Sarah Ferber

Exendin-4 Promotes Liver Cell Proliferation and Enhances the PDX-1-induced Liver to Pancreas Transdifferentiation Process

Over the last few years, evidence has accumulated revealing the unexpected potential of committed mammalian cells to convert to a different phenotype via a process called transdifferentiation or adult cell reprogramming. These findings may have major practical implications because this process may facilitate the generation of functional autologous tissues that can be used for replacing malfunctioning organs. An instructive role for transcription factors in diverting the developmental fate of cells in adult tissues has been demonstrated when adult human liver cells were induced to transdifferentiate to the pancreatic endocrine lineage upon ectopic expression of the pancreatic master regulator PDX-1 (pancreatic and duodenal homeobox gene 1). Since organogenesis and lineage commitment are affected also by developmental signals generated in response to environmental triggers, we have now analyzed whether the hormone GLP-1 (glucogen-like peptide-1) documented to play a role in pancreatic beta cell differentiation, maturation, and survival, can also increase the efficiency of liver to pancreas transdifferentiation. We demonstrate that the GLP-1R agonist, exendin-4, significantly improves the efficiency of PDX-1-mediated transdifferentiation. Exendin-4 affects the transdifferentiation process at two distinct steps; it increases the proliferation of liver cells predisposed to transdifferentiated in response to PDX-1 and promotes the maturation of transdifferentiated cells along the pancreatic lineage. Liver cell reprogramming toward the pancreatic beta cell lineage has been suggested as a strategy for functional replacement of the ablated insulin-producing cells in diabetics. Understanding the cellular and molecular basis of the transdifferentiation process will allow us to increase the efficiency of the reprogramming process and optimize its therapeutic merit.

Pancreatic and duodenal homeobox gene 1 induces hepatic dedifferentiation by suppressing the expression of CCAAT/enhancer‐binding protein β

It is believed that adult tissues in mammals lack the plasticity needed to assume new developmental fates because of the absence of efficient pathways of dedifferentiation. However, the well‐documented ability of the transcription factor pancreatic and duodenal homeobox gene 1 (PDX‐1) to activate pancreatic lineage development and insulin production following ectopic expression in liver suggests a surprising degree of residual plasticity in adult liver cells. This study seeks a mechanistic explanation for the capacity of PDX‐1 to endow liver cells with pancreatic characteristics and function. We demonstrate that PDX‐1, previously shown to play an essential role in normal pancreatic organogenesis and pancreatic β‐cell function and to possess the potential to activate multiple pancreatic markers in liver, can also direct hepatic dedifferentiation. PDX‐1 represses the adult hepatic repertoire of gene expression and activates the expression of the immature hepatic marker α‐fetoprotein. We present evidence indicating that PDX‐1 triggers hepatic dedifferentiation by repressing the key hepatic transcription factor CCAAT/enhancer‐binding protein β. Hepatic dedifferentiation is necessary though not sufficient for the activation of the mature pancreatic repertoire in liver. Conclusion: Our study suggests a dual role for PDX‐1 in liver: inducing hepatic dedifferentiation and activating the pancreatic lineage. The identification of dedifferentiation signals may promote the capacity to endow mature tissues in mammals with the plasticity needed for acquiring novel developmental fates and functions to be implemented in the field of regenerative medicine. (HEPATOLOGY 2007.)

The peptide-hormone glucagon-like peptide-1 activates cAMP and inhibits growth of breast cancer cells

The incretin hormone glucagon-like peptide (GLP)-1 is secreted from intestinal L cells in response to food intake, and promotes insulin secretion and pancreatic β-cell proliferation. Reduced GLP-1 levels are observed in obesity and type 2 diabetes mellitus (T2DM) and are associated with reduced insulin secretion and increased insulin resistance. GLP-1 mediates its activities through activation of a G-protein coupled receptor, which is expressed in the pancreas, as well as other tissues. Long-acting GLP-1 receptor (GLP-1R) agonists, such as exendin-4, are currently approved for the treatment of T2DM. As obesity and T2DM are associated with increased risk of breast cancer, we aimed to explore the effects of GLP-1 and exendin-4, on breast cancer cells. Treatment with GLP-1 or exendin-4 reduced viability and enhanced apoptosis of breast cancer cells but did not affect viability of nontumorigenic cells. Moreover, exendin-4 attenuated tumor formation by breast cancer cells in athymic mice. Treatment with either GLP-1 or exendin-4 elevated cAMP levels, activated the down-stream target CREB, and enhanced CRE promoter transcription, in breast cancer cells. Moreover, inhibition of exendin-4-induced adenylate cyclase activation restored cell viability, thus suggesting cAMP as a principle mediator of exendin-4 anti-tumorigenic activity. While the pancreatic form of the GLP-1R could not be detected in breast cancer cells, several lines of evidence indicated the existence of an alternative GLP-1R in mammary cells. Thus, internalization of GLP-1 into MCF-7 cells was evidenced, infection of MCF-7 cells with the pancreatic receptor enhanced proliferation, and treatment with exendin-(9–39), a GLP-1R antagonist, further increased cAMP levels. Our studies indicate the incretin hormone GLP-1 as a potent inducer of cAMP and an inhibitor of breast cancer cell proliferation. Reduced GLP-1 levels may, therefore, serve as a novel link between obesity, diabetes mellitus, and breast cancer.

Ectopic PDX-1 expression directly reprograms human keratinocytes along pancreatic insulin-producing cells fate.

Background Cellular differentiation and lineage commitment have previously been considered irreversible processes. However, recent studies have indicated that differentiated adult cells can be reprogrammed to pluripotency and, in some cases, directly into alternate committed lineages. However, although pluripotent cells can be induced in numerous somatic cell sources, it was thought that inducing alternate committed lineages is primarily only possible in cells of developmentally related tissues. Here, we challenge this view and analyze whether direct adult cell reprogramming to alternate committed lineages can cross the boundaries of distinct developmental germ layers. Methodology/Principal Findings We ectopically expressed non-integrating pancreatic differentiation factors in ectoderm-derived human keratinocytes to determine whether these factors could directly induce endoderm-derived pancreatic lineage and β-cell-like function. We found that PDX-1 and to a lesser extent other pancreatic transcription factors, could rapidly and specifically activate pancreatic lineage and β-cell-like functional characteristics in ectoderm-derived human keratinocytes. Human keratinocytes transdifferentiated along the β cell lineage produced processed and secreted insulin in response to elevated glucose concentrations. Using irreversible lineage tracing for KRT-5 promoter activity, we present supporting evidence that insulin-positive cells induced by ectopic PDX-1 expression are generated in ectoderm derived keratinocytes. Conclusions/Significance These findings constitute the first demonstration of human ectoderm cells to endoderm derived pancreatic cells transdifferentiation. The study represents a proof of concept which suggests that transcription factors induced reprogramming is wider and more general developmental process than initially considered. These results expanded the arsenal of adult cells that can be used as a cell source for generating functional endocrine pancreatic cells. Directly reprogramming somatic cells into alternate desired tissues has important implications in developing patient-specific, regenerative medicine approaches.

The temporal and hierarchical control of transcription factors-induced liver to pancreas transdifferentiation.

Lineage-specific transcription factors (TFs) display instructive roles in directly reprogramming adult cells into alternate developmental fates, in a process known as transdifferentiation. The present study analyses the hypothesis that despite being fast, transdifferentiation does not occur in one step but is rather a consecutive and hierarchical process. Using ectopic expression of Pdx1 in human liver cells, we demonstrate that while glugacon and somatostatin expression initiates within a day, insulin gene expression becomes evident only 2–3 days later. To both increase transdifferentiation efficiency and analyze whether the process indeed display consecutive and hierarchical characteristics, adult human liver cells were treated by three pancreatic transcription factors, Pdx1, Pax4 and Mafa (3pTFs) that control distinct hierarchical stages of pancreatic development in the embryo. Ectopic expression of the 3pTFs in human liver cells, increased the transdifferentiation yield, manifested by 300% increase in the number of insulin positive cells, compared to each of the ectopic factors alone. However, only when the 3pTFs were sequentially supplemented one day apart from each other in a direct hierarchical manner, the transdifferentiated cells displayed increased mature β-cell-like characteristics. Ectopic expression of Pdx1 followed by Pax4 on the 2nd day and concluded by Mafa on the 3rd day resulted in increased yield of transdifferentiation that was associated by increased glucose regulated c-peptide secretion. By contrast, concerted or sequential administration of the ectopic 3pTFs in an indirect hierarchical mode resulted in the generation of insulin and somatostatin co-producing cells and diminished glucose regulated processed insulin secretion. In conclusion transcription factors induced liver to pancreas transdifferentiation is a progressive and hierarchical process. It is reasonable to assume that this characteristic is general to wide ranges of tissues. Therefore, our findings could facilitate the development of cell replacement therapy modalities for many degenerative diseases including diabetes.

Adult Cell Fate Reprogramming: Converting Liver to Pancreas

Regenerative medicine aims at producing new cells for repair or replacement of diseased and damaged tissues. Embryonic and adult stem cells have been suggested as attractive sources of cells for generating the new cells needed. The leading dogma was that adult cells in mammals, once committed to a specific lineage, become terminally differentiated and can no longer change their fate. However, in recent years increasing evidence has accumulated demonstrating the remarkable ability of some differentiated cells to be converted into a different cell type via a process termed developmental redirection or adult cells reprogramming. For example, abundant human cell types, such as dermal fibroblasts and adipocytes, could potentially be harvested and converted into other, medically important cell types, such as neurons, cardiomyocytes, or pancreatic beta cells. In this chapter, we describe a method of activating the pancreatic lineage and beta-cells function in adult human liver cells by ectopic expression of pancreatic transcription factors. This approach aims to generate custom-made autologous surrogate beta cells for treatment of diabetes, and possibly bypass both the shortage of cadaveric human donor tissues and the need for life-long immune-suppression.

Reprogramming of liver cells into insulin-producing cells.

Tissue replacement is a promising direction for the treatment of diabetes, which will become widely available only when islets or insulin-producing cells that will not be rejected by the diabetic recipients are available in unlimited amounts. The present review addresses the research in the field of generating functional insulin-producing cells by transdifferentiation of adult liver cells both in vitro and in vivo. It presents recent knowledge of the mechanisms which underlie the process and assesses the challenges which should be addressed for its efficient implementation as a cell based replacement therapy for diabetics.

Adult Cell Reprogramming: Using Nonpancreatic Cell Sources to Generate Surrogate Beta Cells for Treatment of Diabetes

Regenerative medicine is designed to produce new cells for repair or replacement of diseased and damaged tissues. Embryonic and adult stem cells have been suggested as attractive sources for generation of new differentiated cells. The leading dogma has maintained that once animal cells are committed to a specific lineage, they become “terminally differentiated” and can no longer change their fate. However, in recent years increasing evidence has demonstrated the remarkable ability of some differentiated cells to convert into a different cell type via a process termed developmental redirection or nuclear reprogramming. For example, abundant human cell types, such as dermal fibroblasts and adipocytes, could potentially be harvested and converted into other, medically important cell types, such as neurons, cardiomyocytes, or pancreatic β cells. In this chapter we review the potential use of adult tissue, specifically liver and bone marrow, to provide a source of tissue for generating functional insulin-producing cells. This approach might generate custom-made autologous surrogate β cells for treatment of diabetes and possibly circumvent both the shortage of cadaveric human donor tissue and the need for life-long immunosuppression.

Use of Extra-Pancreatic Tissues for Cell Replacement Therapy for Diabetes

Pancreatic islet transplantation may constitute the best approach for the long-term control of blood glucose levels in the treatment of diabetes. However, tissue replacement therapy will become widely available as a treatment for diabetic patients only when islets or insulin-producing cells are available in unlimited amounts, and will not be rejected by the diabetic recipients. The present chapter will analyze the option of using adult extrapancreatic tissues for regulated insulin production. Two major approaches could endow adult extra-pancreatic tissues with characteristics and functions that can be used for diabetes cell replacement therapy: First, insulin gene therapy, which involves the ectopic expression of constructs encoding the proinsulin gene or modified proinsulin sequences in adult extra-pancreatic cells from different sources. Second, the induction of a process called developmental redirection of extra-pancreatic tissues into insulin-producing cells. The second approach exploits the instructive roles of pancreatic transcription and soluble factors in controlling pancreas organogenesis in the embryo to dictate the induction of pancreatic lineage and function also in adult tissues. This approach endows adult extra-pancreatic tissues with pancreatic characteristics and function, thus promoting ex vivo differentiation into insulinproducing and secreting cells. Using adult extra-pancreatic tissues may result in the generation of custom made “self” surrogate pancreatic beta cells for the treatment of diabetes, bypassing both the shortage in tissue availability from cadaveric donors and the need for life-long immunosuppression.

The Wnt/β‐catenin pathway determines the predisposition and efficiency of liver‐to‐pancreas reprogramming

Transdifferentiation (TD) is the direct reprogramming of adult cells into cells of alternate fate and function. We have previously shown that liver cells can be transdifferentiated into beta‐like, insulin‐producing cells through ectopic expression of pancreatic transcription factors (pTFs). However, the efficiency of the process was consistently limited to <15% of the human liver cells treated in culture. The data in the current study suggest that liver‐to‐pancreas TD is restricted to a specific population of liver cells that is predisposed to undergo reprogramming. We isolated TD‐predisposed subpopulation of liver cells from >15 human donors using a lineage tracing system based on the Wnt response element, part of the pericentral‐specific promoter of glutamine synthetase. The cells, that were propagated separately, consistently exhibited efficient fate switch and insulin production and secretion in >60% of the cells upon pTF expression. The rest of the cells, which originated from 85% of the culture, resisted TD. Both populations expressed the ectopic pTFs with similar efficiencies, followed by similar repression of hepatic genes. Our data suggest that the TD‐predisposed cells originate from a distinct population of liver cells that are enriched for Wnt signaling, which is obligatory for efficient TD. In TD‐resistant populations, Wnt induction is insufficient to induce TD. An additional step of chromatin opening enables TD of these cells. Conclusion: Liver‐to‐pancreas TD occurs in defined predisposed cells. These cells predisposition is maintained by Wnt signaling that endows the cells with the plasticity needed to alter their transcriptional program and developmental fate when triggered by ectopic pTFs. These results may have clinical implications by drastically increasing the efficacy of TD in future clinical uses. (Hepatology 2018).