Isolation and analysis of discreet human prostate cellular populations

Abstract

The use of lineage tracing in transgenic mouse models has revealed an abundance of subcellular phenotypes responsible for maintaining prostate homeostasis. The ability to use fresh human tissues to examine the hypotheses generated by these mouse experiments has been greatly enhanced by technical advances in tissue processing, flow cytometry and cell culture. We describe in detail the optimization of protocols for each of these areas to facilitate research on solving human prostate diseases through the analysis of human tissue.

Figure 1

Major cell populations found in the prostate. The stroma is a complex mix of smooth muscle, resident fibroblasts and immune/inflammatory infiltrate along with blood vessels and nerves that are not illustrated here. The epithelium is composed of luminal and basal cells. The basal epitheliium includes presumptive stem cells. The luminal epithelial cells are predominantly secretory. Neuroendocrine cells are normally rare but can expand in some forms of advanced cancer. Expansion of intermediate epithelial cells is associated with inflammation in vivo and with ex vivo culturing of primary cells.

Figure 2

Tissue preparation and analysis workflow. (A, B) Pathologist prepares 20–30 gram transition zone section. Ten grams is digested overnight into ~100 million single cells (C) and frozen down into ten separate 10 million cell aliquots in 90% FBS/10% DMSO.

Figure 3

Flow cytometry strategy to fractionate stromal, epithelial, and leukocytic cell populations in a simple prostatectomy specimen. (A) Live/Dead stain identifies viable cells already enriched by scatter plots for further analysis. (B) The three major cellular populations in human prostate are fractionated by CD326 (epithelia) and CD45 (leukocytes) with stroma being negative for both. (C, D) Enzymatic digestion with collagenase (C) preserves CD49f antigen for identification of basal epithelia, which is eliminated with Liberase TH digestion (D). CD26 detection of luminal epithelia is unaffected, but other antigens such as those presented by leukocytes are also cleaved by Liberase digestion.

Figure 4

(A–D) Flow cytometry and immunohistochemistry for chronic inflammatory cells in BPH tissue. Percentages listed are of total CD45⁺ leukocytes in a representative simple prostatectomy sample digested with collagenase. (A) 50% of all viable leukocytes are CD11b⁺ monocytes with 36% also positive for the M2 macrophage marker CD163 (B). Only 0.3% of all CD45⁺ leukocytes were CD4⁺ (C) while 3% were CD8a⁺ (D). (E) Example of a high resolution scanned slide utilizing a Leica SCN400 slide scanner. Cells were immunostained for clustered CD20+ B cells. Screenshot of Ariol software showing a selected area (boxed) of automated quantification of positive cells in red (DAB-stained cells), with blue denoting negative cells (Hematoxylin-stained nuclei).