Date

May 2011

Document Type

Poster

Description

Abstract

Pancreatic endocrine cells are co-located into clusters called the islets of Langerhans that are comprised of glucagon producing alpha cells, insulin secreting beta cells, somatostatin generating delta cells, and other cell types. Type 1 diabetes results from an autoimmune process in which autoreactive T cells destroy the insulin producing beta cells, requiring the patient to inject insulin to regulate their blood glucose levels. Thus far, attempts to cure diabetes via islet transplantation have been limited by insufficient donor supply, inconsistent isolated islet quality, continued autoimmunity, alloimmune rejection, and limited beta cell regeneration. Diabetes research has focused on preventing the autoimmune response, promoting stem cell to beta cell differentiation, and defining the factors that influence beta cell proliferation. Islet research, in turn, has been limited to whole islet studies since, isolating the islet cell subtypes has not been possible. Using a method recently developed for mouse islet cells (Pechhold et al. Nat Biotechnol. 2009 Nov; 27(11):1038-42), that uses intracellular hormone staining and flow cytometry, we are able to sort human islets into populations uniquely expressing glucagon, insulin, or somatostatin. Further, we have developed a human gene array to measure candidate gene expression using a quantitative nuclease protection assay (qNPA). This technique uses 50 base oligomers that specifically recognize RNA from each gene of interest, overcoming limitations caused by the harsh conditions required for intracellular staining. We report gene expression analysis for specific hormones and transcription factors expressed in each islet cell population. We are further modifying this technique to study nonhuman primate islets, and investigate the specific proteome and miRNA profiles for individual islet cell populations. The goal of these studies is to characterize the genetic differences between the islet cell populations and understand which factors control beta cell regeneration and proliferation. We have shown that we can purify adult human islets into individual cellular populations. This is the first step in understanding the genetic and environmental components that regulate increased beta cell proliferation and beta cell mass. In the absence of full-length mRNA for RT-PCR or next generation sequencing, the qNPA technique provides candidate gene expression profiles for these cells.

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Gene Expression Profiling of Islet Cell Subtypes

Abstract

Pancreatic endocrine cells are co-located into clusters called the islets of Langerhans that are comprised of glucagon producing alpha cells, insulin secreting beta cells, somatostatin generating delta cells, and other cell types. Type 1 diabetes results from an autoimmune process in which autoreactive T cells destroy the insulin producing beta cells, requiring the patient to inject insulin to regulate their blood glucose levels. Thus far, attempts to cure diabetes via islet transplantation have been limited by insufficient donor supply, inconsistent isolated islet quality, continued autoimmunity, alloimmune rejection, and limited beta cell regeneration. Diabetes research has focused on preventing the autoimmune response, promoting stem cell to beta cell differentiation, and defining the factors that influence beta cell proliferation. Islet research, in turn, has been limited to whole islet studies since, isolating the islet cell subtypes has not been possible. Using a method recently developed for mouse islet cells (Pechhold et al. Nat Biotechnol. 2009 Nov; 27(11):1038-42), that uses intracellular hormone staining and flow cytometry, we are able to sort human islets into populations uniquely expressing glucagon, insulin, or somatostatin. Further, we have developed a human gene array to measure candidate gene expression using a quantitative nuclease protection assay (qNPA). This technique uses 50 base oligomers that specifically recognize RNA from each gene of interest, overcoming limitations caused by the harsh conditions required for intracellular staining. We report gene expression analysis for specific hormones and transcription factors expressed in each islet cell population. We are further modifying this technique to study nonhuman primate islets, and investigate the specific proteome and miRNA profiles for individual islet cell populations. The goal of these studies is to characterize the genetic differences between the islet cell populations and understand which factors control beta cell regeneration and proliferation. We have shown that we can purify adult human islets into individual cellular populations. This is the first step in understanding the genetic and environmental components that regulate increased beta cell proliferation and beta cell mass. In the absence of full-length mRNA for RT-PCR or next generation sequencing, the qNPA technique provides candidate gene expression profiles for these cells.