RESUMEN
Diabetes mellitus (DM) is a complex metabolic disease with many specifically related complications. Early diagnosis of this disease could prevent the progression to overt disease and its related complications. There are several limitations to using existing biomarkers, and between 24% and 62% of people with diabetes remain undiagnosed and untreated, suggesting a large gap in current diagnostic practices. Early detection of the percentage of insulin-producing cells preceding loss of function would allow for effective therapeutic interventions that could delay or slow down the onset of diabetes. MicroRNAs (miRNAs) could be used for early diagnosis, as well as for following the progression and the severity of the disease, due to the fact of their pancreatic specific expression and stability in various body fluids. Thus, many studies have focused on the identification and validation of such groups or "signatures of miRNAs" that may prove useful in diagnosing or treating patients. Here, we summarize the findings on miRNAs as biomarkers in diabetes and those associated with direct cellular reprogramming strategies, as well as the relevance of miRNAs that act as a bidirectional switch for cell therapy of damaged pancreatic tissue and the studies that have measured and tracked miRNAs as biomarkers in insulin resistance are addressed.
Asunto(s)
Diabetes Mellitus , Resistencia a la Insulina , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Medicina de Precisión , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/genética , Diabetes Mellitus/terapia , Biomarcadores/metabolismo , Resistencia a la Insulina/fisiologíaRESUMEN
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).
Asunto(s)
Linaje de la Célula , Transdiferenciación Celular/genética , Proteínas Wnt/genética , Vía de Señalización Wnt/genética , beta Catenina/genética , Animales , Causalidad , Células Cultivadas , Reprogramación Celular , Hepatocitos/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Páncreas/citología , Sensibilidad y EspecificidadRESUMEN
PURPOSE OF THE REVIEW: Here, we review recent findings in the field of generating insulin-producing cells by pancreatic transcription factor (pTF)-induced liver transdifferentiation (TD). TD is the direct conversion of functional cell types from one lineage to another without passing through an intermediate stage of pluripotency. We address potential reasons for the restricted efficiency of TD and suggest modalities to overcome these challenges, to bring TD closer to its clinical implementation in autologous cell replacement therapy for insulin-dependent diabetes. RECENT FINDINGS: Liver to pancreas TD is restricted to cells that are a priori predisposed to undergo the developmental process. In vivo, the predisposition of liver cells is affected by liver zonation and hepatic regeneration. The TD propensity of liver cells is related to permissive epigenome which could be extended to TD-resistant cells by specific soluble factors. An obligatory role for active Wnt signaling in continuously maintaining a "permissive" epigenome is suggested. Moreover, the restoration of the pancreatic niche and vasculature promotes the maturation of TD cells along the ß cell function. Future studies on liver to pancreas TD should include the maturation of TD cells by 3D culture, the restoration of vasculature and the pancreatic niche, and the extension of TD propensity to TD-resistant cells by epigenetic modifications. Liver to pancreas TD is expected to result in the generation of custom-made "self" surrogate ß cells for curing diabetes.
Asunto(s)
Transdiferenciación Celular/efectos de los fármacos , Hígado/citología , Páncreas/citología , Transdiferenciación Celular/fisiología , Diabetes Mellitus Tipo 1/fisiopatología , Diabetes Mellitus Tipo 1/terapia , Proteínas de Homeodominio/fisiología , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/fisiología , Hígado/fisiología , Páncreas/fisiología , Transactivadores/fisiologíaRESUMEN
BACKGROUND: Insulin producing cells generated by liver cell transdifferentiation, could serve as an attractive source for regenerative medicine. The present study assesses the relationship between DNA methylation pTFs induced liver to pancreas transdifferentiation. RESULTS: The transdifferentiation process is associated with DNA demethylation, mainly at gene regulatory sites, and with increased expression of these genes. Active inhibition of DNA methylation promotes the pancreatic transcription factor-induced transdifferentiation process, supporting a causal role for DNA demethylation in this process. CONCLUSIONS: Transdifferentiation is associated with global DNA hypomethylation, and with increased expression of specific demethylated genes. A combination of epigenetic modulators may be used to increase chromatin accessibility of the pancreatic transcription factors, thus promoting the efficiency of the developmental process.
Asunto(s)
Desmetilación del ADN , Insulinas , Transdiferenciación Celular/genética , Cromatina , ADN , Insulinas/genética , Hígado , Páncreas , Factores de Transcripción/genéticaRESUMEN
Autologous cells replacement therapy by liver to pancreas transdifferentiation (TD) allows diabetic patients to be also the donors of their own therapeutic tissue. Aim: To analyze whether the efficiency of the process is affected by liver donors' heterogeneity with regard to age, gender and the metabolic state. Materials & methods: TD of liver cells derived from nondiabetic and diabetic donors at different ages was characterized at molecular and cellular levels, in vitro. Results: Neither liver cells proliferation nor the propagated cells TD efficiency directly correlate with the age (3-60 years), gender or the metabolic state of the donors. Conclusion: Human liver cells derived from a wide array of ages and metabolic states can be used for autologous cells therapies for diabetics.
Asunto(s)
Transdiferenciación Celular , Páncreas , Adolescente , Adulto , Proliferación Celular , Niño , Preescolar , Humanos , Hígado , Persona de Mediana Edad , Adulto JovenRESUMEN
Background: Liver cells represent an attractive source of cells for autologous regenerative medicine. The present study assesses the liver cells' stability during in vitro expansion, as a prerequisite for therapeutic use. Results: The human liver cell cultures in this study were propagated efficiently in vitro for at least 12 passages. No significant changes in morphology, intracellular ultrastructures and characteristic markers expression were found during in vitro expansion of cells from all analyzed donors. However, expanded cells derived from male donors of >60 years old, lost the Y chromosome. Conclusion: Liver-derived cell cultures adopt a proliferative, stable mesenchymal phenotype, through an epithelial to mesenchymal transition process. The molecular and phenotypic changes of the cells during propagation are uniform, despite the heterogeneity of the different donors. Loss of Y chromosome occurs after cells' propagation in elder male donors.
Asunto(s)
Células Madre Mesenquimatosas , Anciano , Técnicas de Cultivo de Célula , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Transición Epitelial-Mesenquimal , Humanos , Hígado , Masculino , Persona de Mediana Edad , FenotipoRESUMEN
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.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Transdiferenciación Celular/efectos de los fármacos , Proteínas de Homeodominio/metabolismo , Péptidos/farmacología , Transactivadores/metabolismo , Ponzoñas/farmacología , Adulto , Western Blotting , Linaje de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Niño , Preescolar , Exenatida , Citometría de Flujo , Receptor del Péptido 1 Similar al Glucagón , Proteínas de Homeodominio/genética , Humanos , Insulina/genética , Insulina/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Hígado/citología , Hígado/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Glucagón/agonistas , Receptores de Glucagón/metabolismo , Transducción de Señal/efectos de los fármacos , Transactivadores/genéticaRESUMEN
BACKGROUND: Insulin-dependent diabetes is a multifactorial disorder that could be theoretically cured by functional pancreatic islets and insulin-producing cell (IPC) implantation. Regenerative medicine approaches include the potential for growing tissues and organs in the laboratory and transplanting them when the body cannot heal itself. However, several obstacles remain to be overcome in order to bring regenerative medicine approach for diabetes closer to its clinical implementation; the cells generated in vitro are typically of heterogenic and immature nature and the site of implantation should be readily vascularized for the implanted cells to survive in vivo. The present study addresses these two limitations by analyzing the effect of co-implanting IPCs with vasculature promoting cells in an accessible site such as subcutaneous. Secondly, it analyzes the effects of reconstituting the in vivo environment in vitro on the maturation and function of insulin-producing cells. METHODS: IPCs that are generated by the transdifferentiation of human liver cells are exposed to the paracrine effects of endothelial colony-forming cells (ECFCs) and human bone marrow mesenchymal stem cells (MSCs), which are the "building blocks" of the blood vessels. The role of the vasculature on IPC function is analyzed upon subcutaneous implantation in vivo in immune-deficient rodents. The paracrine effects of vasculature on IPC maturation are analyzed in culture. RESULTS: Co-implantation of MSCs and ECFCs with IPCs led to doubling the survival rates and a threefold increase in insulin production, in vivo. ECFC and MSC co-culture as well as conditioned media of co-cultures resulted in a significant increased expression of pancreatic-specific genes and an increase in glucose-regulated insulin secretion, compared with IPCs alone. Mechanistically, we demonstrate that ECFC and MSC co-culture increases the expression of CTGF and ACTIVINßα, which play a key role in pancreatic differentiation. CONCLUSIONS: Vasculature is an important player in generating regenerative medicine approaches for diabetes. Vasculature displays a paracrine effect on the maturation of insulin-producing cells and their survival upon implantation. The reconstitution of the in vivo niche is expected to promote the liver-to-pancreas transdifferentiation and bringing this cell therapy approach closer to its clinical implementation.
Asunto(s)
Transdiferenciación Celular/fisiología , Células Secretoras de Insulina/metabolismo , Hígado/fisiología , Adulto , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Células Secretoras de Insulina/citología , RatonesRESUMEN
Recent advances in pancreatic islet transplantation emphasize the potential of this approach for the long-term control of blood glucose levels as treatment of diabetes. To overcome the organ shortage for cell replacement therapy, efforts are being invested in generating new and abundant sources of insulin-producing cells from embryonic or adult stem cells. We review recent evidence documenting the surprising capacity of the mature liver to serve as a potential source of tissue for generating functional endocrine pancreas. The process of liver-to-pancreas developmental redirection is induced by ectopic expression of pancreatic transcription and differentiation factors. This approach may allow the diabetic patient to be the donor of his or her own therapeutic tissue, thus alleviating both the need for allotransplantations and the subsequent immune suppression.
Asunto(s)
Diabetes Mellitus/terapia , Células Secretoras de Insulina , Hígado/citología , Células Madre Pluripotentes/metabolismo , Adulto , Diferenciación Celular , Diabetes Mellitus/cirugía , Diabetes Mellitus Tipo 2/terapia , Humanos , Inmunosupresores/administración & dosificación , Células Secretoras de Insulina/metabolismo , Trasplante de Islotes Pancreáticos , Factores de Transcripción/metabolismo , Transcripción Genética , Trasplante HomólogoRESUMEN
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.
Asunto(s)
Proteínas de Homeodominio , Hígado/fisiología , Páncreas/fisiología , Animales , Diferenciación Celular , Humanos , Trasplante de Hígado , Trasplante de Páncreas , Trasplante de Células Madre , Transactivadores/biosíntesis , Transactivadores/genéticaRESUMEN
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.
Asunto(s)
Transdiferenciación Celular , Hepatocitos/citología , Células Secretoras de Insulina/citología , Regeneración Hepática , Animales , Hepatocitos/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/trasplanteRESUMEN
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 glucagon 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 ß-cell-like characteristics. Ectopic expression of Pdx1 followed by Pax4 on the 2(nd) day and concluded by Mafa on the 3(rd) 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.
Asunto(s)
Transdiferenciación Celular , Hepatocitos/citología , Hepatocitos/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Factores de Transcripción/metabolismo , Adolescente , Linaje de la Célula/genética , Transdiferenciación Celular/genética , Niño , Preescolar , Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Insulina/biosíntesis , Factores de Transcripción Maf de Gran Tamaño/genética , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Somatostatina/biosíntesis , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Adulto JovenRESUMEN
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.
RESUMEN
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 ß-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 ß-cell-like functional characteristics in ectoderm-derived human keratinocytes. Human keratinocytes transdifferentiated along the ß 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.
Asunto(s)
Diferenciación Celular , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Células Secretoras de Insulina/citología , Queratinocitos/citología , Queratinocitos/metabolismo , Transactivadores/genética , Células 3T3 , Adulto , Animales , Biomarcadores/metabolismo , Técnicas de Cultivo de Célula , Ectodermo/citología , Endodermo/citología , Humanos , Recién Nacido , Ratones , Hormonas Pancreáticas/genética , Ratas , Transcripción Genética/genéticaRESUMEN
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.
Asunto(s)
Diferenciación Celular/fisiología , Células Secretoras de Insulina/fisiología , Hígado/citología , Adulto , Animales , Péptido C/metabolismo , Técnicas de Cultivo de Célula , Linaje de la Célula , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica , Glucosa/metabolismo , Humanos , Hiperglucemia/terapia , Insulina/metabolismo , Células Secretoras de Insulina/citología , Trasplante de Islotes Pancreáticos , Hígado/fisiología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Factores de Transcripción/metabolismoRESUMEN
Reprogramming adult mammalian cells is an attractive approach for generating cell-based therapies for degenerative diseases, such as diabetes. Adult human liver cells exhibit a high level of developmental plasticity and have been suggested as a potential source of pancreatic progenitor tissue. An instructive role for dominant pancreatic transcription factors in altering the hepatic developmental fate along the pancreatic lineage and function has been demonstrated. Here we analyze whether transcription factors expressed in mature pancreatic ß-cells preferentially activate ß-cell lineage differentiation in liver. NKX6.1 is a transcription factor uniquely expressed in ß-cells of the adult pancreas, its potential role in reprogramming liver cells to pancreatic lineages has never been analyzed. Our results suggest that NKX6.1 activates immature pancreatic markers such as NGN-3 and ISL-1 but not pancreatic hormones gene expression in human liver cells. We hypothesized that its restricted capacity to activate a wide pancreatic repertoire in liver could be related to its incapacity to activate endogenous PDX-1 expression in liver cells. Indeed, the complementation of NKX6.1 by ectopic PDX-1 expression substantially and specifically promoted insulin expression and glucose regulated processed hormone secretion to a higher extent than that of PDX-1 alone, without increasing the reprogrammed cells. This may suggest a potential role for NKX6.1 in promoting PDX-1 reprogrammed cells maturation along the ß-cell-like lineage. By contrast, NKX6.1 repressed PDX-1 induced proglucagon gene expression. The individual and concerted effects of pancreatic transcription factors in adult extra-pancreatic cells, is expected to facilitate developing regenerative medicine approaches for cell replacement therapy in diabetics.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas de Homeodominio/metabolismo , Células Secretoras de Insulina/fisiología , Transactivadores/metabolismo , Adolescente , Adulto , Animales , Biomarcadores/metabolismo , Linaje de la Célula , Niño , Preescolar , Glucagón/genética , Glucagón/metabolismo , Proteínas de Homeodominio/genética , Humanos , Células Secretoras de Insulina/citología , Hígado/citología , Transactivadores/genética , Adulto JovenRESUMEN
Type 1 diabetes mellitus (T1DM) results from a specific autoimmune mediated destruction of the pancreatic beta-cells. PDX-1 induced developmentally redirected liver cells were suggested to restore the ablated pancreatic function in chemically induced diabetes. However, developmentally redirected liver cells, may have acquired along with the desired beta-cell characteristics and functions, also undesired sensitivity to autoimmune attack and therefore may be inefficient in ameliorating T1DM. This study analyzes whether subjects with beta-cell autoimmunity could benefit from Ad-CMV-PDX-1 gene therapy. Using the model of cyclophosphamide-accelerated diabetes in non-obese diabetic (CAD-NOD) mice, we report that recombinant adenovirus mediated PDX-1 gene therapy, ameliorates hyperglycemia in CAD-NOD mice. Our data demonstrate that 43% of the overtly diabetic CAD-NOD mice treated with Ad-CMV-PDX-1 became normoglycemic and maintained a stable body weight. Ectopic PDX-1 expression induced pancreatic gene expression and insulin production in the mice livers. The amelioration of hyperglycemia, in PDX-1 treated diabetic mice was associated with an immune modulation manifested by Th1 to Th2 shift in the autoimmune T-cell response to antigens associated with NOD diabetes. Thus, liver-to-pancreas transdifferentiation ameliorates T1DM in a process which is associated with a concomitant modulation of the autoimmune attack. Our findings suggest a beneficial therapeutic effect of the PDX-1 gene therapy for treating autoimmune type 1 diabetes mellitus (T1DM).
Asunto(s)
Autoinmunidad , Diabetes Mellitus Tipo 1/terapia , Terapia Genética/métodos , Vectores Genéticos/uso terapéutico , Proteínas de Homeodominio/genética , Células Secretoras de Insulina/metabolismo , Hígado/efectos de los fármacos , Transactivadores/genética , Adenoviridae/genética , Animales , Cromatografía Líquida de Alta Presión , Ciclofosfamida/inmunología , Ciclofosfamida/farmacología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Insulina/biosíntesis , Insulina/inmunología , Células Secretoras de Insulina/inmunología , Hígado/inmunología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/inmunología , Células Th2/metabolismo , Transactivadores/metabolismoRESUMEN
UNLABELLED: 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 beta-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 alpha-fetoprotein. We present evidence indicating that PDX-1 triggers hepatic dedifferentiation by repressing the key hepatic transcription factor CCAAT/enhancer-binding protein beta. 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.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Diferenciación Celular , Genes Homeobox , Proteínas de Homeodominio/metabolismo , Hígado/citología , Transactivadores/metabolismo , Adulto , Biomarcadores/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Linaje de la Célula , Preescolar , Duodeno/citología , Duodeno/metabolismo , Femenino , Proteínas de Homeodominio/genética , Humanos , Hígado/metabolismo , Masculino , Páncreas/citología , Páncreas/metabolismo , Fenotipo , Transactivadores/genéticaRESUMEN
Shortage in tissue availability from cadaver donors and the need for life-long immunosuppression severely restrict the large-scale application of cell-replacement therapy for diabetic patients. This study suggests the potential use of adult human liver as alternate tissue for autologous beta-cell-replacement therapy. By using pancreatic and duodenal homeobox gene 1 (PDX-1) and soluble factors, we induced a comprehensive developmental shift of adult human liver cells into functional insulin-producing cells. PDX-1-treated human liver cells express insulin, store it in defined granules, and secrete the hormone in a glucose-regulated manner. When transplanted under the renal capsule of diabetic, immunodeficient mice, the cells ameliorated hyperglycemia for prolonged periods of time. Inducing developmental redirection of adult liver offers the potential of a cell-replacement therapy for diabetics by allowing the patient to be the donor of his own insulin-producing tissue.
Asunto(s)
Diferenciación Celular , Diabetes Mellitus Tipo 1/terapia , Ingeniería Genética , Terapia Genética/métodos , Hepatocitos/citología , Proteínas de Homeodominio/genética , Islotes Pancreáticos/citología , Transactivadores/genética , Adenoviridae , Animales , Células Cultivadas , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Glucosa/metabolismo , Hepatocitos/trasplante , Hepatocitos/ultraestructura , Humanos , Inmunohistoquímica , Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Microscopía Electrónica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción Genética/métodosRESUMEN
Pancreatic and duodenal homeobox gene-1 (PDX-1) regulates pancreas development during embryogenesis, whereas in the adult it controls beta-cell function. Here we analyze whether PDX-1 functions as a pancreatic differentiation factor and a bona fide master regulator when ectopically expressed in mature fully differentiated liver in vivo. By ectopic and transient PDX-1 expression in liver in vivo, using the first generation recombinant adenoviruses, we demonstrate that PDX-1 induces in liver a wide repertoire of both exocrine and endocrine pancreatic gene expression. Moreover, PDX-1 induces its own expression (auto-induction), which in turn may explain the long lasting nature of the "liver to pancreas" transdifferentiation. Insulin as well glucagon-producing cells are mainly located in the proximity of hepatic central veins, possibly allowing direct hormone release into the bloodstream, without affecting normal hepatic function. Importantly, we demonstrate that hepatic insulin production triggered by Ad-CMV-PDX-1 recombinant adenovirus administration is functional and prevents streptozotocin-induced hyperglycemia in Balb/c mice even 8 months after the initial treatment. We conclude that PDX-1 plays an important instructive role in pancreas differentiation, not only from primitive gut endoderm but also from mature liver. Transconversion of liver to pancreas may serve as a novel approach for generating endocrine-pancreatic tissue that can replace malfunctioning beta-cells in diabetics.