Publications

BackgroundCell therapy of diabetes aims at restoring the physiological control of blood glucose by transplantation of functional pancreatic islet cells. A potentially unlimited source of cells for such transplantations would be islet cells derived from an in vitro differentiation of human pluripotent stem cells (hESC/hiPSC). The islet-like clusters (ILC) produced by the known differentiation protocols contain various cell populations. Among these, the β-cells that express both insulin and the transcription factor Nkx6.1 seem to be the most efficient to restore normoglycemia in diabetes animal models. Our aim was to find markers allowing selection of these efficient cells.MethodsFunctional Cell-Capture Screening (FCCS) was used to identify markers that preferentially capture the cells expressing both insulin and Nkx6.1, from hESC-derived ILC cells. In order to test whether selection for such markers could improve cell therapy in diabetic mouse models, we used ILC produced from a clinical-grade line of hESC by a refined differentiation protocol adapted to up-scalable bioreactors. Re-aggregated MACS sorted cells were encapsulated in microspheres made of alginate modified to reduce foreign body reaction. Implantation was done intraperitoneally in STZ-treated C57BL/6 immuno-competent mice.ResultsCD49A (integrin alpha1) was identified by FCCS as a marker for cells that express insulin (or C-peptide) as well as Nkx6.1 in ILC derived by hESC differentiation. The ILC fraction enriched in CD49A+ cells rapidly reduced glycemia when implanted in diabetic mice, whereas mice receiving the CD49A depleted population remained highly diabetic. CD49A-enriched ILC cells also produced higher levels of human C-peptide in the blood of transplanted mice. However, the difference between CD49A-enriched and total ILC cells remained small. Another marker, CD26 (DPP4), was identified by FCCS as binding insulin-expressing cells which are Nkx6.1 negative. Depletion of CD26+ cells followed by enrichment for CD49A+ cells increased insulin+/Nkx6.1+ cells fraction to ~70%. The CD26-/CD49A+ enriched ILC exhibited improved function over non-sorted ILC or CD49A+ cells in diabetic mice and maintain prolonged blood C-peptide levels.ConclusionsRefining the composition of ILC differentiated from hPSC by negative selection to remove cells expressing CD26 and positive selection for CD49A expressing cells could enable more effective cell therapy of diabetes.

The potential of reprogrammed β cells derived from pancreatic exocrine cells to treat diabetes has been demonstrated in animal models. However, the precise mechanisms and regulators involved in this process are not clear. Here, we describe a method that allows mechanistic studies of this process in primary exocrine cultures using adenoviral expression vectors. This rapid 5-day protocol, provides the researcher with a highly controlled experimental system in which the effects of different compounds or genetic manipulations can be studied. For complete details on the use and execution of this protocol, please refer to Elhanani et al. (2020).

Background: The transcription factor FoxA2 is a key mediator of endoderm development and pancreas gene expression. Results: DNA methylation is paradoxically associated with active expression of the FoxA2 gene. Conclusion: Developmental control genes such as FoxA2 may be regulated by a novel genetic mechanism. Significance: Clarification of this mechanism may provide a better understanding of how developmental pathways are activated. The transcription factor FoxA2 is a master regulator of endoderm development and pancreatic beta cell gene expression. To elucidate the mechanisms underlying the activation of the FoxA2 gene during differentiation, we have compared the epigenetic status of undifferentiated human embryonic stem cells (hESCs), hESC-derived early endoderm stage cells (CXCR4+ cells), and pancreatic islet cells. Unexpectedly, a CpG island in the promoter region of the FoxA2 gene displayed paradoxically high levels of DNA methylation in expressing tissues (CXCR4+, islets) and low levels in nonexpressing tissues. This CpG island region was found to repress reporter gene expression and bind the Polycomb group protein SUZ12 and the DNA methyltransferase (DNMT)3b preferentially in undifferentiated hESCs as compared with CXCR4+ or islets cells. Consistent with this, activation of FoxA2 gene expression, but not CXCR4 or SOX17, was strongly inhibited by 5-aza-2-deoxycytidine and by knockdown of DNMT3b. We hypothesize that in nonexpressing tissues, the lack of DNA methylation allows the binding of DNA methyltransferases and repressing proteins, such as Polycomb group proteins; upon differentiation, DNMT activation leads to CpG island methylation, causing loss of repressor protein binding. These results suggest a novel and unexpected role for DNA methylation in the activation of FoxA2 gene expression during differentiation.

Heterogeneity, shortage of material, and lack of progenitor-specific cell surface markers are major obstacles to elucidating the mechanisms underlying developmental processes. Here we report a proteomics platform that alleviates these difficulties and demonstrate its effectiveness in fractionating heterogeneous cultures of early endoderm derived from human embryonic stem cells. The approach, designated differential cell-capture antibody array, is based on highly parallel, comparative screening of live cell populations using hundreds of antibodies directed against cell-surface antigens. We used this platform to fractionate the hitherto unresolved early endoderm compartment of CXCR4+ cells and identify several endoderm (CD61+ and CD63+) and non-endoderm (CD271+, CD49F+, CD44+ and B2M+) sub-populations. We provide evidence that one of these sub-populations, CD61+, is directly derived from CXCR4+ cells, displays characteristic kinetics of emergence, and exhibits a distinct gene expression profile. The results demonstrate the potential of the cell-capture antibody array as a powerful proteomics tool for detailed dissection of heterogeneous cellular systems.

GPR40 is a G protein-coupled receptor expressed preferentially in β cells, that has been implicated in mediating free fatty acid-stimulated insulin release. GPR40 RNAi impaired the ability of palmitic acid (PA) to increase both insulin secretion and intracellular calcium ([Ca²⁺]_i). The PA-dependent [Ca²⁺]_i increase was attenuated by inhibitors of Gαq, PLC, and SERCA. Thus GPR40 activates the Gαq pathway, leading to release of Ca²⁺ from the ER. Yet the GPR40-dependent [Ca²⁺]_i rise was dependent on extracellular Ca²⁺ and elevated glucose, and was blocked by inhibition of L-type calcium channels (LTCC) or opening of the K_ATP channel; this suggests that GPR40 promotes Ca²⁺ influx through up-regulation of LTCC pre-activated by glucose and membrane depolarization. Taken together, the data indicate that GPR40 mediates the increase in [Ca ²⁺]_i and insulin secretion through the Gαq-PLC pathway, resulting in release of Ca²⁺ from the ER and leading to up-regulation of Ca²⁺ influx via LTCC.

Since both major forms of diabetes involve inadequate function of pancreatic beta cells, intensive research in ongoing to better understand how beta cells perform their complex role of secreting the hormone insulin in response to physiologic needs. Identification and characterization of pancreatic transcription factors has revealed that they play a crucial role not only in maintenance of mature beta-cell function but also at multiple stages in pancreatic development. Furthermore, recent reports have revealed their potential to convert non-beta cells into insulin-producing cells, which in some cases can function to ameliorate diabetes in experimental animals. The ability to translate these successes to the clinic will require a detailed mechanistic understanding of the molecular basis of action of these proteins. Specific gene regulation in beta cells involves the action of multiple transcription factors recruited to the promoter and functioning synergistically to activate transcription, in part through recruitment of co-activator proteins and components of the basal transcriptional machinery. In addition, the process involves modification of chromatin structure, the details of which are beginning to be elucidated. Our ability to modulate gene expression patterns may lead to developing ways to provide an unlimited supply of functional beta cells for transplantation, permitting a dramatic improvement in therapeutic options for diabetes.

A number of natural phenolic compounds display antioxidant and cell protective effects in cell culture models, yet in some studies show prooxidant and cytotoxic effects. Pancreatic β-cells have been reported to exhibit particular sensitivity to oxidative stress, a factor that may contribute to the impaired β-cell function characteristic of diabetes. The aim of this study was to examine the potential of natural phenolics to protect cultured pancreatic β-cells (βTC1 and HIT) from H₂O₂ oxidative stress. Exposure of cells to H₂O₂ led to significant proliferation inhibition. Contrary to what one should expect, simultaneous exposure to H₂O₂ and the phenolics, quercetin (10-100 μM), catechin (50-500 μM), or ascorbic acid (100-1000 μM), led to amplification of proliferation inhibition. At higher concentrations, these compounds inhibited proliferation, even in the absence of added H₂O₂. This prooxidant effect is attributable to the generation of H₂O₂ through interaction of the added phenolic compounds with as yet undefined componenets of the culture media. On the other hand, inclusion of metmyoglobin (30 μM) in the culture medium significantly reduced the prooxidant impact of the phenolics. Under these conditions, quercetin and catechin significantly protected the cells against oxidative stress when these components were present during the stress period. Furthermore, significant cell protection was observed upon preincubation of cells with chrysin, quercetin, catechin, or caffeic acid (50 μM, each) prior to application of oxidative stress. It is concluded that provided artifactual prooxidant effects are avoided, preincubation of β-cells with relatively hydrophobic natural phenolics can confer protection against oxidative stress.

By screening for genes expressed differentially in pancreatic beta cells, we have isolated a cDNA encoding GRFβ, a novel 178-amino acid protein whose N terminus is identical to that of GRF1, a calcium-dependent guanine nucleotide exchange factor, and whose C terminus is unrelated to known proteins. We show that both GRF1 and GRFβ are expressed selectively in beta cell lines, pancreatic islet cells and brain. Treatment of beta cell lines (βTC1 and HIT) with calcium ionophore led to a significant elevation in activity of the Ras signal transduction pathway, as determined by phosphorylation of extracellular signal-related kinase (ERK). Transfection of beta cells with a plasmid encoding a dominant negative variant of GRF1 led to 70% reduction in ERK phosphorylation, consistent with a role for GRF1 in calcium-dependent Ras signaling in these cells. To examine the possible function of GRFβ, cultured cells were transfected with a GRFβ expression vector. This led to a significant reduction in both GRF1-dependent ERK phosphorylation and AP1-dependent reporter gene activity. The results suggest that GRF1 plays a role in mediating calcium-dependent signal transduction in beta cells and that GRFβ represents a novel dominant negative modulator of Ras signaling.

The E2A protein is a mammalian transcription factor of the helix-loop- helix family which is implicated in cell-specific gene expression in several cell lineages. Mouse E2A contains two independent transcription activation domains, ADI and ADII; whereas ADI functions effectively in a variety of cultured cell lines, ADII shows preferential activity in pancreatic beta cells. To analyze this preferential activity in an in vivo setting, we adapted u system involving transient gene expression in microinjected zebra fish embryos. Fertilized one- to four-cell embryos were coinjected with an expression plasmid and a reporter plasmid. The expression plasmids used encode the yeast Gal4 DNA-binding domain (DBD) alone, or Gal4 DBD fused to ADI, ADII, or VP16. The reporter plasmid includes the luciferase gene linked to a promoter containing repeats of UASg, the Gal4-binding site. Embryo extracts prepared 24 h after injection showed significant luciferase activity in response to each of the three activation domains. To determine the cell types in which the activation domains were functioning, a reporter plasmid encoding β-galactosidase and then in situ staining of whole embryos were used. Expression of ADI led to activation in all major groups of cell types of the embryo (skin, sclerotome, myotome, notochord, and nervous system). On the other hand, ADII led to negligible expression in the sclerotome, notochord, and nervous system and much more frequent expression in the myotome. Parallel experiments conducted with transfected mammalian cells have confirmed that ADII shows significant activity in myoblast cells but little or no activity in neuronal precursor cells, consistent with our observations in zebra fish. This transient-expression approach permits rapid in vivo analysis of the properties of transcription activation domains: the data show that ADII functions preferentially in cells of muscle lineage, consistent with the notion that certain activation domains contribute to selective gene activation in vivo.

Human immunodeficiency virus (HIV) infection of the brain leads to massive neuronal damage, resulting in the AIDS (acquired immunodeficiency syndrome) dementia complex (ADC). A recent study using transgenic mice indicates that neurons possess transcription factors capable of activating the HIV promoter. To identify these, we transfected two types of primary cultures of rat neurons with HIV promoter-reporter gene constructs. The two κB regulatory sites in the HIV long terminal repeat (LTR) are shown to be essential for strong promoter activity. Two proteins present in neurons, BETA and an NF-κB-like protein, can bind the κB sites. These proteins are shown to belong to distinct families of transcription factors. Mutation analysis and transfection of a dominant negative NF-κB mutant, indicate that the neuronal NF-κB-like activity mediates HIV promoter activation. cDNA cloning, biochemical and immunological analyses indicate that neuronal NF-κB is similar to NF-κB of other tissues. Transfections of primary neuron cultures with an HIV promoter-β-galactosidase construct show that within these cultures, neurons are indeed the cells that highly activate the HIV promoter. Thus, analogous to the situation in T-lymphocytes and macrophages, NF-κB is an activator of HIV transcription in neurons.

The cis-acting DNA element known as the E box (consensus sequence CAxxTG) plays an important role in the transcription of a number of cell-specifically expressed genes. The rat insulin I gene, for example, contains two such sequences (IEB1 and IEB2) that are recognized specifically by a characteristic β cell nuclear factor insulin enhancer factor 1 (IEF1). To define the role of these elements better, we tested for cooperative interactions between the IEB sequences. Transfection experiments were performed with a series of plasmids containing the elements separated by different distances. Transcriptional activity in vivo is only modestly affected (less than two-fold) when the distances between the IEB elements are changed by a half-integral number of double-helical turns. Surprisingly, plasmids bearing four and six copies of the IEB motif showed sharply reduced activity as compared to those with two copies. In vitro DNA-binding studies revealed that this effect was not due to inability of IEF1 to bind to multiple copies of IEB. Moreover, multiple copies of the IEB sequence were able to inhibit activity of a cis-linked Moloney sarcoma virus (MSV) or insulin enhancer upon transfection to β cells but not to other cell types. The above data are consistent with the view that β cells contain a cell-specific repressor molecule capable of binding to multiple copies of IEB and thereby inhibiting transcription. This interpretation was further strengthened by in vivo competition and trans-activation experiments. The β-cell-specific repressor activity identified by these studies may play an important role in mediating gene expression in insulin-producing cells, perhaps by regulating the access of helix-loop-helix transcription factors to E-box sequence elements.

Transcription of a number of mammalian genes is controlled in part by closely-related DNA elements sharing a CAxxTG consensus sequence (E boxes). In this report, we survey cell extracts from a variety of mammalian cell lineages for ability to bind to the E box denoted IEB1/×E1, which plays an important role in expression of both insulin and immunoglobulln x genes. Insulin enhancer factor 1 (IEF1), a binding activity previously identified in β cells, was also present in pituitary endocrine cells but absent in 7 other mammalian cell lines tested. A distinct binding activity, lymphold enhancer factor 1 (LEF1), was observed in several lymphold cell lines, but was absent from all non-lymphoid cells tested. IEF1 and LEF1 were distinct according to electrophoretic mobility, and DNA binding specificity. As previously reported, both β cell and lymphoid cell factors are recognized by antibodies to helix-loop-helix (HLH) proteins, Indicating that they may contain functional helix-loop-helix dimerization domains. To directly demonstrate this, we showed that the binding factors are able to interact In vitro with the HLH domain of a characterized HLH protein. These results support the notion that HLH proteins play a key role in cell-specific transcrlptional regulation in cells from endocrine and lymphocyte lineages.

Rat brain mRNA enriched for tubulin and actin sequences was used to prepare double stranded cDNA. A library of recombinant clones was constructed by inserting the dsDNA into the Pstl site of pBR322 plasmid and transformation of E.coli x1776 host. Clones bearing sequences coding for tubulin and actin were identified and characterized.

A system was established in which nuclear preparations from rat brains were capable of protein synthesis under cell free conditions. The electrophoretic pattern of the synthesized proteins was similar to that found in vivo provided that the reaction mixture contained pH 5 precipate factors derived from the high speed supernatant fraction of brain. In the absence of the pH 5 factors, using nuclear preparations from brains of 2 day old rats, approximately 1.5% and 2% of the newly synthesized proteins were identified as tubulin and actin, respectively. In the presence of pH 5 factors, protein synthesis was stimulated and the proportion of the newly synthesized tubulin and actin increased to 26% and 11%, respectively. In contrast to nuclear fractions from 2 day old rats, when nuclei from brains of 1 month old rats were tested in the presence of pH 5 factors, the proportion of tubulin and actin synthesized was lower and amounted to 10% and 4%, respectively. The age dependent change in the relative amount of the tubulin and actin synthesized is in good agreement with the translational pattern shown by brain polyribosomes in a brain cell free system as well as with the pattern obtained with brain mRNA translated in a wheat germ cell free system. Nuclei enriched for either neuronal or glial populations synthesized similar proportions of tubulin and actin in vitro. We conclude that the reduction in the synthesis of tubulin and actin during the postnatal development of the rat brain occurs in both neuronal and glial cells.

The distribution of phosphoprotein phosphatase (PPPase) and estrogen-induced protein (IP) from 19- to 20-day-old rat uteri before and after fractionation of uterine cytosol, by ammonium sulfate, and by preparative cellulose acetate gel electrophoresis was studied. There was a lack of significant difference between the specific activity of PPPase or its electrophoretic mobility in control extracts and those of extracts made 1 hour after injection of the rats with 5 mcg estradiol-17beta. Most of the recovered PPPase activity appeared in the fraction precipitating between 0-50% saturation with ammonium sulfate. Most of the IP is found at 50-80% saturation, and less than 10% of the PPPase activity. A single peak of PPPase activity was shown at a mobility of .5 relative to bovine serum albumin with electrophoresis of the 50-80% ammonium sulfate fraction. A peak of IP with mobility of 1.2 was also shown. ''The results are incompatible with the view that IP mediates estrogen action by virtue of its PPPase activity.''.