Irit Meivar-Levy

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.)

New organs from our own tissues: liver-to-pancreas transdifferentiation

Recent advances in pancreatic islet transplantation emphasize the potential of this approach for the long-term control of blood glucose levels in diabetic patients. However, tissue-replacement therapy will become widely available as a treatment for diabetes only when new sources of islets and insulin-producing cells are found. Here, we review recent evidence that documents the potential of mature liver as a source of tissue for generating a functional endocrine pancreas, by ectopic expression of pancreatic transcription and differentiation factors. When key events in the transconversion process have been identified, using the liver as a source of pancreatic tissue might provide a valuable approach for replacing impaired beta cell function in diabetics.

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.

Human Liver Cells Expressing Albumin and Mesenchymal Characteristics Give Rise to Insulin-Producing Cells

Activation of the pancreatic lineage in the liver has been suggested as a potential autologous cell replacement therapy for diabetic patients. Transcription factors-induced liver-to-pancreas reprogramming has been demonstrated in numerous species both in vivo and in vitro. However, human-derived liver cells capable of acquiring the alternate pancreatic repertoire have never been characterized. It is yet unknown whether hepatic-like stem cells or rather adult liver cells give rise to insulin-producing cells. Using an in vitro experimental system, we demonstrate that proliferating adherent human liver cells acquire mesenchymal-like characteristics and a considerable level of cellular plasticity. However, using a lineage-tracing approach, we demonstrate that insulin-producing cells are primarily generated in cells enriched for adult hepatic markers that coexpress both albumin and mesenchymal markers. Taken together, our data suggest that adult human hepatic tissue retains a substantial level of developmental plasticity, which could be exploited in regenerative medicine approaches.

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.

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

Replenishment of regulated insulin secretion is the ultimate goal of therapy for type 1 diabetes mellitus (Cozar-Castellano and Stewart in Proc Natl Acad Sci USA 102:7781–7782, 2005; Efrat and Russ in Trends Endocrinol Metab TEM 23:278–285, 2012; Ricordi et al. in Regen Med 7:41–48, 2012). β cell replacement by pancreas transplantation or by islet cell implantation is both restricted by severe shortage of tissue supply from cadaver donors, and by the requirement for extensive, lifelong suppression of the immune system. Therefore, tissue replacement therapy will become widely available as a treatment for diabetic patients only when islets or insulin-producing cells will be available in unlimited amounts and preferentially will not be rejected by the diabetic recipients. Efforts have been put forth to generate transplantable islets in vitro from embryonic stem cells (D’Amour et al. in Nat Biotechnol 23:1534–1541, 2005, Nat Biotechnol 24:1392–1401, 2006; Rezania et al. in Stem Cells (Dayton, Ohio) 31:2432–2442, 2013; Schulz et al. in PloS One 7:e37004, 2012) or induced pluripotent cells (Alipio et al. in Proc Natl Acad Sci USA 107:13426–13431, 2010; Rezania et al. in Stem Cells (Dayton, Ohio) 31:2432–2442, 2014) as well as from self-renewing cell populations that reside outside or within the adult pancreas (Baeyens et al. in Diabetologia 48:49–57, 2005; Bonner-Weir et al. in Proc Natl Acad Sci USA 97:7999–8004, 2000; Courtney et al. in Diabetes Obes Metab 13:47–52, 2011; Zhou et al. in Nature 455:627–632, 2008). The present chapter will address the research in the field of generating functional insulin-producing cells by transdifferentiation of adult extra-pancreatic tissues. Adult cell reprogramming or transdifferentiation employs the instructive roles of pancreatic transcription factors in controlling pancreas organogenesis in the embryo to dictate the induction of pancreatic lineage and function in like manner in adult cells of differentiated tissues. The transient ectopic expression of pancreatic transcription factors in extra-pancreatic tissues induces the expression of otherwise silent genetic information that is characteristic to that of the pancreas. This in turn endows the adult extra-pancreatic tissues with pancreatic characteristics and function, thus promoting ex vivo differentiation into insulin-producing and secreting cells. The direct conversion of somatic cells into alternative committed lineages—transdifferentiation, has opened up tremendous opportunities for regenerative medicine. Our laboratory is the first to suggest the capacity of the pancreatic master regulator, Pdx-1 to induce functional liver to pancreas transdifferentiation in the year 2000 (Ferber et al. in Nat Med 6:568–572, 2000). The present chapter reviews 15 years of research in the field of generating insulin-producing cells by transdifferentiation from our and other laboratories. Transdifferentiation of adult extra-pancreatic tissues may result in the generation of custom made “self” surrogate β cells for the treatment of diabetes, overcoming both the shortage in tissue availability from cadaveric donors and the subsequent need for antirejection treatments.