Seung-Hee Lee

Human β-cell Precursors Mature Into Functional Insulin-producing Cells in an Immunoisolation Device: Implications for Diabetes Cell Therapies

Background. Islet transplantation is limited by the need for chronic immunosuppression and the paucity of donor tissue. As new sources of human β-cells are developed (e.g., stem cell-derived tissue), transplanting them in a durable device could obviate the need for immunosuppression, while also protecting the patient from any risk of tumorigenicity. Here, we studied (1) the survival and function of encapsulated human β-cells and their progenitors and (2) the engraftment of encapsulated murine β-cells in allo- and autoimmune settings. Methods. Human islets and human fetal pancreatic islet-like cell clusters were encapsulated in polytetrafluorethylene devices (TheraCyte) and transplanted into immunodeficient mice. Graft survival and function was measured by immunohistochemistry, circulating human C-peptide levels, and blood glucose levels. Bioluminescent imaging was used to monitor encapsulated neonatal murine islets. Results. Encapsulated human islet-like cell clusters survived, replicated, and acquired a level of glucose responsive insulin secretion sufficient to ameliorate hyperglycemia in diabetic mice. Bioluminescent imaging of encapsulated murine neonatal islets revealed a dynamic process of cell death followed by regrowth, resulting in robust long-term allograft survival. Further, in the non-obese diabetic (NOD) mouse model of type I diabetes, encapsulated primary β-cells ameliorated diabetes without stimulating a detectable T-cell response. Conclusions. We demonstrate for the first time that human β-cells function is compatible with encapsulation in a durable, immunoprotective device. Moreover, our study suggests that encapsulation of β-cells before terminal differentiation will be a successful approach for new cell-based therapies for diabetes, such as those derived from stem cells.

Islet Specific Wnt Activation in Human Type II Diabetes

The Wnt pathway effector gene TCF7L2 has been linked to type II diabetes, making it important to study the role of Wnt signaling in diabetes pathogenesis. We examined the expression of multiple Wnt pathway components in pancreases from normal individuals and type II diabetic individuals. Multiple members of the Wnt signaling pathway, including TCF7L2, Wnt2b, β-catenin, pGSK3β, TCF3, cyclinD1, and c-myc, were undetectable or expressed at low levels in islets from nondiabetic individuals, but were also upregulated specifically in islets of type II diabetic patients. Culture of pancreatic tissue and islet isolation led to Wnt activation that was reversed by the Wnt antagonist sFRP, demonstrating that Wnt activation in that setting was due to soluble Wnt factors. These data support a model in which the Wnt pathway plays a dynamic role in the pathogenesis of type II diabetes and suggest manipulation of Wnt signaling as a new approach to β-cell-directed diabetes therapy.

Pharmacological induction of pancreatic islet cell transdifferentiation: relevance to type I diabetes

Type I diabetes (T1D) is an autoimmune disease in which an immune response to pancreatic β-cells results in their loss over time. Although the conventional view is that this loss is due to autoimmune destruction, we present evidence of an additional phenomenon in which autoimmunity promotes islet endocrine cell transdifferentiation. The end result is a large excess of δ-cells, resulting from α- to β- to δ-cell transdifferentiation. Intermediates in the process of transdifferentiation were present in murine and human T1D. Here, we report that the peptide caerulein was sufficient in the context of severe β-cell deficiency to induce efficient induction of α- to β- to δ-cell transdifferentiation in a manner very similar to what occurred in T1D. This was demonstrated by genetic lineage tracing and time course analysis. Islet transdifferentiation proceeded in an islet autonomous manner, indicating the existence of a sensing mechanism that controls the transdifferentiation process within each islet. The finding of evidence for islet cell transdifferentiation in rodent and human T1D and its induction by a single peptide in a model of T1D has important implications for the development of β-cell regeneration therapies for diabetes.

HNF4α antagonists discovered by a high-throughput screen for modulators of the human insulin promoter.

Hepatocyte nuclear factor (HNF)4α is a central regulator of gene expression in cell types that play a critical role in metabolic homeostasis, including hepatocytes, enterocytes, and pancreatic β cells. Although fatty acids were found to occupy the HNF4α ligand-binding pocket and were proposed to act as ligands, there is controversy about both the nature of HNF4α ligands as well as the physiological role of the binding. Here, we report the discovery of potent synthetic HNF4α antagonists through a high-throughput screen for effectors of the human insulin promoter. These molecules bound to HNF4α with high affinity and modulated the expression of known HNF4α target genes. Notably, they were found to be selectively cytotoxic to cancer cell lines in vitro and in vivo, although in vivo potency was limited by suboptimal pharmacokinetic properties. The discovery of bioactive modulators for HNF4α raises the possibility that diseases involving HNF4α, such as diabetes and cancer, might be amenable to pharmacologic intervention by modulation of HNF4α activity.

Id3 upregulates BrdU incorporation associated with a DNA damage response, not replication, in human pancreatic β-cells

Elucidating mechanisms of cell cycle control in normally quiescent human pancreatic β-cells has the potential to impact regeneration strategies for diabetes. Previously we demonstrated that Id3, a repressor of basic Helix-Loop-Helix (bHLH) proteins, was sufficient to induce cell cycle entry in pancreatic duct cells, which are closely related to β-cells developmentally. We hypothesized that Id3 might similarly induce cell cycle entry in primary human β-cells. To test this directly, adult human β-cells were transduced with adenovirus expressing Id3. Consistent with a replicative response, β-cells exhibited BrdU incorporation. Further, Id3 potently repressed expression of the cyclin dependent kinase inhibitor p57Kip2, a gene which is also silenced in a rare β-cell hyperproliferative disorder in infants. Surprisingly, however, BrdU positive β-cells did not express the proliferation markers Ki67 and pHH3. Instead, BrdU uptake reflected a DNA damage response, as manifested by hydroxyurea incorporation, γH2AX expression and 53BP1 subcellular relocalization. The uncoupling of BrdU uptake from replication raises a cautionary note about interpreting studies relying solely upon BrdU incorporation as evidence of β-cell proliferation. The data also establish that loss of p57Kip2 is not sufficient to induce cell cycle entry in adult β-cells. Moreover, the differential responses to Id3 between duct and β-cells reveal that β-cells possess intrinsic resistance to cell cycle entry not common to all quiescent epithelial cells in the adult human pancreas. The data provide a much needed comparative model for investigating the molecular basis for this resistance in order to develop a strategy for improving replication competence in β-cells.

Phenothiazine Neuroleptics Signal to the Human Insulin Promoter as Revealed by a Novel High-Throughput Screen

A number of diabetogenic stimuli interact to influence insulin promoter activity, making it an attractive target for both mechanistic studies and therapeutic interventions. High-throughput screening (HTS) for insulin promoter modulators has the potential to reveal novel inputs into the control of that central element of the pancreatic β-cell. A cell line from human islets in which the expression of insulin and other β-cell-restricted genes are modulated by an inducible form of the bHLH transcription factor E47 was developed. This cell line, T6PNE, was adapted for HTS by transduction with a vector expressing green fluorescent protein under the control of the human insulin promoter. The resulting cell line was screened against a library of known drugs for those that increase insulin promoter activity. Members of the phenothiazine class of neuroleptics increased insulin gene expression upon short-term exposure. Chronic treatment, however, resulted in suppression of insulin promoter activity, consistent with the effect of phenothiazines observed clinically to induce diabetes in chronically treated patients. In addition to providing insights into previously unrecognized targets and mechanisms of action of phenothiazines, the novel cell line described here provides a broadly applicable platform for mining new molecular drug targets and central regulators of β-cell differentiated function.

Adult human β-cell neogenesis?

Prospects for inducing endogenous β‐cell regeneration in the pancreas, one of the most attractive approaches to reverse type 1 and type 2 diabetes, have gained substantially from recent evidence that cells in the adult pancreas exhibit more plasticity than previously recognized. There are two major pathways to β‐cell regeneration, β‐cell replication and β‐cell neogenesis. Substantial evidence for a role for both processes exists in different models. While β‐cell replication clearly occurs during development and early in life, the potential for replication appears to decline substantially with age. In contrast, we have demonstrated that the exocrine compartment of the adult human pancreas contains a facultative stem cell that can differentiate into β‐cells under specific circumstances. We have favoured the idea that, similar to models described in liver regeneration, β‐cell mass can be increased either by neogenesis or replication, depending on the intensity of different stimuli or stressors. Understanding the nature of endocrine stem/progenitor cells and the mechanism by which external stimuli mobilize them to exhibit endocrine differentiation is central for success in therapeutic approaches to induce meaningful endogenous β‐cell neogenesis.

The Id3/E47 axis mediates cell-cycle control in human pancreatic ducts and adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) has a 5-year survival rate of less than 5%, and therapeutic advances have been hampered by gaps in our understanding of cell-cycle control in the adult pancreas. Previously, we reported that basic Helix-Loop-Helix (bHLH) transcription factors regulate cell fate specification in the pancreas. In the present study, we found that a repressor of bHLH activity, Id3, was profoundly upregulated in ductal cells in murine models of pancreatitis and pancreatic intraepithelial neoplasia (PanIN). Id3 was also pervasively expressed in neoplastic lesions in human PDA in situ. We hypothesized that an imbalance in bHLH versus Id activity controlled cell growth in PDA. Consistent with this model, cell-cycle progression in PDA cells was impeded by siRNA-mediated depletion of Id3 or overexpression of the bHLH protein E47. The precursors of human PDA are normally quiescent duct cells which do not proliferate in response to high serum or growth factors. The finding that Id3 was expressed in pancreatitis, as well as PDA, suggested that Id3 might induce cell-cycle entry in ducts. To test this hypothesis, primary human pancreatic duct cells were transduced with an adenovirus-expressing Id3. Remarkably, Id3 expression alone was sufficient to trigger efficient cell-cycle entry, as manifested by expression of the proliferation markers Ki67, phospho-cyclin E, and phospho-histone H3. Collectively, the data establish dysregulation of the Id/bHLH axis as an early and sustained feature of ductal pathogenesis and mark this axis as a potential therapeutic target for intervention in pancreatitis and PDA. Mol Cancer Res; 9(6); 782–90. ©2011 AACR.

β-cell replication and islet neogenesis following partial pancreatectomy

Partial pancreatectomy is one of the most commonly used models in the study of β-cell regeneration. The mechanism by which regeneration occurs in this model has been controversial, with some claiming that islet and β-cell neogenesis is important, while others claim that β-cell replication is predominant. Here, we combined a time course analysis with continuous BrdU administration to study β-cell regeneration following partial pancreatectomy. While exocrine cells in regenerating areas were highly proliferative and positive for BrdU, islets in regenerating areas were negative for BrdU one week after partial pancreatectomy, suggesting that they were derived from preexisting islets rather than being neogenic. The insulin-positive cells in ducts that have been reported by others and taken as evidence of β-cell neogenesis were present in regenerating regions of the pancreas, but were relatively uncommon and were not highly proliferative, suggesting that they could not account for significant islet neogenesis. Consistent with a lack of islet neogenesis, regenerating areas following a second partial pancreatectomy were devoid of islets. β-cell replication was detectable at a high frequency two weeks following partial pancreatectomy and was present at a similar frequency in both regenerating and preexisting regions of the pancreas. In summary, our data indicate that islet neogenesis following partial pancreatectomy does not occur.

Efficient β-cell regeneration by a combination of neogenesis and replication following β-cell ablation and reversal of pancreatic duct ligation.

Achieving efficient β‐cell regeneration is a major goal of diabetes research. Previously, we found that a combination of β‐cell ablation and pancreatic duct ligation led to β‐cell regeneration by direct conversion from α‐cells. Here, we studied the effect of surgical reversal of the duct ligation, finding that there was a wave of β‐cell replication following reversal. The combination of β‐cell neogenesis prior to reversal of the duct ligation and β‐cell replication following reversal resulted in efficient β‐cell regeneration and eventual recovery of function. This provides an important proof of principle that efficient β‐cell regeneration is possible, even from a starting point of profound β‐cell ablation. This has important implications for efforts to promote β‐cell regeneration. Stem Cells 2013;31:2388–2395