Elizabeth Geras-Raaka

Human pancreatic precursor cells secrete FGF2 to stimulate clustering into hormone-expressing islet-like cell aggregates

Development of the endocrine pancreas includes a series of early events wherein precursor cells cluster, that is migrate to form cell aggregates, which subsequently differentiate into islets of Langerhans. We show that PANC-1 cells, a human pancreatic cell line, differentiates into hormone-producing islet-like cell aggregates after exposure to a defined serum-free medium. These cells were used to provide the following evidence that fibroblast growth factor (FGF)2 is a paracrine chemoattractant during PANC-1 cell clustering: (i) FGF2 is secreted and remains bound to the extracellular matrix from where it may diffuse to form chemoattractive gradients; (ii) a subset of cells expresses FGF receptors (FGFRs) -1, -2, -3, and -4; (iii) inhibition of FGFR tyrosine kinase inhibits cell clustering; and (iv) FGF2 neutralizing antibody inhibits clustering. In addition, adult human islet-derived precursor cells, which cluster and differentiate in a manner similar to PANC-1 cells, also secrete FGF2 and express FGFRs. We conclude that FGF2, acting as a paracrine chemoattractant, stimulates clustering of precursor cells, an early step leading to islet-like cell aggregate formation. Similar processes may occur during development of the islet of Langerhans in humans.

Human Islet‐Derived Precursor Cells Are Mesenchymal Stromal Cells That Differentiate and Mature to Hormone‐Expressing Cells In Vivo

Islet transplantation offers improved glucose homeostasis in diabetic patients, but transplantation of islets is limited by the supply of donor pancreases. Undifferentiated precursors hold promise for cell therapy because they can expand before differentiation to produce a large supply of functional insulin‐producing cells. Previously, we described proliferative populations of human islet‐derived precursor cells (hIPCs) from adult islets. To show the differentiation potential of hIPCs, which do not express insulin mRNA after at least 1,000‐fold expansion, we generated epithelial cell clusters (ECCs) during 4 days of differentiation in vitro. After transplantation into mice, 22 of 35 ECC preparations differentiated and matured into functional cells that secreted human C‐peptide in response to glucose. Transcripts for insulin, glucagon, and somatostatin in recovered ECC grafts increased with time in vivo, reaching levels approximately 1% of those in adult islets. We show that hIPCs are mesenchymal stromal cells (MSCs) that adhere to plastic, express CD73, CD90, and CD105, and can differentiate in vitro into adipocytes, chondrocytes, and osteocytes. Moreover, we find a minor population of CD105+/CD73+/CD90+ cells in adult human islets (prior to incubation in vitro) that express insulin mRNA at low levels. We conclude that hIPCs are a specific type of pancreas‐derived MSC that are capable of differentiating into hormone‐expressing cells. Their ability to mature into functional insulin‐secreting cells in vivo identifies them as an important adult precursor or stem cell population that could offer a virtually unlimited supply of human islet‐like cells for replacement therapy in type 1 diabetes.

Endocrine Precursor Cells from Mouse Islets Are Not Generated by Epithelial-to-Mesenchymal Transition of Mature Beta Cells

We previously presented evidence that proliferative human islet precursor cells may be derived in vitro from adult islets by epithelial-to-mesenchymal transition (EMT) and show here that similar fibroblast-like cells can be derived from mouse islets. These mouse cell populations exhibited changes in gene expression consistent with EMT. Both C-peptide and insulin mRNAs were undetectable in expanded cultures of mouse islet-derived precursor cells (mIPCs). After expansion, mIPCs could be induced to migrate into clusters and differentiate into hormone-expressing islet-like aggregates. Although early morphological changes suggesting EMT were observed by time-lapse microscopy when green fluorescent protein-labeled beta cells were placed in culture, the expanded precursor cell population was not fluorescent. Using two mouse models in which beta cells were permanently made either to express alkaline phosphatase or to have a deleted M(3) muscarinic receptor, we provide evidence that mIPCs in long term culture are not derived from beta cells.

Reprogramming Adult Human Dermal Fibroblasts to Islet-Like Cells by Epigenetic Modification Coupled to Transcription Factor Modulation

In this article, we describe novel conditions for culture, expansion, and transdifferentiation of primary human dermal fibroblasts (hDFs) to induce expression of transcription factors (TFs) and hormones characteristic of the islets of Langerhans. We show that histones associated with the insulin gene are hyperacetylated and that insulin gene DNA is less methylated in islet cells compared to cells that do not express insulin. Using two compounds that alter the epigenetic signature of cells, romidepsin (Romi), a histone deacetylase inhibitor, and 5-Azacytidine (5-AzC), a chemical analogue of cytidine that cannot be methylated, we show that hDFs exhibit a distinctive regulation of expression of TFs involved in islet development as well as of induction of glucagon and insulin. Overexpression of Pdx1, a TF important for islet differentiation, and silencing of musculoaponeurotic fibrosarcoma oncogene homolog B, a TF that is expressed in mature glucagon-producing cells, result in induction of insulin to a higher level compared to Romi and 5-AzC alone. The cells obtained from this protocol exhibit glucose-stimulated insulin secretion and lower blood glucose levels of diabetic mice. These data show that fully differentiated nonislet-derived cells could be made to transdifferentiate to islet-like cells and that combining epigenetic modulation with TF modulation leads to enhanced insulin expression.

Trypsin and thrombin accelerate aggregation of human endocrine pancreas precursor cells

Human islet‐derived precursor cells (hIPCs) and human pancreatic ductal carcinoma (PANC‐1) cells can be induced to form aggregates that subsequently differentiate into hormone‐expressing islet‐like cell aggregates (ICAs). We show that challenge of hIPCs or PANC‐1 cells with thrombin or trypsin resulted in stimulation of signaling via the inositol‐tris‐phosphate second messenger pathway leading to rapid, transient increases in cytosolic calcium ion concentration in the majority of the cells. Because we found that hIPCs, PANC‐1 cells, human fetal pancreas, and human adult islets express two protease‐activated receptors (PARs), PAR‐1 and PAR‐2, we tested whether the effects of thrombin and trypsin were mediated, at least in part, by these receptors. Peptide agonists that are relatively specific for PAR‐1 (SFLLRN‐amide) or PAR‐2 (SLIGRL‐amide) stimulated increases in inositol phosphates and cytosolic calcium ion concentration, and increased the phosphorylation of Rho, a small G‐protein associated with cytoskeletal changes affecting cellular morphology and migration. Most importantly, we show that these agonists increased the rate of hIPC aggregation leading to the formation of more viable, smaller ICAs. Our data show that thrombin and trypsin accelerate aggregation, an early stage of hIPC differentiation in vitro, and imply that pancreatic trypsin and thrombin may be involved in islet development in vivo. J. Cell. Physiol. 206: 322–328, 2006. Published 2005 Wiley‐Liss, Inc.

Persistent cAMP signaling by thyrotropin (TSH) receptors is not dependent on internalization

Evidence was presented that thyrotropin [thyroid‐stimulating hormone (TSH)]‐stimulated persistent cAMP signaling is dependent on receptor (with G‐protein a subunits and adenylyl cyclase) internalization. Because it is not clear whether G proteins and adenylyl cyclase internalize with receptors, we tested whether persistent cAMP signaling by TSH receptor (TSHR) is dependent on internalization. We measured persistent TSHR signaling as an accumulation of cAMP in HEK‐EM293 cells permanently expressing human TSHRs incubated with isobutylmethylxanthine for 30 min after washing the cells to remove unbound TSH, and TSHR internalization by fluorescence microscopy using Alexa‐tagged TSH and binding assays using 125I‐TSH. TSHRs, but not the closely related lutropin or follitropin receptors, exhibit persistent cAMP signaling. TSHRs were not internalized by 30 min incubation with unlabeled TSH; however, expression of β‐arrestin‐2 promoted TSHR internalization that was inhibited by dynasore, a dynamin inhibitor. Expression of β‐arrestin‐2 had no effect on TSHR cAMP signaling, dynasore inhibited TSHR cAMP signaling in the absence or presence of TSHR internalization, and expression of a dominantnegative mutant dynamin, which inhibited internalization, had no effect on persistent cAMP signaling. Persistent cAMP signaling was specifically inhibited by a small molecule TSHR antagonist. We conclude that TSHRs do not have to be internalized to exhibit persistent cAMP signaling.—Neumann, S., Geras‐Raaka, E., Marcus‐Samuels, B., Gershengorn, M. C. Persistent cAMP signaling by thyrotropin (TSH) receptors is not dependent on internalization. FASEB J. 24, 3992–3999 (2010). www.fasebj.org

Thyrotropin Receptor Stimulates Internalization-independent Persistent Phosphoinositide Signaling

The thyrotropin [thyroid-stimulating hormone (TSH)] receptor (TSHR) is known to acutely and persistently stimulate cAMP signaling and at higher TSH concentrations to acutely stimulate phosphoinositide signaling. We measured persistent signaling by stimulating TSHR-expressing human embryonic kidney-EM293 cells with TSH and measuring cAMP or inositol monophosphate (IP1) production, a measure of phosphoinositide signaling, 60 min or longer after TSH removal. In contrast to persistent cAMP production, persistent IP1 production increased progressively when TSH exposure was increased from 1 to 30 min, whereas the rates of decay of persistent signaling were similar. A small-molecule agonist and a thyroid-stimulating antibody also caused persistent IP1 and cAMP signaling. A small-molecule inverse agonist and a neutral antagonist inhibited TSH-stimulated persistent IP1 production, whereas the inverse agonist but not the neutral antagonist inhibited persistent cAMP production. As with persistent cAMP production, persistent IP1 production was not affected when TSHR internalization was inhibited or enhanced. Moreover, Alexa546-TSH-activated TSHR internalization was not accompanied by Gαq coupling protein internalization. Thus, transient exposure to high concentrations of TSH causes persistent phosphoinositide and cAMP signaling that is not dependent on internalization. To our knowledge, this is the first demonstration of persistent activation by any G protein-coupled receptor (GPCR) via the Gαq pathway and of two G protein-mediated pathways by any GPCR.

Insulin but Not Glucagon Gene is Silenced in Human Pancreas‐Derived Mesenchymal Stem Cells

We previously characterized human islet‐derived precursor cells (hIPCs) as a specific type of mesenchymal stem cell capable of differentiating to insulin (INS)‐ and glucagon (GCG)‐expressing cells. However, during proliferative expansion, INS transcript becomes undetectable and then cannot be induced, a phenomenon consistent with silencing of the INS gene. We explored this possibility by determining whether ectopic expression of transcription factors known to induce transcription of this gene in β cells, pancreatic and duodenal homeobox factor 1 (Pdx1), V‐maf musculoaponeurotic fibrosarcoma oncogene homolog A (Mafa), and neurogenic differentiation 1 (Neurod1), would activate INS gene expression in long‐term hIPC cultures. Coexpression of all three transcription factors had little effect on INS mRNA levels but unexpectedly increased GCG mRNA at least 100,000‐fold. In contrast to the endogenous promoter, an exogenous rat INS promoter was activated by expression of Pdx1 and Mafa in hIPCs. Chromatin immunoprecipitation (ChIP) assays using antibodies directed at posttranslationally modified histones show that regions of the INS and GCG genes have similar levels of activation‐associated modifications but the INS gene has higher levels of repression‐associated modifications. Furthermore, the INS gene was found to be less accessible to micrococcal nuclease digestion than the GCG gene. Lastly, ChIP assays show that exogenously expressed Pdx1 and Mafa bind at very low levels to the INS promoter and at 20‐ to 25‐fold higher levels to the GCG promoter in hIPCs. We conclude that the INS gene in hIPCs is modified epigenetically (“silenced”) so that it is resistant to activation by transcription factors. STEM CELLS 2009; 27:2703–2711

Measurement of changes in cellular calcium metabolism in response to thyrotropin-releasing hormone

Publisher Summary Elevation of the concentration of free calcium ion in the cytoplasm of cells ([Ca 2+ ] i ) is an early event following the binding of many stimuli to plasma membrane receptors. The free (or ionized) calcium then plays an important role as a coupling factor leading to the activation of the final cellular response (“stimulus-response coupling”). The elevation of [Ca 2+ ] i is caused by the mobilization (or redistribution) of cellular calcium, enhanced influx of extracellular Ca 2+ , inhibition of the efflux of intracellular calcium, or a combination of these processes. The chapter describes the methods that can be employed to study these various aspects of cellular calcium homeostasis, including the direct measurement of [Ca 2+ ] i . It discusses studies of two pituitary cell types in vitro ––mammotropic (GH3) and thyrotropic (TtT) cells in culture. The examples presented to illustrate the use of these methods are from studies that attempt to define the mechanism of the action of thyrotropin releasing hormone (TRH) in these two cell types.

Heritability of fat accumulation in white adipocytes.

Since individual cells from freshly isolated white adipose tissue (WAT) exhibit variable levels of fat accumulation, we attempted to determine which factor(s) cause this variation. We used primary WAT cells from adult mice and the mouse 3T3-L1 cell-line of preadipocytes for these studies. Cells were labeled with BODIPY (boron-dipyrromethene) lipid probe, a marker for fat accumulation in live cells, and sorted on a fluorescence-activated cell sorter into two populations exhibiting low or high BODIPY fluorescence intensity. After more than 12 doublings as dedifferentiated cells in growth medium, the sorted populations were exposed to adipogenic medium for 7 days and analyzed for BODIPY accumulation and mRNA expression of adipogenic markers. WAT-derived cells initially sorted to have low or high BODIPY fluorescence intensity maintained a similar low or high lipid phenotype after redifferentiation. Cell surface TSH receptor expression, which is known to increase when preadipocytes are differentiated, correlated with BODIPY staining in all states. mRNA levels of Pparγ, Srebp1c, aP2, and Pref1, key regulators of adipogenesis, and leptin, Glut4, Fasn, and Tshr, markers of adipocyte differentiation, correlated with the levels of fat accumulation. Overexpression of Pparγ in 3T3-L1 cells, as expected, caused cells from low- and high-BODIPY populations to accumulate more fat. More importantly, prior to differentiation, the endogenous Pparγ promoter exhibited higher levels of acetylated histone H3, an activatory modification, in high-BODIPY- compared with low-BODIPY-derived populations. We conclude that fat accumulation is a heritable trait in WAT and that epigenetic modification on the Pparγ promoter contributes to this heritability.