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Evidence of stress in ? cells obtained with laser capture microdissection from pancreases of brain dead donors.

ABSTRACT: Isolated islets used for transplantation are known to be stressed, which can result from the circumstances of death, in particular brain death, the preservation of the pancreas with its warm and cold ischemia, from the trauma of the isolation process, and the complex events that occur during tissue culture. The current study focused upon the events that occur before the islet isolation procedure. Pancreases were obtained from brain dead donors (n = 7) with mean age 50 (11) and normal pancreatic tissue obtained at surgery done for pancreatic neoplasms (n = 7), mean age 69 (9). Frozen sections were subjected to laser capture microdissection (LCM) to obtain ?-cell rich islet tissue, from which extracted RNA was analyzed with microarrays. Gene expression of the 2 groups was evaluated with differential expression analysis for genes and pathways. Marked changes were found in pathways concerned with endoplasmic reticulum stress with its unfolded protein response (UPR), apoptotic pathways and components of inflammation. In addition, there were changes in genes important for islet cell identity. These findings advance our understanding of why islets are stressed before transplantation, which may lead to strategies to reduce this stress and lead to better clinical outcomes.

SUBMITTER: Ebrahimi A

PROVIDER: S-EPMC5345752 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Evidence of stress in β cells obtained with laser capture microdissection from pancreases of brain dead donors.

Ebrahimi Aref A Jung Min-Ho MH Dreyfuss Jonathan M JM Pan Hui H Sgroi Dennis D Bonner-Weir Susan S Weir Gordon C GC

Islets 20170301 2

Isolated islets used for transplantation are known to be stressed, which can result from the circumstances of death, in particular brain death, the preservation of the pancreas with its warm and cold ischemia, from the trauma of the isolation process, and the complex events that occur during tissue culture. The current study focused upon the events that occur before the islet isolation procedure. Pancreases were obtained from brain dead donors (n = 7) with mean age 50 (11) and normal pancreatic ...[more]

PMID: 28252345

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Project description:BackgroundSuccessful achievement of early folliculogenesis is crucial for female reproductive function. The process is finely regulated by cell-cell interactions and by the coordinated expression of genes in both the oocyte and in granulosa cells. Despite many studies, little is known about the cell-specific gene expression driving early folliculogenesis. The very small size of these follicles and the mixture of types of follicles within the developing ovary make the experimental study of isolated follicular components very difficult.The recently developed laser capture microdissection (LCM) technique coupled with microarray experiments is a promising way to address the molecular profile of pure cell populations. However, one main challenge was to preserve the RNA quality during the isolation of single cells or groups of cells and also to obtain sufficient amounts of RNA.Using a new LCM method, we describe here the separate expression profiles of oocytes and follicular cells during the first stages of sheep folliculogenesis.ResultsWe developed a new tissue fixation protocol ensuring efficient single cell capture and RNA integrity during the microdissection procedure. Enrichment in specific cell types was controlled by qRT-PCR analysis of known genes: six oocyte-specific genes (SOHLH2, MAEL, MATER, VASA, GDF9, BMP15) and three granulosa cell-specific genes (KL, GATA4, AMH).A global gene expression profile for each follicular compartment during early developmental stages was identified here for the first time, using a bovine Affymetrix chip. Most notably, the granulosa cell dataset is unique to date. The comparison of oocyte vs. follicular cell transcriptomes revealed 1050 transcripts specific to the granulosa cell and 759 specific to the oocyte.Functional analyses allowed the characterization of the three main cellular events involved in early folliculogenesis and confirmed the relevance and potential of LCM-derived RNA.ConclusionsThe ovary is a complex mixture of different cell types. Distinct cell populations need therefore to be analyzed for a better understanding of their potential interactions. LCM and microarray analysis allowed us to identify novel gene expression patterns in follicular cells at different stages and in oocyte populations.

Project description:Thirty-five adult (8-16 week old) C57BL/6 mice (which had undergone previous behavioral protocols) were anesthetized with isoflurane, and the brains were removed quickly, embedded in OCT, and frozen in a dry ice-ethanol mixture. The interval between decapitation and complete freezing was always less than 3 minutes. The brain was warmed up to -20oC, and cut into 10-14 m sections with a cryostat (Leica). Serial coronal sections were kept on slides in 100% ethanol until sectioning was complete. Tissue was then processed with hematoxylin and eosin (H&E) staining for cell layer visualization, followed by dehydration with increasing concentrations of ethanol and then xylene. We focused on two cell types, the Purkinje (Pk) and granule cells (gc) of the flocculus. We used an Arcturus PixCell II laser capture microdissection (LCM) scope to locate and capture cells from the appropriate areas, obtaining ten samples per cell type per mouse. We estimate that each Pk sample contained about 50-100 Purkinje cells, and each gc sample contained a few thousand granule cells. Granule cell samples (dorsal flocculus) were captured before Purkinje cell samples, to reduce contamination of the Purkinje cell samples by granule cell material. Samples were removed from the LCM caps with RNEasy lysis buffer (Qiagen) containing 1% -mercaptoethanol, then frozen at -80oC. For each mouse, all the samples of a given cell type were pooled together; samples from different mice were not pooled. Total RNA samples were isolated using RNEasy kits (Qiagen), then amplified in two rounds of in vitro transcription (IVT) using the Ambion MessageAmp aRNA kit. IVT-amplified samples were hybridized to microarrays if the end product after the second round of amplification was of concentration greater than or equal to 0.2 g/l, and at least 10 times the negative control signal (measured after two rounds of amplification of a blank sample). Due to the small starting sample sizes, samples often were rejected due to insufficient quantity for hybridization: thus, 18/35 granule cell samples and 23/35 Purkinje cell samples survived this quality control process, and were run on arrays (41 arrays total). Mus musculus spotted cDNA microarrays (MM arrays) containing ~42,000 spots were obtained from the Stanford Functional Genomics facility (complete information at http://www.microarray.org/), in order to analyze gene expression in each cell type. We used a type II experimental design, where all experimental samples were hybridized against a common reference sample for multi-way comparison. The reference sample comprised mRNA extracted from neonatal and adult brain and liver, and amplified twice by IVT. We used standard protocols for cDNA labeling, as well as array hybridization, washing, scanning, and data analysis (http://cmgm.stanford.edu/pbrown/protocols/index.html).

Dataset Information

Evidence of stress in ? cells obtained with laser capture microdissection from pancreases of brain dead donors.

Publications

Evidence of stress in β cells obtained with laser capture microdissection from pancreases of brain dead donors.

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