Pancreatic And Duodenal Homeobox Gene-1 Research Articles

256-OR: Using Human Induced Pluripotent Stem Cell Derived Organoids to Identify New Pathologies in Patients with Pdx1 Mutations

Pancreas and gastrointestinal (GI) tract development is guided by several transcription factors, including pancreatic and duodenal homeobox gene-1 (PDX1). While it is well known that PDX1is essential for pancreatic development and beta cell function, its role in GI development and function is not fully understood. We identified a patient with a homozygous point mutation in PDX1 causing total loss of PDX1 protein resulting in pancreatic agenesis. We differentiated induced pluripotent stem cells from the patient into human antral gastric (HAGO) and intestinal (HIO) organoids as a novel diagnostic approach to identify new GI pathologies. Immunofluorescence staining revealed that portions of the PDX1-null HAGOs lost the gastric marker claudin18 and increased intestinal villin1 expression, signifying that regions of the antrum are converting to an intestinal phenotype. Similarly, PDX1-null HIOs lost the intestinal marker cadherin17 and increased claudin18, indicating that regions of the intestine are undergoing gastric metaplasia. Examination of functional markers revealed an increase in acid producing parietal cells, complete lack of gastrin producing endocrine cells in HAGOs, and an increase in gastric surface mucin in HIOs. These in vitro results were further validated using endoscopy biopsies from two patients with PDX1 mutations, which showed a similar disruption in gastric and intestinal identity. CRISPR/Cas9-mediated correction of the PDX1 mutation reversed the observed PDX1-null metaplasic phenotypes in HAGOs and HIOs and also allowed for the successful generation of pancreatic tissue indicating unambiguously that the PDX1 mutation is responsible for these pathologies. Based on these findings, these PDX1-null patients will have additional screening to monitor the progression of metaplasia into gastric and intestinal cancer. This study further demonstrates the utility of organoids in improving patient diagnoses and treatment. Disclosure M. Krishnamurthy: None. T.R. Broda: None. D.O. Kechele: None. X. Zhang: None. J.J. Palermo: None. M.H. Collins: None. I.H. Thomas: None. A. Heider: None. A. Dauber: None. J. Wells: None. Funding American Diabetes Association (1-19-PDF-036 to M.K.)

330-LB: Using Human Induced Pluripotent Stem Cell-Derived Organoids to Identify New Pathologies in Patients with PDX1 Mutations

Pancreas and gastrointestinal (GI) tract development is guided by several transcription factors, including Pancreatic and duodenal homeobox gene-1 (PDX1). While it is well known that PDX1 is essential for pancreas development, its role in GI development and function in humans has not been investigated. We have identified two patients with a homozygous point mutation in PDX1 (c.188delC(p.P63fs)), causing total loss of protein function. These patients have additional GI complications however endoscopy and histological analysis of gastric and ?intestinal biopsies did not? reveal any pathologies. We used patient derived GI organoids from induced pluripotent stem cells (iPSCs) as a novel diagnostic approach and identified a myriad of undiagnosed GI pathologies. Analyses of human antral gastric organoids (HAGO) using immunofluorescence staining demonstrated a loss of gastric marker? claudin18 (CLDN18) and increased intestinal villin expression in PDX1-null? organoids, signifying that regions of the antrum are converting to an intestinal?phenotype. Similarly, human intestinal organoids (HIO) demonstrated a loss of intestinal marker ?Cadherin17 and an increase in gastric marker? CLDN18, indicating that regions of the intestine are undergoing gastric metaplasia. We also observed an increase in parietal cells in HAGOs, a lack of gastrin hormone in HAGO, and an increase in mucin production in HIOs. Based on this data we re-analyzed antral and duodenal biopsies of both patients and confirmed gastric and intestinal metaplasia increased parietal cells, decreased gastrin and increased mucin production. Because of our findings, these patients will have additional screening to monitor for development of gastric and intestinal cancer. This clearly demonstrates the utility of organoids in improving patient diagnoses and developing new clinical treatment plans. Disclosure M. Krishnamurthy: None. T.R. Broda: None. X. Zhang: None. J.J. Palermo: None. I.H. Thomas: None. A. Heider: None. D.B. Steien: None. A. Dauber: Consultant; Self; Novo Nordisk Inc. Research Support; Self; Novo Nordisk Inc. J. Wells: None. Funding American Diabetes Association (1-19-PDF-036 to M.K.)

Treatment of diabetes mellitus with microencapsulated fetal human liver (FH-B-TPN) engineered cells

Transplantation of whole human pancreases or isolated islets into patients with type 1 diabetes mellitus has been severely hampered by the scarcity of cadaveric human donor organs, which mandates search for insulin producing cells/tissue source alternatives. Recent progress in stem cell biology has started looking into functionally competent, insulin-secreting progenitor cells. It had been previously observed that induced expression of the β-cell transcriptional factor of the pancreatic and duodenal homeobox gene1 (PDX1), in human hepatocytes, may activate multiple features of the β-cell phenotype. These “FH-B-TPN” cells were shown to release insulin in response to physiological glucose stimulation both, in vitro and in vivo. However, because FH-B-TPNs lack the expression of a number of β-cell or non β-cell genes, and are associated with low insulin content, we aimed to determine whether these cells, upon physical manipulation and envelopment within “clinical grade” alginate-based microcapsules, would reverse hyperglycemia after graft into diabetic animal models.

A Short-activating RNA Oligonucleotide Targeting the Islet β-cell Transcriptional Factor MafA in CD34+ Cells

Upon functional loss of insulin producing islet β-cells, some patients with diabetes become dependent on life-long insulin supplementation therapy. Bioengineering surrogate insulin producing cells is an alternative replacement strategy. We have developed a novel approach using short-activating RNA oligonucleotides to differentiate adult human CD34+ cells into insulin-secreting cells. By transfecting RNA to increase transcript levels of the master regulator of insulin biosynthesis, v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), several pancreatic endodermal genes were upregulated during the differentiation procedure. These included Pancreatic and duodenal homeobox gene-1 (PDX1), Neurogenin 3, NeuroD, and NK6 homeobox 1 (NKx6-1). Differentiated CD34+ cells also expressed glucokinase, glucagon-like peptide 1 receptor (GLP1R), sulfonylurea receptor-1 (SUR1) and phogrin—all essential for glucose sensitivity and insulin secretion. The differentiated cells appropriately processed C-peptide and insulin in response to increasing glucose stimulation as shown by enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting analysis, western blotting, and immunofluorescence staining. We provide a new approach using short-activating RNA in developing insulin producing surrogate cells for treating diabetes.

Differentiation of CD34+ cells using small activating RNA targeting the islet ß-cell transcriptional factor MafA

U functional loss of insulin producing islet β-cells, some diabetic patients become dependent on life-long insulin supplementation therapy. Bioengineering surrogate insulin producing cells is an alternative replacement strategy. We have developed a novel cell culture methodology using RNA oligonucleotides to differentiate adult human hematopoietic CD34+ cells into insulin secreting cells. By transfecting RNA to increase transcript levels of the master regulator of insulin biosynthesis, v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), several pancreatic endodermal genes were upregulated during the differentiation procedure. These included Pancreatic and duodenal homeobox gene-1 (PDX1), Neurogenin 3, NeuroD, and NK6 homeobox 1 (NKx6.1). Differentiated CD34+ cells also expressed glucokinase, Glucagon-like peptide-1 (GLP1), Sulfonylurea receptor-1 (SUR1) and phogrin all essential for glucose sensitivity and insulin secretion. The differentiated cells appropriately processed c-peptide and insulin in response to increasing glucose stimulation as shown by enzyme-linked immunosorbent assay (ELISA), fluorescence activated cell-sorting (FACS) analysis, Western blotting and immunofluorescence staining. We provide a new approach to exploit human CD34+ cells that may be useful in developing insulin producing surrogate cells for treating diabetes.

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.

Establishment of a non-invasive mouse reporter model for monitoring in vivo pdx-1 promoter activity

It is well known that pancreatic and duodenal homeobox gene-1 (PDX-1) plays a crucial role in β-cell differentiation, and maintaining mature β-cell function. Thus, it is important to understand how pdx-1 gene is regulated under various pathophysiological conditions in vivo. In this study, to non-invasively and quantitatively monitor pdx-1 promoter activity in vivo, we constructed a pdx-1 promoter-SEAP-IRES-GFP reporter plasmid. In this construct, the −4.6 kb pdx-1 promoter region sufficient for driving β-cell-selective PDX-1 expression was inserted to the upstream of the secreted alkaline phosphatase (SEAP) reporter gene. It is noted here that the pdx-1 promoter-mediated SEAP activity can be distinguished from endogenous alkaline phosphatase activity. First, we transfected the construct in mouse β-cell line MIN6 and human hepatocellular carcinoma cell line HepG2. SEAP activity was readily detected in the media of MIN6 cells, but not in HepG2 cells. These results indicate that this construct specifically reports β-cell-specific pdx-1 promoter activity in a cell culture system. Based on these in vitro findings, we next generated transgenic mice using the same construct. SEAP activity was readily detected in serum of the transgenic mice, but not in their littermate mice. Furthermore, SEAP activity was detected in protein extract from the transgenic pancreas and slightly from the transgenic duodenum, but not from the liver, and brain. These results indicate that serum SEAP activity likely represents in vivo pdx-1 promoter activity. This transgenic mouse model would be useful to non-invasively monitor in vivo pdx-1 promoter activity and to screen new molecules which regulate PDX-1 expression.

Enriched human pancreatic ductal cultures obtained from selective death of acinar cells express pancreatic and duodenal homeobox gene-1 age-dependently.

Adult pancreatic ductal cells are believed to be islet precursors. Our aim was to obtain an enriched human ductal cell population in defined culture conditions, and to characterize these cultures for the presence of pancreatic developmental transcription factors. Non-endocrine adult human pancreatic digest was cultured for 4 days in serum-containing and serum-free media. During this time, analysis was done for phenotypic changes, cell death, and expression of islet and islet precursor markers. Culture in serum-supplemented and serum-free media gave similar recoveries of an enriched ductal population after 4 days. Extensive cell death due to apoptosis and necrosis was also observed over this time period. A donor-age dependent expression of pancreatic and duodenal homeobox gene-1 (PDX-1) in ductal cells was seen at 4 days whereby donors <25 yr expressed significantly more than donors >25 yr. Analysis of gene expression by RT-PCR showed the presence of islet developmental transcription factors neuroD, Nkx6.1, and PDX-1, as well as mature islet hormones. While acinar-ductal transdifferentiation of some cells cannot be ruled out, we provide evidence that the predominant mechanism for the derivation of enriched human ductal cultures in our culture conditions is selective acinar cell death. Furthermore, we have shown that ductal cultures from younger donors exhibit greater plasticity through expression of PDX-1, and may be of greater value in attempts to induce islet neogenesis. The presence, however, of insulin and glucagon mRNA indicates that contaminating endocrine cells remain in these cultures and underscores the need to use caution when assessing differentiation potential.

Analysis of expression profiles of islet-associated transcription and growth factors during beta-cell neogenesis from duct cells in partially duct-ligated mice.

Beta-cell neogenesis from pancreatic duct cells has been reported to occur in duct-ligated rat. Nevertheless, detailed process of this phenomenon has not been clarified. To clarify the mechanism of beta-cell neogenesis, a partial pancreatic duct ligation mouse model was created. Proliferation of duct cells, beta-cell neogenesis, and expression of transcription factors and differentiation/growth factors were studied by immunohistochemistry, cDNA array, and RT-PCR methods. In the duct-ligated portion of the pancreas, newly formed islet-like cell clusters (ICCs) were observed arising from the ducts on day 7 and afterward. Transcription factors, such as pancreatic and duodenal homeobox gene-1 (PDX-1), paired box factor 6 (Pax6), islet1 and Nkx2.2-positive cells, and protein gene product 9.5 (PGP9.5) were also induced in duct lining cells. By cDNA microarray analysis, expression of insulin-like growth factor-1 (IGF-1) and transforming growth factor beta1 (TGF-beta1) were above control levels on day 5, and RT-PCR showed an increase from day 5 to day 28. IGF-1 and activin A-positive cells were detected in ducts. In addition, expression of betacellulin (BTC), heparin-binding epidermal growth factor-like growth factor (HB-EGF), and TGF-alpha were also increased from day 3 or 5. These findings suggest that beta-cell or endocrine precursors are localized among duct lining cells. Induction of several islet cell-associated transcription factors and differentiation and/or growth factors may play important roles during beta-cell neogenesis in this model.

Functional, Persistent, and Extended Liver to Pancreas Transdifferentiation

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.

Transforming growth factor beta induction of insulin gene expression is mediated by pancreatic and duodenal homeobox gene-1 in rat insulinoma cells.

Although transforming growth factor-beta (TGF-beta) stimulates pancreatic islet cells to synthesize and secret insulin, the mechanism underlying this effect is not known. To investigate this question, we examined the insulin promoter activity focusing on a transcription factor, pancreatic and duodenal homeobox gene-1 (PDX-1) that binds to the A3 element of the rat insulin promoter. Studies performed using the rat insulinoma cell line, INS-1 showed that TGF-beta stimulation of endogenous insulin mRNA expression correlated with increased activity of a reporter construct containing the insulin promoter. A potential mechanism for this increase arose from, electrophoretic mobility shift assay showing that the nuclear extract from TGF-beta treated cells contained higher levels of A3 binding activity. Western blot analysis confirmed that PDX-1 was increased in the nuclear extract from INS-1 cells treated with TGF-beta. As expected, a mutant insulin promoter that lacked the PDX-1 binding site was not stimulated by TGF-beta. In summary, the results of these studies show that TGF-beta stimulates the transcription of insulin gene and this action is mediated by the transcription factor, PDX-1.

Possible Involvement of Atypical Protein Kinase C(PKC) in Glucose-Sensitive Expression of the Human Insulin Gene: DNA-Binding Activity and Transcriptional Activity of Pancreatic and Duodenal Homeobox Gene-1(PDX-1) Are Enhanced via Calphostin C-Sensitive but Phorbol 12-Myristate 13-Acetate(PMA) and Goe 6976-Insensitive Pathway.

Pancreatic and duodenal homeobox gene-1 (PDX-1) is a transcription factor which regulates the insulin gene expression. In this study, we tried to elucidate the role of PDX-1 in the glucose-induced transcriptional activation of the human insulin gene promoter in MIN6 cells. Electrophoretic mobility shift assay (EMSA) and chloramphenicol acetyltransferase (CAT) assay demonstrated that both DNA-binding activity and transcriptional activity of PDX-1 were increased with 20 mmol/l glucose more than with 2 mmol/l glucose. The DNA-binding activity of PDX-1 induced by high glucose was blocked by phosphatase treatment, suggesting the involvement of PDX-1 phosphorylation in this event. In an in vitro phosphorylation study, PDX-1 was phosphorylated by protein kinase C (PKC), but not by cAMP dependent protein kinase (PKA) or mitogen-activated protein kinase (MAPK). Furthermore, increased PDX-1 function induced by high glucose was blocked by calphostin C, an inhibitor of all PKC isoforms, but unaffected by phorbol 12-myristate 13-acetate (PMA), an activator of classical and novel PKC, or Gö 6976, an inhibitor of classical and novel PKC, which suggested that the PKC family which activated PDX-1 in MIN6 cells was atypical PKC. Western blot and immunocytochemical studies with anti-PKC zeta antibody confirmed the presence of PKC zeta, one of the isoforms of atypical PKC, in MIN6 cells. Furthermore, PKC zeta activity was significantly increased by glucose stimulation. These results suggest that high glucose increased DNA-binding activity of PDX-1 by activating atypical PKC including PKC zeta, resulting in transcriptional activation of the human insulin gene promoter.