isolation-culture-and-imaging-of-human-fetal-pancreatic-cell-clusters

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

University of California, San Diego

Previously, we had shown that inhibition of PLC activity impaired the ability of insulin to activate ERK in 3T3-L1 adipocytes. In this study, we confirmed that the insulin receptor and PLC-gamma1 are physically associated in hIRcB fibroblasts, insulin stimulates PLC-gamma1 enzyme activity, and inhibition of PLC activity impairs activation of ERK. We subsequently investigated whether PLC-gamma1 is required for insulin-stimulated mitogenesis. First, inhibition of PLC activity using U73122 impairs the ability of insulin to stimulate DNA synthesis. Second, disruption of the interaction of the insulin receptor with PLC-gamma1 by microinjection of SH2 domains derived from PLC-gamma1 or Grb2 but not Shc similarly blocks insulin-induced DNA synthesis. Third, microinjection of neutralizing antibodies to PLC-gamma1 blocks DNA synthesis, but nonneutralizing antibodies do not. The blockade in all three cases is rescued by synthetic diacylglycerols but not by inositol-1,4,5-trisphosphate, indicating a requirement for PLC enzyme activity. These experimental data point to a requirement for PLC-gamma1 in insulin-stimulated mitogenesis in hIRcB cells.

PLC-gamma1 enzyme activity is required for insulin-induced DNA synthesis.

Based on recent studies showing that PLCgamma associates to insulin receptor, we investigated its role in insulin stimulation of glucose transport in brown adipocytes. Insulin stimulation induced rapid PLCgamma association to phosphorylated insulin receptor, and activation of PLCgamma, as assessed by the mobilization of Ca(2+) from intracellular stores and by the production of the second messenger DAG. Both events are dependent on activation of PI3-kinase. Inhibition of PLCgamma activity either with the chemical compound U73122 or with an inhibitor peptide precluded insulin stimulation of glucose uptake, GLUT4 translocation, and actin reorganization, as wortmannin did. In contrast, the inactive analog U73343 did not have an inhibitory effect. Furthermore, translocation of GLUT4-GFP in response to insulin was completely abolished by cotransfection with a PLCgamma-inactive mutant in HeLa cells, a cell model sensitive to insulin that express PLCgamma. U73122 did not affect PI3-kinase nor Akt activation, but impaired PKCzeta activation by insulin, as wortmannin did. PLC activity renders two products, IP(3) and DAG, and DAG can be metabolized to PA by the action of DAG-kinase. Using the compound R54494, a DAG-kinase inhibitor, insulin-induced PKCzeta activation was also suppressed, this activity being restored by addition of PA. In summary, these data indicate that PLCgamma, activated at least partially by PI3-kinase, is a link between insulin receptor and PKCzeta through the production of PA and could mediate insulin-induced glucose uptake and GLUT4 translocation.

PLCgamma participates in insulin stimulation of glucose uptake through activation of PKCzeta in brown adipocytes.

There is increasing evidence that protein kinase C (PKC) isoforms modulate insulin-signaling pathways in both positive and negative ways. Recent reports have indicated that the novel PKCdelta mediates some of insulin's actions in muscle and liver cells. Many studies use the specific inhibitor rottlerin to demonstrate the involvement of PKCdelta. In this study, we investigated whether PKCdelta might play a role in 3T3-L1 adipocytes. We found that PKCdelta is highly expressed in mouse adipose tissue and increased on 3T3-L1 adipocyte differentiation, and insulin-stimulated glucose transport is blocked by rottlerin. The phosphorylation state and activity of PKCdelta are not altered by insulin, but the protein translocates to membranes following insulin treatment. In contrast to the results with rottlerin, inhibition of PKCdelta activity or expression has no effect on glucose transport in adipocytes, unlike muscle cells. Lastly, we found that rottlerin lowers adenosine triphosphate levels in 3T3-L1 cells by acting as a mitochondrial uncoupler, and this is responsible for the observed inhibition of glucose transport.

Rottlerin inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes by uncoupling mitochondrial oxidative phosphorylation.

Hyperplasia of middle-ear mucosa (MEM) during otitis media (OM) is thought to be partially mediated by the actions of growth factors and their receptors. The intracellular pathway leading from the small G-protein Ras to the extracellular regulated kinases (Erks) often links growth factor stimulation to cellular proliferation. This study assessed whether this pathway is involved in MEM hyperplasia during bacterial OM via the activation of Erk1/Erk2 in MEM of an in vivo rat bacterial OM model. Activation was maximal at 1 and 6 h and at 1 week after introduction of bacteria into the middle ear. Additionally, an in vitro model of rat MEM in bacterial OM was treated with farnesyl transferase inhibitor 277 or the Mek inhibitor U0126. MEM explants treated with either inhibitor demonstrated significant suppression of bacterially induced growth. These data support a role for Ras and Erk signaling in MEM hyperplasia during bacterial OM.

Participation of Ras and extracellular regulated kinase in the hyperplastic response of middle-ear mucosa during bacterial otitis media.

The SDF-1alpha/CXCR4 ligand/chemokine receptor pair is required for appropriate patterning during ontogeny and stimulates the growth and differentiation of critical cell types. Here, we demonstrate SDF-1alpha and CXCR4 expression in fetal pancreas. We have found that SDF-1alpha and its receptor CXCR4 are expressed in islets, also CXCR4 is expressed in and around the proliferating duct epithelium of the regenerating pancreas of the interferon (IFN) gamma-nonobese diabetic mouse. We show that SDF-1alpha stimulates the phosphorylation of Akt, mitogen-activated protein kinase, and Src in pancreatic duct cells. Furthermore, migration assays indicate a stimulatory effect of SDF-1alpha on ductal cell migration. Importantly, blocking the SDF-1alpha/CXCR4 axis in IFNgamma-nonobese diabetic mice resulted in diminished proliferation and increased apoptosis in the pancreatic ductal cells. Together, these data indicate that the SDF-1alpha-CXCR4 ligand receptor axis is an obligatory component in the maintenance of duct cell survival, proliferation, and migration during pancreatic regeneration.

The stromal cell-derived factor-1alpha/CXCR4 ligand-receptor axis is critical for progenitor survival and migration in the pancreas.

Hyperplasia of the middle ear mucosa contributes to the sequelae of acute otitis media. Understanding the signal transduction pathways that mediate hyperplasia could lead to the development of new therapeutic interventions for this disease and its sequelae. Endotoxin derived from bacteria involved in middle ear infection can contribute to the hyperplastic response. The p38 mitogen-activated protein kinase (MAPK) is known to be activated by endotoxin as well as cytokines and other inflammatory mediators that have been documented in otitis media. We assessed the activation of p38 in the middle ear mucosa of an in vivo rat bacterial otitis media model. Strong activity of p38 was observed 1 to 6 h after bacterial inoculation. Activity continued at a lower level for at least 7 days. The effects of p38 activation were assessed using an in vitro model of rat middle ear mucosal hyperplasia in which mucosal growth is stimulated by nontypeable Haemophilus influenzae during acute otitis media. Hyperplastic mucosal explants treated with the p38 alpha and p38 beta inhibitor SB203580 demonstrated significant inhibition of otitis media-stimulated mucosal growth. The results of this study suggest that intracellular signaling via p38 MAPK influences the hyperplastic response of the middle ear mucosa during bacterial otitis media.

Role of p38 mitogen-activated protein kinase in middle ear mucosa hyperplasia during bacterial otitis media.

Activins regulate the growth and differentiation of a variety of cells. During pancreatic islet development, activins are required for the specialization of pancreatic precursors from the gut endoderm during midgestation. In this study, we probed the role of activin signaling during pancreatic islet cell development and regeneration. Indeed, we found that both activins and activin receptors are upregulated in duct epithelial cells during islet differentiation. Interestingly, the expression of endogenous cellular inhibitors of activin signaling, follistatin and Cripto, were also found to be augmented. Inhibition of activins significantly enhanced survival and expansion of pancreatic epithelial cells but decreased the numbers of differentiated beta-cells. Our results suggest that the homeostasis of growth and terminal differentiation requires a precise context-dependent regulation of activin signaling. Follistatin participates in this process by promoting expansion of precursor cells during pancreas growth.

Inhibition of activin signaling induces pancreatic epithelial cell expansion and diminishes terminal differentiation of pancreatic beta-cells.

Gut peptide YY (PYY) plays an important role in regulating metabolism and is expressed during the ontogeny of the pancreas. However, its biological role during endocrine cell formation is not fully understood, and its role, if any, during pancreatic regeneration in the adult has not yet been explored. The knowledge of factors involved in beta cell renewal in adult animals is clearly relevant for the design of treatment strategies for type 1 diabetes. We therefore sought to determine if observations during fetal pancreas formation also apply to pancreatic growth in adult animals. Indeed, we have found marked expansion of the PYY-expressing population during pancreatic regeneration. In addition, we demonstrate the presence of cells co-expressing PYY and the critical pancreatic transcription factor pancreatic duodenal homeobox1 (PDX-1). Interestingly, these cells also co-expressed specific islet hormones during pancreatic development and re-growth, suggesting a developmental relationship. Furthermore, we have found that PYY can act in concert with IGF-1 to stimulate cellular responsiveness in pancreatic epithelial cells in vitro. Our data suggest that PYY may be a mediator of islet cell development, as well as a cofactor for growth factor responses, not only during fetal pancreas formation but also during regeneration in adult animals.

PYY in the expanding pancreatic epithelium.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) are key signaling molecules for pancreas development. Although FGFR3 is a crucial developmental gene, acting as a negative regulator of bone formation, its participation remains unexplored in pancreatic organogenesis. We found that FGFR3 was expressed in the epithelia in both mouse embryonic and adult regenerating pancreata but was absent in normal adult islets. In FGFR3 knockout mice, we observed an increase in the proliferation of epithelial cells in neonates, leading to a marked increase in islet areas in adults. In vitro studies showed that FGF9 is a very potent ligand for FGFR3 and activates extracellular signal-related kinases (ERKs) in pancreatic cell lines. Moreover, FGFR3 blockade or FGFR3 deficiency led to increased proliferation of pancreatic epithelial cells in vivo. This was accompanied by an increase in the proportion of potential islet progenitor cells. Thus, our results show that FGFR3 signaling inhibits the expansion of the immature pancreatic epithelium. Consequently, this study suggests that FGFR3 participates in regulating pancreatic growth during the emergence of mature islet cells.

FGFR3 is a negative regulator of the expansion of pancreatic epithelial cells.

Limited organ availability is an obstacle to the widespread use of islet transplantation in type 1 diabetic patients. To address this problem, many studies have explored methods for expanding functional human islets in vitro for diabetes cell therapy. We previously showed that islet cells replicate after monolayer formation under the influence of hepatocyte growth factor and selected extracellular matrices. However, under these conditions, senescence and loss of insulin expression occur after >15 doublings. In contrast, other groups have reported that islet cells expanded in monolayers for months progressed through a reversible epithelial-to-mesenchymal transition, and that on removal of serum from the cultures, islet-like structures producing insulin were formed (1). The aim of the current study was to compare the two methods for islet expansion using immunostaining, real-time quantitative PCR, and microarrays at the following time points: on arrival, after monolayer expansion, and after 1 week in serum-free media. At this time, cell aliquots were grafted into nude mice to study in vivo function. The two methods showed similar results in islet cell expansion. Attempts at cell differentiation after expansion by both methods failed to consistently recover a beta-cell phenotype. Redifferentiation of beta-cells after expansion is still a challenge in need of a solution.

Limited capacity of human adult islets expanded in vitro to redifferentiate into insulin-producing beta-cells.

The growth and renewal of epithelial tissue is a highly orchestrated and tightly regulated process occurring in different tissue types under a variety of circumstances. We have been studying the process of pancreatic regeneration in mice. We have identified a cell surface protein, named EP1, which is expressed on the duct epithelium during pancreatic regeneration. Whereas it is not detected in the pancreas of normal mice, it is found in the intestinal epithelium of normal adult mice, as well as during pancreatic repair following cerulein-induced destruction of the acinar tissue. The distinctive situations in which EP1 is expressed, all of which share in common epithelial cell growth in the gastrointestinal tract, suggest that EP1 is involved in the growth and renewal of epithelial tissues in both the intestine and the pancreas.

Epithelial progenitor 1, a novel factor associated with epithelial cell growth and differentiation.

The chemokine receptor CXCR4 and ligand SDF-1α are expressed in fetal and adult mouse islets. Neutralization of CXCR4 has previously been shown to diminish ductal cell proliferation and increase apoptosis in the IFNγ transgenic mouse model in which the adult mouse pancreas displays islet regeneration. Here, we demonstrate that CXCR4 and SDF-1α are expressed in the human fetal pancreas and that during early gestation, CXCR4 colocalizes with neurogenin 3 (ngn3), a key transcription factor for endocrine specification in the pancreas. Treatment of islet like clusters (ICCs) derived from human fetal pancreas with SDF-1α resulted in increased proliferation of epithelial cells in ICCs without a concomitant increase in total insulin expression. Exposure of ICCs in vitro to AMD3100, a pharmacological inhibitor of CXCR4, did not alter expression of endocrine hormones insulin and glucagon, or the pancreatic endocrine transcription factors PDX1, Nkx6.1, Ngn3 and PAX4. However, a strong inhibition of β cell genesis was observed when in vitro AMD3100 treatment of ICCs was followed by two weeks of in vivo treatment with AMD3100 after ICC transplantation into mice. Analysis of the grafts for human C-peptide found that inhibition of CXCR4 activity profoundly inhibits islet development. Subsequently, a model pancreatic epithelial cell system (CFPAC-1) was employed to study the signals that regulate proliferation and apoptosis by the SDF-1α/CXCR4 axis. From a selected panel of inhibitors tested, both the PI 3-kinase and MAPK pathways were identified as critical regulators of CFPAC-1 proliferation. SDF-1α stimulated Akt phosphorylation, but failed to increase phosphorylation of Erk above the high basal levels observed. Taken together, these results indicate that SDF-1α/CXCR4 axis plays a critical regulatory role in the genesis of human islets.

The SDF-1α/CXCR4 axis is required for proliferation and maturation of human fetal pancreatic endocrine progenitor cells.

Here, we demonstrate that the fractalkine (FKN)/CX3CR1 system represents a regulatory mechanism for pancreatic islet β cell function and insulin secretion. CX3CR1 knockout (KO) mice exhibited a marked defect in glucose and GLP1-stimulated insulin secretion, and this defect was also observed in vitro in isolated islets from CX3CR1 KO mice. In vivo administration of FKN improved glucose tolerance with an increase in insulin secretion. In vitro treatment of islets with FKN increased intracellular Ca(2+) and potentiated insulin secretion in both mouse and human islets. The KO islets exhibited reduced expression of a set of genes necessary for the fully functional, differentiated β cell state, whereas treatment of wild-type (WT) islets with FKN led to increased expression of these genes. Lastly, expression of FKN in islets was decreased by aging and high-fat diet/obesity, suggesting that decreased FKN/CX3CR1 signaling could be a mechanism underlying β cell dysfunction in type 2 diabetes.

Article	Year
PLC-gamma1 enzyme activity is required for insulin-induced DNA synthesis. Endocrinology\| PubMed ID: 11796522	2002
PLCgamma participates in insulin stimulation of glucose uptake through activation of PKCzeta in brown adipocytes. Experimental cell research\| PubMed ID: 12169270	2002
Rottlerin inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes by uncoupling mitochondrial oxidative phosphorylation. Endocrinology\| PubMed ID: 12239100	2002
Participation of Ras and extracellular regulated kinase in the hyperplastic response of middle-ear mucosa during bacterial otitis media. The Journal of infectious diseases\| PubMed ID: 12447762	2002
The stromal cell-derived factor-1alpha/CXCR4 ligand-receptor axis is critical for progenitor survival and migration in the pancreas. The Journal of cell biology\| PubMed ID: 14638861	2003
Role of p38 mitogen-activated protein kinase in middle ear mucosa hyperplasia during bacterial otitis media. Infection and immunity\| PubMed ID: 15271927	2004
Inhibition of activin signaling induces pancreatic epithelial cell expansion and diminishes terminal differentiation of pancreatic beta-cells. Diabetes\| PubMed ID: 15277382	2004
PYY in the expanding pancreatic epithelium. Endocrine\| PubMed ID: 17185798	2006
FGFR3 is a negative regulator of the expansion of pancreatic epithelial cells. Diabetes\| PubMed ID: 17192470	2007
Limited capacity of human adult islets expanded in vitro to redifferentiate into insulin-producing beta-cells. Diabetes\| PubMed ID: 17327439	2007
Epithelial progenitor 1, a novel factor associated with epithelial cell growth and differentiation. Endocrine\| PubMed ID: 20960269	2010
The SDF-1α/CXCR4 axis is required for proliferation and maturation of human fetal pancreatic endocrine progenitor cells. PloS one\| PubMed ID: 22761699	2012
The fractalkine/CX3CR1 system regulates β cell function and insulin secretion. Cell\| PubMed ID: 23582329	2013

Ayse G. Kayali

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