Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Summary

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

Abstract

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-naïve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.

The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

Introduction

There is a tremendous need for better human model systems of prostate cancer. In particular, relevant human model systems of normal, non-malignant prostate tissues which can be genetically manipulated to directly discern the role of specific genes in the initiation of prostate cancer would be incredibly informative. The advent of the genomic era has identified numerous genes which may have a role in cancer formation. A lack of experimental human model systems, however, severely impairs our ability to functionally test and characterize candidate prostate cancer susceptibility genes. An ideal model system would facilitate the rapid and more rapid functional an....

Protocol

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the University of Chicago Institutional Animal Care and Use Committee (IACUC, protocol numbers 72066 and 72231). All surgery was performed under anesthesia, and all efforts were made to minimize suffering. The human embryonic stem cell line WA01 (H1; NIH-registration #0043) was acquired from WiCell (Madison, WI) and cultured using the feeder-independent protocol using mTeSR1 media (Stem Cell Technologies; Vancouver, B.C.). ES cells were used within ten passages of thawing.

Results

Building on the exciting report by Taylor, et al., our lab has developed an engrafting protocol using the commonly used H1 (NIH-designated WA01, genetically male) human embryonic stem cell line.¹ This line has been rigorously tested for quality control and is karyotypically normal.¹³ When cultured appropriately, hES cells can be maintained, expanded, and cryopreserved in an undifferentiated and pluripotent state using a feeder-free culture method (feeder-free systems commercially availa.......

Discussion

Tissue recombination using UGSM is an incredibly useful technique to investigate the development of the prostate and the molecular events leading to prostate cancer initiation. The inductive potential of UGSM has been used for numerous applications in prostate research; these include enhancing tumor take of prostate cell lines and tumors, studying stromal-epithelial interactions, and forming cross-species prostate recombinants.^7,17-20 Proper preparation of UGSM, however, is critical to experimental succ.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hank’s Balanced Salt Solution (HBSS)	GIBCO	14170
DMEM/F12	GIBCO	11330
R1881	Sigma	965-93-5	Mix to 1 ug/ml in Ethanol (1,000x stock)
NEAA	GIBCO	11140
Pen-Strep Solution	GIBCO	15070	100x stock
Matrigel	BD Biosciences	354230
KETASET (ketamine hydrochloride)	Fort Dodge Animal Health	NDC 0856-2013-01	100 mg/ml; dilute 1:10 in sterile saline
AnaSed (xylazine)	VET-A-MIX, Inc.	NADA 139-236	20 mg/ml; dilute 1:10 in sterile saline
Trypsin	BD Biosciences	215240
Collagenase	Sigma	C2014
Ketoprofen	Fort Dodge	NDC 0856-4396-01	100 mg/ml; dilute 1:1,000 in sterile saline
Altalube eye ointment	Altaire Pharmaceuticals, Inc.	NLC 56641-19850
Leica MZ16 F Stereomicroscope	Leica		Any good dissecting scope can be used.
Vannas spring scissors	Fine Science Tools	15001-08
Syringe	Hamilton	84855
Hamilton Needle, Small RN, 28 gauge, 0.5inches, Point Style #3 (Blunt)	Hamilton	7803-02	Custom Needle
Ethanol Prep Pads	Fisher Scientific	06-669-62
Sterile Gauze Pads	Fisher Scientific	22-415-469
Ethicon Vicryl Suture (4-0 FS-2)	MedVet International	J392H	Needle-in, dissolvable suture
Autoclip 9 mm Wound Clips	Becton Dickenson	427631
PVP Iodine Prep Pads	Fisher Scientific	06-669-98
Dissector scissor with blunt end	Fine Science Tools	14072-10
Dumont fine tip forceps	Fine Science Tools	11252-50
Needle holder with Scissor	Fine Science Tools	12002-14