Biomedical subjects

Shimon Efrat

Publications and source records attributed to Shimon Efrat.

13 recordsLinked to original sources

PubMed2006

Expansion and redifferentiation of adult human pancreatic islet cells.

Beta-cell replacement represents the ultimate cure for type 1 diabetes, however it is limited by availability of organ donors. Adult human islets are difficult to propagate in culture, and efforts to expand them result in dedifferentiation. Here we describe conditions for expansion of adult human islet cells, as well as a way for their redifferentiation. Most cells in islets isolated from human pancreata were induced to replicate within the first week of culture in expansion medium. Cells were propagated for 16 population doublings, without a change in replication rate or noticeable cell mortality, representing an expansion of over 65,000-fold. Replication was accompanied by a decrease in expression of key beta-cell genes. Shift of the cells to differentiation medium containing betacellulin resulted in redifferentiation, as manifested by restoration of beta-cell gene expression and insulin content. These methods may allow transplantation of functional islet cells from single donors into multiple recipients.

Adult↗

PubMed2005

Baculovirus p35 increases pancreatic beta-cell resistance to apoptosis.

beta-cells die by apoptosis in type 1 diabetes as a result of autoimmune attack mediated by cytokines, and in type 2 diabetes by various perpetrators including human islet amyloid polypeptide (hIAPP). The cascade of apoptotic events induced by cytokines and hIAPP is mediated through caspases and reactive oxygen species. The baculovirus p35 protein is a potent anti-apoptotic agent shown to be effective in a variety of species and able to inhibit a number of apoptotic pathways. Here, we aimed at determining the protective potential of p35 in beta-cells exposed to cytokines and hIAPP, as well as the effects of p35 on beta-cell function. The p35 gene was introduced into betaTC-tet cells, a differentiated murine beta-cell line capable of undergoing inducible growth-arrest. Both proliferating and growth-arrested cells expressing p35 manifested increased resistance to cytokines and hIAPP, compared with control cells, as judged by cell viability, DNA fragmentation, and caspase-3 activity assays. p35 was significantly more protective in growth-arrested, compared with proliferating, cells. No significant differences were observed in proliferation and insulin content between cells expressing p35 and control cells. In contrast, p35 manifested a perturbing effect on glucose-induced insulin secretion. These findings suggest that p35 could be incorporated as part of a multi-pronged approach of immunoprotective strategies to provide protection from recurring autoimmunity for transplanted beta-cells, as well as in preventive gene therapy in type 1 diabetes. p35 may also be protective from beta-cell damage caused by hIAPP in type 2 diabetes.

Amyloid↗

PubMed2005

Differentiation of human liver-derived, insulin-producing cells toward the beta-cell phenotype.

beta-Cell transplantation is viewed as a cure for type 1 diabetes; however, it is limited by the number of pancreas donors. Human stem cells offer the promise of an abundant source of insulin-producing cells, given the existence of methods for manipulating their differentiation. We have previously demonstrated that the expression of the beta-cell transcription factor pancreatic duodenal homeobox 1 (PDX-1) in human fetal liver cells activates multiple aspects of the beta-cell phenotype. These cells, termed FH-B-TPN cells, produce insulin, release insulin in response to physiological glucose levels, and replace beta-cell function in diabetic immunodeficient mice. However, they deviate from the normal beta-cell phenotype by the lack of expression of a number of beta-cell genes, the expression of non-beta-cell genes, and a lower insulin content. Here we aimed to promote differentiation of FH-B-TPN cells toward the beta-cell phenotype using soluble factors. Cells cultured with activin A in serum-free medium upregulated expression of NeuroD and Nkx2.2 and downregulated paired box homeotic gene 6 (PAX-6). Glucokinase and prohormone convertase 1/3 were also upregulated, whereas pancreatic polypeptide and glucagon as well as liver markers were downregulated. Insulin content was increased by up to 33-fold, to approximately 60% of the insulin content of normal beta-cells. The cells were shown to contain human C-peptide and release insulin in response to physiological glucose levels. Cell transplantation into immunodeficient diabetic mice resulted in the restoration of stable euglycemia. The cells continued to express insulin in vivo, and no cell replication was detected. Thus, the manipulation of culture conditions induced a significant and stable differentiation of FH-B-TPN cells toward the beta-cell phenotype, making them excellent candidates for beta-cell replacement in type 1 diabetes.

Activins↗

PubMed2004

Inhibition of islet amyloid polypeptide fibril formation: a potential role for heteroaromatic interactions.

The formation of amyloid fibril is associated with major human diseases, including Alzheimer's disease, prion diseases, and type 2 diabetes. Methods for efficient inhibition of amyloid fibril formation are therefore highly clinically important. A principal approach for the inhibition of amyloid formation is based on the use of modified molecular recognition elements. Here, we demonstrate efficient inhibition of amyloid formation of the type 2 diabetes-related human islet amyloid polypeptide (hIAPP) by a modified aromatic peptide fragment and a small aromatic polyphenol molecule. A molecular recognition assay using peptide array analysis suggested that molecular recognition between hIAPP and its core amyloidogenic module is mediated by aromatic rather than hydrophobic interactions. To study the possible effect of aromatic interactions on inhibition of hIAPP fibril formation, we have used peptide and small molecule inhibitors. The addition of a nonamyloidogenic peptide analogue of the core module NFGAILSS, in which phenylalanine was substituted with tyrosine (NYGAILSS), resulted in substantial inhibition of fibril formation by hIAPP. The inhibition was significantly stronger than the one achieved using a beta-sheet breaker-conjugated peptide NFGAILPP. On the basis of the molecular arrangement of the tyrosine-phenylalanine interaction, we suggest that the inhibition stems from the geometrical constrains of the heteroaromatic benzene-phenol interaction. In line with this notion, we demonstrate remarkable inhibition of hIAPP fibril formation and cytotoxicity toward pancreatic beta-cells by a small polyphenol molecule, the nontoxic phenol red compound. Taken together, our results provide further experimental support for the potential role of aromatic interactions in amyloid formation and establish a novel approach for its inhibition.

Amino Acid Substitution↗

PubMed2004

Generation of surrogate beta cells from tissue stem cells.

beta-Cell replacement represents a promising approach for the treatment of type 1 diabetes, but is limited by donor shortage and recurring autoimmunity. Stem and progenitor cells, which can be expanded and differentiated in vitro, may offer an abundant source of cells for transplantation. The difficulty of expanding mature beta cells and their pancreatic precursors has turned the attention to the potential of cells from other tissues to function as surrogate beta cells. Recent research has shown that cells from a number of tissues can be induced to produce, process, and store insulin, release it in response to physiologic signals, and replace beta-cell function in rodents. The change in cell phenotype has been induced by dominant transcription factor genes ectopically expressed in the cells, or following cell exposure to differentiation factors in vitro or in vivo. Propagation of these cells in tissue culture provides an opportunity for further modifying them to enhance their resistance to immune destruction.

Animals↗

PubMed2004

Regulation of insulin secretion: insights from engineered beta-cell lines.

Abnormalities in insulin secretion are involved in a number of diseases, including type 1 and type 2 diabetes, persistent hyperinsulinemic hypoglycemia of infancy, and insulinoma. Understanding the mechanisms that regulate insulin secretion may allow the development of new therapies for these diseases as well as contribute to our ability to engineer insulin-producing cells for cell replacement therapy of type 1 diabetes. Glucose phosphorylation in beta-cells has been viewed as a key regulatory event in coupling insulin secretion to extracellular glucose concentrations. Work with transformed rodent beta-cell lines as well as recent findings from human progenitor cells induced to differentiate into insulin-producing cells has provided new insights into the role of glucose phosphorylating enzymes in the regulation of insulin secretion.

Animals↗

PubMed2004

Generation of insulin-producing cells from stem cells for cell replacement therapy of type 1 diabetes.

Type 1 diabetes mellitus is caused by an autoimmune destruction of pancreatic islet beta cells, leading to insulin deficiency. Beta-cell replacement is considered the optimal treatment for type 1 diabetes, however it is severely limited by the shortage of human organ donors. An effective cell-replacement strategy depends on the development of an abundant supply of beta cells and their protection from recurring immune destruction. Stem/progenitor cells, which can be expanded in tissue culture and induced to differentiate into multiple cell types, represent an attractive source for generation of cells with beta-cell properties: insulin biosynthesis, storage, and regulated secretion in response to physiologic signals. Embryonic stem cells have been shown to spontaneously differentiate into insulin-producing cells at a low frequency, and this capacity could be further enhanced by tissue culture conditions, soluble agents, and expression of dominant transcription factor genes. Progenitor cells from fetal and adult tissues, such as liver and bone marrow, have also been shown capable of differentiation towards the beta-cell phenotype in vivo, or following expression of dominant transcription factors in vitro. These approaches offer novel ways for generation of cells for transplantation into patients with type 1 diabetes.

Animals↗

PubMed2003

Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells.

Beta-cell replacement is considered to be the most promising approach for treatment of type 1 diabetes. Its application on a large scale is hindered by a shortage of cells for transplantation. Activation of insulin expression, storage, and regulated secretion in stem/progenitor cells offers novel ways to overcome this shortage. We explored whether fetal human progenitor liver cells (FH) could be induced to differentiate into insulin-producing cells after expression of the pancreatic duodenal homeobox 1 (Pdx1) gene, which is a key regulator of pancreatic development and insulin expression in beta cells. FH cells possess a considerable replication capacity, and this was further extended by introduction of the gene for the catalytic subunit of human telomerase. Immortalized FH cells expressing Pdx1 activated multiple beta-cell genes, produced and stored considerable amounts of insulin, and released insulin in a regulated manner in response to glucose. When transplanted into hyperglycemic immunodeficient mice, the cells restored and maintained euglycemia for prolonged periods. Quantitation of human C-peptide in the mouse serum confirmed that the glycemia was normalized by the transplanted human cells. This approach offers the potential of a novel source of cells for transplantation into patients with type 1 diabetes.

Animals↗

PubMed2003

Adenovirus early region 3 antiapoptotic 10.4K, 14.5K, and 14.7K genes decrease the incidence of autoimmune diabetes in NOD mice.

Genes in the early region 3 (E3) of the adenovirus genome allow the virus to evade host immune responses by interfering with major histocompatibility (MHC) class I-mediated antigen presentation and tumor necrosis factor-alpha (TNF-alpha)- or Fas-induced apoptosis of infected cells. Autoimmune type 1 diabetes (T1D) is inhibited in NOD mice transgenically expressing all E3 genes under control of a rat insulin promoter (RIPE3/NOD). For dissecting the protective mechanisms afforded by various E3 genes, they were subdivided into RIP-driven transgene constructs. Strong T1D protection mediated at the beta-cell level characterized DL704/NOD mice lacking the E3 gp19K gene suppressing MHC class I expression but retaining the 10.4K, 14.5K, and 14.7K genes inhibiting Fas- or TNF-alpha-induced apoptosis and TNF-alpha-induced NF-kB activation. Much weaker protection characterized DL309/NOD mice expressing the gp19K but not the 10.4K, 14.5K, and 14.7K genes. While RIPE3/NOD splenocytes had an unexpected decrease in ability to adoptively transfer T1D, splenocytes from both the DL704 and DL309 stocks efficiently did so. These findings indicate that all E3 genes must be expressed to inhibit the diabetogenic potential of NOD immune cells. They also demonstrate that the antiapoptotic E3 genes most effectively protect pancreatic beta-cells from diabetogenic immune responses.

Adenoviridae↗

PubMed2002

Cell replacement therapy for type 1 diabetes.

Replacement of the insulin-producing pancreatic islet beta cells represents the ultimate treatment for type 1 diabetes. Recent advances in islet transplantation underscore the urgent need for developing alternatives to human tissue donors, which are scarce. Two possible approaches are the expansion of differentiated beta cells by reversible immortalization and the generation of insulin-producing cells from embryonic or adult stem cells. It is possible that new insights into endocrine pancreas development will ultimately lead to manipulation of progenitor-cell fate towards the beta-cell phenotype of insulin production, storage and regulated secretion. Both allogeneic and autologous surrogate beta cells are likely to require protection from recurring autoimmunity. This protection might take the form of tolerization, cell encapsulation, or cell engineering with immunoprotective genes. If successful, these approaches could lead to widespread cell replacement therapy for type 1 diabetes.

Adult↗

PubMed2002

Growth factor-dependent proliferation of the pancreatic beta-cell line betaTC-tet: an assay for beta-cell mitogenic factors.

The ability to expand normal pancreatic islet beta cells in culture would significantly advance the prospects of cell therapy for diabetes. A number of growth factors can stimulate limited islet cell replication, however other factors may exist which are more effective beta-cell-specific mitogens. The search for novel beta-cell growth factors has been hampered by the lack of a beta-cell-specific proliferation assay. We developed a simple and sensitive assay for beta-cell growth factors based on a conditionally-transformed mouse beta-cell line (betaTC-tet). These cells express the SV40 T antigen (Tag) oncoprotein under control of the tetracycline (Tc) operon regulatory system. In the presence of Tc, Tag expression is tightly shut off and the cells undergo complete growth arrest. Here we show that the growth-arrested cells can proliferate in response to growth factors in the absence of Tag. Using this assay, a number of growth factors previously shown to be mitogenic to a mixed islet cell population were found to induce proliferation of pure beta cells. We conclude that growth-arrested betaTC-tet cells can be employed in a survey of factors from various sources for identifying novel factors with beta-cell mitogenic activity.

Animals↗

PubMed2002

Preventing type 1 diabetes mellitus: the promise of gene therapy.

Type 1 (insulin-dependent) diabetes mellitus is an autoimmune disease that has no cure. Closed-loop insulin administration strategies and approaches for replacement of the insulin-producing beta cells may offer improved treatments, which could delay or prevent diabetes complications. In the long run, however, prevention of type 1 diabetes in susceptible individuals represents the best chance for reducing the toll of the disease. Prevention of type 1 diabetes will require reliable methods for early diagnosis of predisposition to the disease, using improved genetic and serological screening on a wide scale. Identification of the primary antigenic target(s) for autoimmunity will allow intervention in prediabetes stages aimed at the induction of antigen-specific tolerance. In addition to manipulation of the immune system, the susceptibility of beta cells to autoimmunity could be reduced. A number of genes have been shown to increase beta-cell resistance to immune effector molecules in animal models and cultured beta-cell lines. These genes could be used for preventive gene therapy of type 1 diabetes mellitus if expressed in beta cells prior to the onset of autoimmune destruction. This prospect depends on the development of safe and efficient vectors, and approaches for cell-specific targeting of these vectors to beta cells in vivo.

Animals↗

PubMedDate not supplied

Beta-cell expansion for therapeutic compensation of insulin resistance in type 2 diabetes.

Insulin resistance is the primary cause of type 2 diabetes. However, if compensated by increased insulin production, insulin resistance by itself does not lead to overt disease. Type 2 diabetes develops when this compensation is insufficient, due to defects in beta-cell function and in regulation of the beta-cell mass. beta-Cell transplantation, as well as approaches that replenish or preserve the endogenous beta-cell mass, may facilitate the treatment of type 2 diabetes in patients requiring exogenous insulin.

Diabetes Mellitus, Type 2↗