Isolation and analysis of discreet human prostate cellular populations

Strand, Douglas W.; Aaron, LaTayia; Henry, Gervaise H.; Franco, Omar E.; Hayward, Simon W.

doi:10.1016/j.diff.2015.10.013

Cited by 20 publications

(28 citation statements)

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“…The specimens were transition zone samples from 5ARI-treament naïve patients who underwent open simple prostatectomy for obstructive symptoms due to large volume BPH (Figure S1A). Given the difficulties in acquiring rapid autopsy tissues from age-matched men with congruent clinical information, the best age-matched, small to medium volume transition zone samples available came from patients being treated by transurethral resection of the prostate (TURP) and from patients undergoing robotic assisted prostatectomy for low Gleason grade, peripheral zone localized tumors (Strand et al, 2015). As shown by representative FACS plots in Figure 1A, we observed a significantly higher percentage of CD45 + leukocytes in larger volume prostates.…”

Section: Resultsmentioning

confidence: 99%

Non-Cell-Autonomous Regulation of Prostate Epithelial Homeostasis by Androgen Receptor

et al. 2016

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Summary Prostate inflammation has been suggested as an etiology for benign prostatic hyperplasia (BPH). We show that decreased expression of the androgen receptor (AR) in luminal cells of human BPH specimens correlates with a higher degree of regional prostatic inflammation. However, the cause-and-effect relationship between the two events remains unclear. We investigated specifically whether attenuating AR activity in prostate luminal cells induces inflammation. Disrupting luminal cell AR signaling in mouse models promotes cytokine production cell-autonomously, impairs epithelial barrier function, and induces immune cell infiltration, which further augments local production of cytokines and chemokines including Il-1 and Ccl2. This inflammatory microenvironment promotes AR-independent prostatic epithelial proliferation, which can be abolished by ablating IL-1 signaling or depleting its major cellular source, the macrophages. This study demonstrates that disrupting luminal AR signaling promotes prostate inflammation, which may serve as a mechanism for resistance to androgen-targeted therapy for BPH.

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Section: Resultsmentioning

confidence: 99%

Non-Cell-Autonomous Regulation of Prostate Epithelial Homeostasis by Androgen Receptor

et al. 2016

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“…Details for each specimen and its usage in associated figures are shown inTable 1.Tissue processing. Fresh tissue samples less than 24 hours post-mortem were transported in ice-cold saline and immediately dissected into portions for 1) flash freezing in liquid nitrogen, 2) fixation in 10% formalin followed by paraffin embedding, and 3) a 4 hour enzymatic digestion into single cells at 37ºC using 5 mg/ml collagenase type I (Life Technologies), 10µM ROCK inhibitor Y-27632 (StemRD), 1nM DHT (Sigma), 1mg DNAse I, and 1% antibiotic/antimycotic solution (100X, Corning) in HBSS51,52 . Single cells were filtered, resuspended, and incubated with antibodies for flow cytometry.…”

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confidence: 99%

A cellular anatomy of the normal adult human prostate and prostatic urethra

Henry

Malewska

Joseph

et al. 2018

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A cellular anatomy of normal human organs is essential for solving the cellular origins of disease. We report the first comprehensive cellular atlas of the young adult human prostate and prostatic urethra using an iterative process of single cell RNA sequencing and flow cytometry on ~98,000 cells taken from different anatomical regions. Two previously unrecognized epithelial cell types were identified by KRT13 and SCGB1A1 expression and found to be highly similar to hillock and club cells of the proximal lung. It was demonstrated by immunohistochemistry that prostate club and hillock cells are similarly concentrated in the proximal prostate. We also optimized a new flow cytometry antibody panel to improve cell type-specific purification based on newly established cellular markers. The molecular classification, anatomical distribution, and purification methods for each cell type in the human prostate create a powerful new resource for experimental design in human prostate disease.

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“…Fresh tissue samples were transported in ice‐cold saline and immediately dissected into portions for (i) flash freezing in liquid nitrogen; (ii) fixation in 10% formalin followed by paraffin embedding; and (iii) an overnight enzymatic digestion into single cells at 37°C using 1.5 μg/mL collagenase type I (Life Technologies), 10 μM ROCK inhibitor Y‐27632 (StemRD), 1 nM DHT (Sigma), 1% antibiotic/antimycotic solution (100X, Corning), and 1% Amphotericin B (250 μg/mL, Life Technologies) in HBSS …”

Section: Methodsmentioning

confidence: 99%

“…Human prostate basal epithelia (CD45 − /CD31 − /CD326 + /CD49f Hi /CD26 − ), luminal epithelia (CD45 − /CD31 − /CD326 + /CD49f LO /CD26 + ), stromal cells (CD45 − /CD31 − /CD326 − ), and leukocytes (CD45 + /CD31 − /CD326 − ) were analyzed and isolated by fluorescence activated cell sorting (FACS) in the UT Southwestern CRI Flow Cytometry Core as previously published . Antibody sources, catalog numbers, and concentrations are listed in Table .…”

Section: Methodsmentioning

confidence: 99%

Molecular pathogenesis of human prostate basal cell hyperplasia

et al. 2017

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Background Understanding the molecular pathogenesis of distinct phenotypes in human benign prostatic hyperplasia (BPH) is essential to improving therapeutic intervention. Current therapies target smooth muscle and luminal epithelia for relief of lower urinary tract symptoms (LUTS) due to BPH, but basal cell hyperplasia (BCH) remains untargeted. The incidence of has been reported at 8–10%, but a molecular and cellular characterization has not been performed on this phenotype. Methods Using freshly digested tissue from surgical specimens, we performed RNA-seq analysis of flow cytometry-purified basal epithelia from 3 patients with and 4 patients without a majority BCH phenotype. qPCR was performed on 28 genes identified as significant from 13 non-BCH and 7 BCH specimens to confirm transcriptomic analysis. IHC was performed on several non-BCH and BCH specimens for 3 proteins identified as significant by transcriptomic analysis. Results A total of 141 human BPH specimens were analyzed for the presence of BCH. Clinical characteristics of non-BCH and BCH cohorts revealed no significant differences in age, PSA, prostate volume, medical treatment, or comorbidities. Quantitation of cellular subsets by flow cytometry in 11 BCH patients vs. 11 non-BCH patients demonstrated a significant increase in the ratio of basal to luminal epithelia in patients with BCH (P <0.05), but no significant differences in the total number of leukocytes. RNA-seq data from flow cytometry isolated basal epithelia from patients with and without BCH were subjected to gene set enrichment analysis of differentially expressed genes, which revealed increased expression of members of the epidermal differentiation complex. Transcriptomic data were complemented by immunohistochemistry for members of the epidermal differentiation complex, revealing a morphological similarity to other stratified squamous epithelial layers. Conclusions Increased expression of epidermal differentiation complex members and altered epithelial stratification resembles the progression of other metaplastic diseases. These data provide insight into the plasticity of the human prostate epithelium and suggest a classification of basal cell hyperplasia as a metaplasia.

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Isolation and analysis of discreet human prostate cellular populations

Cited by 20 publications

References 100 publications

Non-Cell-Autonomous Regulation of Prostate Epithelial Homeostasis by Androgen Receptor

Non-Cell-Autonomous Regulation of Prostate Epithelial Homeostasis by Androgen Receptor

A cellular anatomy of the normal adult human prostate and prostatic urethra

Molecular pathogenesis of human prostate basal cell hyperplasia

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