led to the presence of immunoreactive insulin by 3 weeks in parenchymal hepatocytes (). However, by 6 weeks most of the insulin immunofluorescence had disappeared () and by 12 weeks, insulin was no longer detectable in parenchymal hepatocytes. This transient induction of insulin production in hepatocytes was sufficient to suppress the elevated PEPCK expression (Supplemental Figure 5
), a key gluconeogenic gene, in diabetic mice back to that of a nondiabetic control.
Ngn3-Btc induces two waves of insulin expression – first in parenchymal hepatocytes and later in periportal neo-islets
Second wave of insulin production occurs in neo-islets in the periportal region
Ngn3-Btc-induced reversal of hyperglycemia occurred within days, and was associated with the production of immunoreactive insulin early in the hepatocytes. However, the normalization of blood glucose, plasma insulin and insulin content in the liver was sustained beyond 6 months after Ngn3-Btc even after insulin immunofluorescence had disappeared from the hepatocytes ( and ). Further examination revealed that as the insulin expression was waning in parenchymal hepatocytes (size ~20-40 μM), there was the emergence of clusters of cells that were significantly smaller than hepatocytes in the periportal region that stained strongly positive for immunoreactive insulin 6 weeks after Ngn3-Btc (). Over time, there was a gradual increase in the size, number and density of these periportal insulin-positive cell clusters (). As in Ngn3-Btc-treated mice, in Ngn3-only animals without Btc, the disappearance of insulin-positive hepatocytes coincided with the appearance of periportal insulin-positive cell clusters (), indicating that Btc was not required for the induction of these cells, though the addition of Btc did augment the response. Neither the first nor the second wave of insulin production was observed in any of the empty vector-treated livers ().
On higher magnification, the periportal insulin-positive cells in Ngn3-Btc-treated mice have a granular cytoplasm that stains positive with anti-insulin antibody (). Another notable feature of these periportal islet-like clusters of insulin-positive cells is their size (7-10 μm) and high nuclear-cytoplasmic ratio, a morphological characteristic of hepatic oval cells. Most of the portal triads appeared to harbor some insulin-positive clusters, though the size in terms of the number of cells per cluster varied (Supplemental Figure 6
). Significantly, very few of these types of cells occurred in the portal region in the empty vector-treated livers, none of which stained positive for insulin (). We also detected immunoreactive proinsulin only in the liver of Ngn3-Btc- () and Ngn3-only-treated mice () that was present in islet-like periportal cell clusters. The presence of proinsulin in the periportal cells is consistent with the insulin being produced, and not taken up, by these cells in response to Ngn3 treatment.
The Ngn3-Btc-induced first and second wave of insulin production in different cell types in the liver was highly reproducible, not only in multiple experiments in wild-type STZ-diabetic mice, but also in STZ-diabetic mip-GFP mice, using GFP as a surrogate marker for insulin production (see Supplemental Text and Supplemental Figure 7
Neo-islets express multiple islet hormones
The first wave of insulin expression in hepatocytes in mice treated with Ngn3, with or without Btc, was not associated with detectable amounts of non-insulin islet hormones either by immunostaining or by RT-PCR of RNA obtained by LCM (see later). On the other hand, these other islet hormones were easily detected in the periportal neo-islets; Gcg, PP and Sst-expressing cells appeared along with or just before the appearance of insulin positive cells in the periportal region of both Ngn3-Btc () and Ngn3-only (Supplemental Figure 8D-L
) treated mice. Indeed, a unique observation is the occurrence of individual hormone-expressing cells in these neo-islets suggesting that the Ngn3, with or without Btc, initiates a complete islet development program in the periportal cells in the liver. We note, however, that some of the insulin-positive cell clusters weakly co-express other hormones, a feature also seen in regenerating pancreatic islets (Fernandes et al., 1997
; Guz et al., 2001
). In contrast to the spatial organization of normal rodent islets, which present with a central core of β-cells surrounded by a peripheral rim of α-cells and scattered PP and δ-cells, the periportal neo-islets are loosely clustered with intervening nonhormone-producing cells that have not committed to the islet cell lineage (, and Supplemental Figures 8F, 8I and 8L
). None of the islet hormones were detected in the livers of empty vector treated diabetic mice (Supplemental Figure 8A-C
) or in the non-diabetic control mice (data not shown), in keeping with the mRNA data (). Therefore, in response to Ngn3 gene transfer, the neo-islets that have arisen from periportal cells express insulin as well as other major islet hormones. We next examined the lineage origin of these cells.
Ngn3-Btc-induced neo-islets express individual islet hormones
Bone marrow-derived cells are not the source of hepatic neo-islets
To determine whether the insulin-positive cell clusters arose from bone marrow-derived cells, we performed lineage tracing with bone marrow transfer experiments (see Supplemental Text
), which excluded both bone marrow-derived fusion cells and other bone marrow-derived cells in the liver (Supplemental Figure 9
), including circulating blood cells, Kupfer cells and stellate cells, as the source of the insulin-producing cells.
Lineage tracing establishes that oval cells express albumin in their lineage history
We designed lineage tracing experiments to determine whether parenchymal hepatocytes and oval cells, which are not derived from bone marrow cells, are involved in the two waves of insulin production that occur after Ngn3-Btc treatment. Lineage tracing of hepatocytes is well established. However, to date, there is no known transgenic reporter mouse that can be used for tracing the origin of oval cells. The timing of albumin expression during mouse embryonic development suggests that albumin promoter-driven reporter gene expression could be used as a lineage marker not only for hepatocytes, but also for oval cells. Embryologically, the liver is formed from the developing foregut. It is noteworthy that the foregut endoderm expresses albumin in the foregut precursor cells as early as e9.5 days of mouse embryo development, prior to cell aggregation and liver formation (Cascio and Zaret, 1991
). Hence, the early albumin-expressing foregut endoderm encompasses precursor cells that develop into multiple cell types in the liver, except for those derived from bone marrow. While albumin expression has been used for tracking parenchymal hepatocytes, whether transgenic albumin-promoter driven reporter gene expression can be used for lineage tracing of oval cells has not been explored.
To determine if such a strategy would work, we used bigenic mice resulting from a cross between a ROSA-Stop-Lox-eGFP reporter mouse and an albumin promoter-Cre mouse. In these animals, all cells that expressed albumin anytime in their lineage history also express eGFP. As there are very few oval cells in untreated mouse liver, we utilized the model of D-galactosamine-induced oval cell proliferation (Lemire et al., 1991
; Hsiao et al., 2000
) in which oval cells appear in the periportal region within days of treatment. We sacrificed the bigenic mice 10 days after D-galactosamine treatment and identified oval cells in the periportal region by their typical morphology. These oval cells all express eGFP, indicating that hepatic oval cells are derived from cells that expressed albumin in their lineage history (Supplemental Figure 10
) and that these mice are indeed a useful reporter model to trace not only the hepatocyte lineage (Magnuson, 1992
), but also the oval cell lineage, a finding that has not been appreciated in the past.
Cells expressing the first wave of insulin come from hepatocytes and the second wave from periportal cells
Having established that, like parenchymal hepatocytes, oval cells express albumin in their lineage history, we used ROSA-Stop-Lox-eGFP/albumin-Cre bigenic mice to trace the origin of both parenchymal hepatocytes and oval cells. We rendered these mice diabetic by STZ and treated them with either an empty vector or Ngn3-Btc. The diabetic bigenic mice displayed Ngn3-Btc-induced diabetes reversal as in wild-type diabetic mice. We sacrificed the mice at various times afterwards to assess if the insulin-positive cell clusters were eGFP positive. All parenchymal hepatocytes were eGFP positive (), including hepatocytes that were insulin-positive within 3 weeks of Ngn3-Btc treatment (). At later time points when insulin immunostaining had disappeared from the parenchymal hepatocytes, like D-galactosamine-induced oval cells, all periportal insulin-positive oval cell clusters were also eGFP positive ().
Lineage tracing demonstrates that cells expressing the first wave of insulin come from hepatocytes and the second wave from periportal oval cells
Use of bigenic mice indicates that both the first wave and second wave of insulin-positive cells express albumin in their lineage history. However, the experiment did not reveal whether albumin was currently being produced by these cells. We therefore performed a parallel gene transfer experiment in which we assessed liver sections for immunoreactive albumin and insulin expression by immunofluorescence. We found that while immunoreactive albumin was indeed present in the typical parenchymal hepatocytes that also produced insulin 3 weeks after Ngn3-Btc treatment ( and Supplemental Figure 11
), it was absent from the insulin-positive periportal small cells that had the typical morphology of oval cells (). These complementary experiments further support the conclusion that parenchymal hepatocytes constitute the initial wave (at 3 weeks) of insulin-producing cells. Importantly, they indicate that the periportal insulin-positive cells originate not
from hepatocytes, but from periportal cells that appear to be oval cells, based not only on their location and morphology, but also by the absence of immunoreactive albumin.
Ngn3-induced periportal neo-islets appear to originate from oval cells
To further investigate the origin of the insulin-producing cells induced by Ngn3 with or without Btc, we next examined the liver of diabetic mice at different times after treatment by immunohistochemistry. We found little change in morphology within a week of Ngn3-Btc treatment (data not shown). At 3 weeks, we observed the appearance of small cells (~7-10 μm in diameter) with a high nuclear-cytoplasmic ratio in the periportal areas and radiating outwards (), which became more restricted to only the periportal regions by 6 weeks and beyond ( and Supplemental Figures 12A-B
). The classic morphological characteristics of periportal location, size, and high nuclear-cytoplasmic ratio, identify these newly appearing cells as oval cells. Once again, there are more of these cells in the liver of Ngn3-Btc-treated as compared to Ngn3-only-treated mice. These cells appeared in abundance only in the Ngn3-treated (Supplemental Figures 12A-B
) and Ngn3-Btc-treated livers (), and rarely in the empty vector-treated () or nondiabetic livers (not shown). They displayed immunoreactivity toward an oval cell-specific monoclonal antibody A6. The pattern of all insulin-producing cells being A6-positive leads us to conclude that the neo-islets are derived from oval cells. At 3 weeks, there was little insulin immunostaining in these oval cell clusters (). By 6 weeks and later, however, groups of strongly insulin-positive cells appeared among the oval cell clusters, co-expressing A6 reactivity in both Ngn3-Btc ( and Supplemental Figure 13
) and Ngn3-only- treated (Supplemental Figure 14
Ngn3-Btc-induced periportal neo-islets originate from oval cells
Oval cells express other antigens, e.g., CK-19 and Thy1, which are also expressed in other cell types such as biliary cells (Crosby et al., 1998
) and hematopoietic progenitors (Miller and Lipton, 1983
), respectively. However, the concomitant expression of CK-19 and Thy1 has been reported only in hepatic oval cells (Petersen et al., 1998
). Indeed, we detected both CK-19 (, Supplemental Figure 13 and 14
) and Thy1 (Supplemental Figure 12C-F
) immunoreactivity in all insulin-producing periportal cells at all time points. While this unique staining pattern further establishes the oval cell origin of the newly formed islets, we note that as transgenic reporter mouse models that genetically track oval cell lineage exclusively are not currently available, we cannot rule out the possibility that other albumin lineage positive cells, not originating from the bone marrow, could have de-differentiated and acquired an oval cell morphology and expression pattern after Ngn3 treatment. It is interesting that CK-19 has also been observed in insulin-positive cells during islet neogenesis (Gao et al., 2005
) and in islet progenitors in the pancreas during islet regeneration (Xu et al., 2008
). Therefore, the Ngn3-induced neo-islets are more similar to neo-islets in the pancreas than they are to mature pancreatic islets.
We analyzed the global expression profile of LCM-captured Ngn3-Btc-induced neo-islets and mature pancreatic islets, as well as hepatocytes and non-transduced oval cells by cDNA microarray. Correspondence analysis, an unbiased method whereby both genes expressed and individual samples can be projected into the same multi-dimensional space and analyzed simultaneously (Fellenberg et al., 2001
), reveals that induced periportal neo-islets cluster much more closely with pancreatic islets than either one with oval cells or hepatocytes (, also see Supplemental Methods
Ngn3 induces islet neogenesis via transdetermination of oval cells and not transdifferentiation of hepatocytes
Ngn3 induces a full-fledged β-cell transcription program in proliferating periportal cells but an aborted transcription cascade in hepatocytes
To address the mechanism that underlies the differential response of parenchymal hepatocytes vs. periportal cells to Ngn3 expression, we analyzed the expression of different transcription factors that characterize normal β-cells. We performed LCM to obtain RNA specifically from small periportal cells and from larger parenchymal hepatocytes at different times after Ngn3-only or Ngn3-Btc gene transfer, using LCM-RNA from normal pancreatic islets as controls. By RT-PCR, we readily detected Ins1 mRNA in parenchymal hepatocytes at 3 weeks, but the transcript almost completely disappeared by 6 weeks () with both Ngn3-only and Ngn3-Btc treatment. Ins2 mRNA was barely detectable and only at the 6 week time point in these hepatocytes with Ngn3-Btc treatment. The time course of insulin mRNA expression correlates with that of immunoreactive insulin staining presented in and . We failed to detect Ins1 or Ins2 expression at 12 weeks in hepatocytes, despite the continued expression of vector-driven Ngn3. We also did not detect any of the other islet hormone transcripts in the parenchymal hepatocytes at any of the time points. None of the islet-defining transcription factors are expressed in the hepatocytes at 3 weeks. By 6 weeks, the major early β-cell transcription factors (Pdx1, Neurod1 and Isl1) can be detected in the hepatocytes of both Ngn3-Btc and Ngn3-only treatment, while Pax6 could be detected in the hepatocytes with Ngn3-Btc and not with Ngn3-only treatment. However, the transcriptional cascade in hepatocytes appears to be aborted at this stage with failure of expression of the downstream transcription factors Pax4, Nkx2.2, or Nkx6.1 ().
In contrast to hepatocytes, the small periportal cells express transcripts for both Ins1 and Ins2 robustly along with the other islet hormones, Gcg, PP and Sst, whether or not Btc was included in the regimen (). Thus, the transcript expression profile agrees with the immunostaining results ( and Supplemental Figure 8
). Importantly, there is also a robust expression of the entire transcription cascade of developing islet-cells, including Pdx1, Neurod1, Isl1, Pax6, Nkx2.2 and Nkx6.1 with both Ngn3-Btc- and Ngn3-only treatment, though Pax4 was expressed only with Ngn3-Btc treatment. It has been reported that Btc by itself may increase Pax4 expression (Brun et al., 2008
). Interestingly, the endogenous Ngn3 transcript is not detected, as would be predicted from the fact that Ngn3 represses its own expression (Smith et al., 2004
), since the vector-derived Ngn3 was expressed in all the time points in both hepatocytes and oval cells. Furthermore, the level of expression of the major transcription factors is comparable to that seen in normal pancreatic islets (). All the transcription factors along with Ins1 and Ins2 continue to be expressed at 12 weeks. The two waves of Ngn3-Btc-induced cellular insulin expression, from hepatocytes at 3-6 weeks and oval cell-derived periportal cells at 6 weeks and later, and their relation to the blood glucose of the treated mice are depicted in . Therefore, the appearance of insulin transcripts and protein at the 6 week time point appears to coincide with a switch of cell fate of the oval cells to a β-cell specific transcription signature, normally found only in pancreatic β-cells. We note that transcripts for the β-cell-specific molecules, the potassium channel Kcnj11
and the prohormone convertase Pcsk1
, like some of the transcription factors downstream to Isl1 and Pax6, were also expressed in the neo-islets but not in the hepatocytes (). Also of note was that Thy1 was expressed only in the neo-islets, consistent with these cells being derived from oval cells (Petersen et al., 1998
). Interestingly, albumin, which was expressed at high level in hepatocytes, was weakly positive in some of the oval cell-derived RNA samples. We cannot exclude the possibility of contamination from adjoining hepatocytes, as we did not detect albumin in oval cells by immunostaining (); albumin protein is generally thought to be absent in oval cells, however, very low levels of albumin transcript have been described (Nagy et al., 1994
). We also note that not all oval cells picked by LCM, especially early on, had switched their cell-fate, as revealed by insulin and A6 immunostaining in and Supplemental Figures 13 and 14
Taken together, our results indicate that the hepatic oval cells appear to have differentiated into the endocrine pancreas, a lineage that is different from their original determination, viz., biliary cells or hepatocytes, a process that is consistent with transdetermination (McClure and Schubiger, 2007
). Since transdetermination tends to occur in actively proliferating cells, we assayed the proliferation activity of the periportal oval cell clusters. The strong signals for both BrdU labeling and PCNA staining at 3 weeks in the periportal oval cells indicate that many are in the S-phase of the cell cycle (, and Supplemental Figure 15
), consistent with active proliferation. Interestingly, the proliferation rate is twice as high in the oval cells in the Ngn3+Btc regimen as compared with the Ngn3-only regimen. In contrast, BrdU and PCNA-positive cells were rarely identified among the neighboring parenchymal hepatocytes with either treatment. The absence of cell proliferation and an aborted transcription cascade in the hepatocytes go along with their failure to transdifferentiate into β-cells, even though they can be induced to produce insulin transiently. On the other hand, our results indicate that oval cells have undergone a transdetermination process (); in response to induced Ngn3 expression, they display a complete β-cell transcription profile, express islet-specific molecules that lead to reversal of diabetes.
Model for Ngn3-induced transdetermination of hepatic oval cells