Unknown,Transcriptomics,Genomics,Proteomics

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Isolation and characterization of progenitor-like cells from human renal proximal tubules

ABSTRACT: The adult kidney has a remarkable capacity for self-renewal upon damage. Whether this regeneration is mediated by dedifferentiating surviving cells or as recently suggested by stem cells has not been unequivocally settled. The stem cell concept is however hampered by lack of consensus regarding the histological localization of and defining markers for these cells. Here, we demonstrate that aldehyde dehydrogenase (ALDH) activity may be used for isolation of cells with progenitor-like characteristics from adult human renal cortical tissue. Gene expression profiling of the isolated ALDHhigh and ALDHlow cell fractions followed by immunohistochemical interrogation of renal tissues enabled us to delineate a tentative progenitor cell population with a scattered distribution in the epithelial layer of the proximal tubules (PT). The cells differed from the surrounding cells by expression of CD133, CD24, vimentin, KRT7, KRT19, and BCL2, and were negative for PT-specific markers. Based on functional and bioinformatic analyses as well as an immunophenotypical resemblance to cells of regenerating tubules we suggest that these cells are endowed with a more robust phenotype, allowing increased resistance to acute renal injury, enabling rapid repopulation of the tubules. The rare intratubular cells also displayed marked transcriptional and immunophenotypical similarities to that of cortical adenomas and papillary renal cell carcinomas, indicating that these renal neoplasms arise through oncogenic transformation of this subset of PT cells. Total RNA extracted from three pairs of ALDH-high and ALDH-low cell fractions were hybridized to Illumina humanHT-12 v3.0 Expression BeadChips (Illumina Inc) at the SCIBLU Genomics Centre at Lund University Sweden (http://www.lth.se/sciblu).

ORGANISM(S): Homo sapiens

SUBMITTER: Martin Johansson

PROVIDER: E-GEOD-23911 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) has a poor prognosis with less than 1-year median survival. Platinum-based chemotherapy (cisplatin or carboplatin) remains the first-line treatment for HNSCC. The cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self renewing cell populations that constitute the bulk of the tumor. A small population of CSCs exists within HNSCC that are relatively resistant to chemotherapy and clinically predicted to mediate tumor recurrence. These CSCs are identified by high cell-surface expression of CD44 and high intracellular activity of aldehyde dehydrogenase (ALDH) and termed ALDHhighCD44high. We investigated the molecular pathways active in ALDHhighCD44high cells, which remain poorly studied. Additionally, we performed a molecular examination of cisplatin-resistant ALDHhighCD44high cells, which has not been reported. Two HNSCC cell lines, UM-SCC-1 and UM-SCC-22b, were utilized in this study. For microarray analysis, UM-SCC-22b cells were treated for 5 days in vitro with 2uM cisplatin and analyzed by flow cytometry, sorted and submitted for microarray analysis of ALDHhighCD44high and ALDHlowCD44low cells from untreated and cisplatin treated cells. Four separate flow cytometry experiments were performed using Affymetrix Human Gene ST 2.1 microarrays. Microarray data was analyzed using R/Bioconductor. Files were preprocessed by Robust Multiarray Average (RMA) with background substraction, quantile normalization, and median polish (oligo package). Data was fitted with robust probe level linear models to all the probesets (oligo package). Experiment and processing batch differences were accounted for using 'ComBat' within the SVA package. Differentially expressed genes were identified using univariate comparisons after fitting data to a linear model (limma package). Initial statistics were determined using an empirical Bayesian model. Multiple testing comparisons were adjusted using Benjamini and Hochberg (aka FDR). Probes with an adjusted p-value <0.05 were considered statistically significant. Unsupervised hierarchical clustering with complete linkage and Euclidean distance was performed on only statistically significant probes. In four separate experiments, the head and neck squamous cell carcinoma cell line UM-SCC-22b were cultured for 5 days with or without 2uM (micromolar) cisplatin in 6-well plates. Media was replaced every other day. Control and cisplatin treated cells were trypsinized, procesed, and stained for CD44 cell-surface expression and intracellular aldehyde dehydrogenase (ALDH) activity to identify cancer stem cells (ALDH+CD44+). CSCs and non-CSCs (ALDH-CD44-) were collected by flow cytometry from both groups. Total RNA was collected from each fraction (ALDH+CD44+, ALDH-CD44-), treatment (control, cisplatin), and experiment (#1-4). A total of 16 samples were analyzed. One set of 4 (experiment #4) were analyzed on a Human Gene ST 2.1 strip and the rest on a Human Gene ST 2.1 plate. Differential gene expression was determined with R/Bioconductor with Robust Multiarray Average (RMA) and fitting the data to linear models (limma). Experimental and processing batch effects were accounted for using ComBat. Four sets of univariate comparisons were made: 1) Cisplatin ALDH+CD44+ vs Control ALDH+CD44+; 2) Control ALDH+CD44+ vs Control ALDH-CD44-; 3) Cisplatin ALDH+CD44+ vs Cisplatin ALDH-CD44-; 4) Cisplatin ALDH-CD44- vs Control ALDH-CD44-. Multiple testing comparisons were adjusted using Benjamini and Hochberg (aka FDR). Probes with an adjusted p-value <0.05 were considered statistically significant.

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Isolation and characterization of progenitor-like cells from human renal proximal tubules

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