Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Generation of Stem Cell-Derived Î² Cells from Type 1 Diabetic Patients

ABSTRACT: We recently reported the scalable in vitro production of functional stem cell-derived Î² cells. Here we extend this approach to generate SC-Î² cells from Type 1 diabetic patients (T1D), a cell type that is destroyed during disease progression and has not been possible to extensively study. These cells express Î² cell markers, respond to glucose both in vitro and in vivo, prevent alloxan-induced diabetes in mice, and respond to anti-diabetic drugs. Furthermore, we use an in vitro disease model to demonstrate the cells respond to different forms of Î² cell stress. Using these assays, we find no major differences in T1D SC-Î² cells compared to SC-Î² cells derived from non-diabetic patients (ND). These results show that T1D SC-Î² cells can be used for the treatment of diabetes, drug screening, and the study of Î² cell biology. Differentiated cells were sorted and processed for RNA isolation using the MARIS protocol published previously (PMID: 24516164.) Human induced pluripotent stem cell (hiPSC) line were differentiated into SC-beta cells or dysfunctional, polyhormonal cells (PH). Four biological replicates were assessed with differentiation to both SC-beta and PH cells. Those data were normalized together with and compared to existing, previously published data from Hrvatin et al. (PMID: 24516164) and Pagliuca et al. (PMID: 25303535) from human islet-derived insulin+ cells, undifferentiated HUES8 hES cells, SC-beta cells derived from HUES8 and PH cells derived from HUES8 according to previously published protocols.

ORGANISM(S): Homo sapiens

SUBMITTER: Jeffrey Millman

PROVIDER: E-GEOD-70901 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Like humans, the NOD mouse and other diabetes susceptible rat strains, T1D in BB rats is dependent on the major histocompatibility complex (MHC, insulin dependent diabetes mellitus locus 1, Iddm1) located on chromosome 20. In rats this is the HLA-DQB1 homologue RT1-B, specifically the RT1u haplotype. Our studies employ congenic derivatives of the BB rat, the DRlyp/lyp and DR+/+ strains, which differ only by the 2 Mb lyp (lymphopenia, Iddm2) region on chromosome 4. TID in the lymphopenic DRlyp/lyp rat is spontaneous and onset occurs in 100% of animals during adolescence (65.3+/-6.3 days) due to a recessive mutation within GIMAP5 (GTPase, IMAP family member 5). Gimap5 is a mitochondrial GTP-binding protein necessary for post-thymic T cell survival. The spontaneously diabetic phenotype observed in DRlyp/lyp rats is thought to be elicited through deficiency in CD4+CD25+ TREG cells as T1D in lymphopenic BB rats can be rescued through adoptive transfer of this population. Genetic variation in GIMAP5 has been associated with the development of protein-tyrosine phosphatase-2 (IA-2) autoantibodies in human T1D [28] and is significantly associated with systemic lupus erythematosus (SLE). The non-lymphopenic DR+/+ strain possesses wild-type GIMAP5 alleles and does not develop spontaneous T1D, however, T1D is inducible through administration of lymphotoxic anti-RT6 monoclonal antibody and immune activating polyinosinic polycytidylic acid (poly I:C; a ligand of toll-like receptor 3), or through viral depletion of CD4+CD25+ regulatory T (TREG) cells. Such treatments do not induce T1D in the related Wistar-Furth (WF) rats and suggest the presence of an underlying diabetic predisposition in BB rats that is phenotypically manifested upon loss of immune regulation. DRlyp/lyp and DR+/+ rats possess characteristics that make them ideal for the identification of pathways relevant to the development of T1D since they possess absolute or conditional susceptibility to T1D. The Gimap-/- Iddm2 locus conveys upon DRlyp/lyp rats a deficiency in immune regulatory capacity and the spontaneous T1D phenotype. Consistent with the concept that autoimmunity involves both a lack of self-tolerance as well as target organ-specific factors, we have discovered that DRlyp/lyp and DR+/+ (BB) rats share an islet specific stress. This is reflected by the expression of immune mediators, including the chemokine eotaxin that recruits eosinophils, certain T cell subsets, dendritic cells, and mast cells, by islet Î² cells early in life, before infiltration of immune cells into the islet (insulitis). While BB and WF rats share Iddm1 (RT1u/u MHC), islet eotaxin expression in not observed in WF islets and thus is associated with the T1D susceptibility of BB rats. Further supporting the hypothesis that additional genetic factors, perhaps those working at the level of the pancreatic Î² cell, are necessary for the development of T1D are the observations that 1) The generation Fischer 344 (F344) rats either homozygous for Gimap5-/- or homozygous for both RT1u/u and Gimap5-/- fail to develop T1D; and 2) genetic crosses between BB rats and non diabetic strains have identified numerous Iddm loci independent of Iddm1 and Iddm2. In this study, using islet gene expression profiling, direct serum-level measurement, quantitative real-time PCR and targeted histological follow-up, we investigate pancreatic islet gene expression in BB rat at 5 weeks (+/-5 days) which is prior to insulitis in DRlyp/lyp rats. We compare DR+/+, JB++, JBlyp/lyp and DRlyp/lyp islet activity to that of WF and F344 rats to investigate any potential for reduction in capability to deal with oxidative stress in BB rats compared to non-BB and whether any differences in that capability map to known Iddm regions.

Project description:Both genetic and environmental factors are implicated in Type 1 Diabetes (T1D). Since environmental factors can trigger epigenetic changes, we hypothesized that variations in histone posttranslational modifications (PTMs) at the promoter/enhancer regions of T1D susceptible genes may be associated with T1D. We therefore evaluated histone PTM variations at known T1D susceptible genes in blood cells from T1D patients versus healthy non-diabetic controls, and explored their connections to T1D. We used the chromatin-immunoprecipitation-linked-to-microarray approach to profile key histone PTMs, including H3-lysine-4 trimethylation (H3K4me3), H3K27me3, H3K9me3, H3K9 acetylation (H3K9Ac) and H4K16Ac at genes within the T1D susceptible loci in lymphocytes, and H3K4me3, H3K9me2, H3K9Ac and H4K16Ac at the IDDM1 region in monocytes of T1D patients and healthy controls separately. We screened for potential variations in histone PTMs using computational methods to compare datasets from T1D and controls. Interestingly, we observed marked variations in H3K9Ac levels at the upstream regions of HLA-DRB1 and HLA-DQB1 within the IDDM1 locus in T1D monocytes relative to controls. Additional experiments with THP-1 monocytes demonstrated increased expression of HLA-DRB1 and HLA-DQB1 in response to interferon- and TNF-treatment that were accompanied by changes in H3K9Ac at the same promoter regions as that seen in the patient monocytes. These results suggest that the H3K9Ac status of HLA-DRB1 and HLA-DQB1, two genes highly associated with T1D, may be relevant to their regulation and transcriptional response towards external stimuli. Thus, the promoter/enhancer architecture and chromatin status of key susceptible loci could be important determinants in their functional association to T1D susceptibility. We evaluated histone PTM variations at known T1D susceptible genes in blood cells from T1D patients versus healthy non-diabetic controls, and explored their connections to T1D. We used the ChIP-chip approach to profile key histone PTMs, including histone H3K4me3, H3K27me3, H3K9me3, H3K9 acetylation (H3K9Ac) and H4K16Ac, at genes within the T1D susceptible loci in lymphocytes.

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Generation of Stem Cell-Derived Î² Cells from Type 1 Diabetic Patients

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