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1Department of Surgery, Section of Urology, University of Chicago, 2Committee on Cancer Biology, University of Chicago
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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.
Keywords: Stem Cell Biology, Issue 76, Medicine, Biomedical Engineering, Bioengineering, Cancer Biology, Molecular Biology, Cellular Biology, Anatomy, Physiology, Surgery, Embryonic Stem Cells, ESCs, Disease Models, Animal, Cell Differentiation, Urogenital System, Prostate, Urogenital Sinus, Mesenchyme, Stem Cells, animal model
Cai, Y., Kregel, S., Vander Griend, D. J. Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells. J. Vis. Exp. (76), e50327, doi:10.3791/50327 (2013).
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).
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 analyses of cancer susceptibility genes in combination with appropriate transgenic rodent model systems. Furthermore, such a human model system would enable molecular characterization of the signaling mechanisms of prostate carcinogenesis toward the discovery and validation of novel therapies to prevent prostate cancer formation.
Human embryonic stem cells (hESCs) are capable of forming human prostatic tissues as xenografts. In 2006 Taylor, et al. reported that hESCs can be induced to form prostatic epithelia in vivo when re-combined with rodent urogenital sinus mesenchyme (UGSM) within a time period of 8-12 weeks.1 These studies were based upon previous work by the Cunha lab showing that rodent embryonic UGSM can promote prostatic differentiation of stem cells and embryonic epithelial cell populations in vivo.2,3 The prostate develops from an embryonic anlagen termed the urogenital sinus (UGS), and prior to embryonic day 17 (mouse E17; day E18 in the rat) the UGS can be removed and physically divided into epithelium (UGSE) and mesenchyme (UGSM).4 This tissue recombination approach has significantly enhanced our understanding of prostate development and carcinogenesis, particularly growth factor and hormonal signaling pathways and the molecular relationships between prostate stroma and epithelium.5-8 This method involves the ex vivo combination of UGSM with stem or epithelial cells from the same or distinct species and these cellular/tissue recombinants are implanted and grown and xenografts within mouse host.4,9 After a period of in vivo growth, the implant contains prostate epithelial glandular structures embedded in stromal tissue. Further staining can be conducted to determine whether such structures are truly prostatic and of human origin.10,11
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.
1. Isolation of the Urogenital Sinus from Mouse or Rat Embryos
2. Separation of the Urogenital Sinus Mesenchyme
3. Transplantation of Tissue Recombinant Underneath the Renal Capsule
4. Post-operative Analgesia and Monitoring
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 available via Stem Cell Technologies; Vancouver B.C.).14 Our protocol employs single cell suspensions of hESCs combined with single cell suspensions of E18 rat UGSM and injected under the renal capsule of adult male immunocompromised mice. As important controls, USGM implanted alone yields no discernible growth, while hESCs implanted without UGSM form large teratomas (Figure 2B).15 In our experience, implantation of UGSM without contaminating rat epithelial cells never results in tissue growth, while recombination of UGSM plus hESCs results in robust growth over 80% of the time (after 8 weeks sizes range from 1 mm to 5 mm, with over 80% of the tissue containing glandular epithelium). In recombinants containing both hESCs and UGSM, early glandular formation is observed after 4 weeks, and after 8 weeks fully formed, prostate specific antigen (PSA)-positive human prostate epithelium is formed (Figure 3). These glands contain Androgen Receptor (AR)-positive luminal-secretory cells which are not present one week after host castration. Importantly, these glands are positive for the human-specific and prostate-specific protein PSA. Further staining documents that such human glands appear to be non-malignant since they do not express Alpha-methylacyl-CoA racemase (AMACR), which is a prostate cancer-specific marker.16
Figure 1. Isolation of the Urogenital Sinus from an E18.5 Rat Embryo. A. E18.5 rat embryo. The white solid line indicates the position to bisect the embryo. B. E18.5 male rat embryo. The black dotted lines indicate the bladder, urogenital sinus, rectum, and developing testes. C. E18.5 female rat embryo. The black dotted lines indicate the bladder, urogenital sinus and uterus. D. The bladder and urogenital sinus removed from the E18.5 rat embryo. The while solid line indicates the position to remove the bladder. The black dotted line and white arrow indicate the epithelial tube. The white arrow head indicates the urogenital sinus mesenchyme.
Figure 2. Ultrasound imaging and gross morphology of in vivo tissue xenografts derived from UGSM and hESCs. A. Ultrasound imaging of growing xenografts allow live-animal analyses during the course of the experiment. Image was captured 8 weeks post-surgery using a Vevo 2100 small animal ultrasound (Visualsonics; Toronto, ON). Circled area represents the growing tissue xenograft on the kidney surface. B. At the experimental endpoint, hES cells combined with rat UGSM (rUGSM) form a visible tissue mass on the kidney surface (left panel); hES cells implanted alone form large teratomas as expected (middle panel); and UGSM implanted alone fails to form any discernible structure (right panel).
Figure 3. Formation of Human Prostate Epithelia from the Human Embryonic Stem Cell Line WA01(H1). ES cells were recombined with rodent UGSM and implanted under the renal capsule of intact male nude mice. After a growth period of 8 weeks, human prostate glandular epithelium is formed as documented by AR, p63, and human-restricted PSA staining. One week following host castration, the AR-positive, PSA-positive luminal layer is not present, documenting characteristic androgen-dependency. Prostate tissues are non-malignant as demonstrated by a lack of AMACR staining. In addition, ChrA-positive neuroendocrine cells were detectable. Non-malignant human prostate tissue was used as a control for AR, PSA, and p63; a prostate tumor was used as a positive control for cancer-specific AMACR.
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 success as contaminating rodent epithelium will result in glandular formation and can confound results. Thus, the separation of the UGSM from the UGSE (Step 2.4) is by far the most critical and also most tricky step in the protocol. To control for such contamination, the use of techniques to discern between species is strongly encouraged, as well as an additional experimental arm using UGSM alone to document no glandular formation from cellular contaminants.12
The recent development of feeder-free hES culturing reagents and methods allow pure populations hES cells to be cultured, expanded and cryopreserved.14 Moreover, the expanded use of lentiviral gene delivery allows the stable genomic incorporation and expression of genes of interest.21,22 These combined advances allow for the genetic manipulation of pure hES cultures and, when combined with the inductive potential of prostatic embryonic UGSM, will permit the in vivo generation of human prostatic glands expressing specific genes of interest. Moreover, this technique would be amenable to induced-pluripotent stem cell lines (iPSCs) derived from adult hosts and from genetically-modified cells.23,24 Using defined proteins, we show that such hESC-derived human prostate epithelium appear very similar to adult human prostate epithelia, but at a global molecular level there is sure to be some differences in gene expression which should be taken into consideration. To account for this, investigators should use an expanded sample size and consider laser-capture microdissection (LCM)-based approaches and comparative gene expression analyses platforms to validate the molecular profiles of their tissues compared to adult human tissues. Nonetheless, the formation of human prostate glandular epithelium using a serially-cultured and feeder-free stem cell line has the potential to facilitate numerous molecular studies pertaining to prostate function and cancer initiation.
Our technique utilizes single cell suspensions of both UGSM and hESCs, which enables the use of lentiviral infection and flow sorting approaches, as well as more accurate control of cellular quantities. This can, however, diminish the inductive potential of UGSM. An alternate approach has been to use non-dissociated UGSM (after Step 2.5), before collagenase digestion) and either direct incubation with ES cells, or embedding cells within a collagen solution.4 To implant these tissues under the renal capsule, an alternate technique would be to make an incision in the renal capsule, create a small pocket under the renal capsule on top of the kidney, and physically place the cell mass within that pocket.4,20
The authors have no conflict of interest with the work presented.
We wish to acknowledge the support of the University Of Chicago Section Of Urology led by Dr. Arieh Shalhav, and the Director of Urologic Research Dr. Carrie Rinker-Schaeffer. We would also like to acknowledge the support of the University of Chicago Comprehensive Cancer Center (UCCCC) led by Dr. Michelle Le Beau. We also with to thank expert technical assistance of the Human Tissue Resource Center core facility led by Dr. Mark Lingen, and the assistance of Leslie Martin and Mary Jo Fekete. We also thank the Immunohistochemistry Core Facility run by Terri Li. This work was funded by the University of Chicago Department of Surgery, the Section of Urology; an American Cancer Society Institutional Research Grant (ACS-IRG, #IRG-58-004); a Cancer Center Support Grant (P30 CA14599); The Brinson Foundation; the Alvin Baum Family Fund; The University of Chicago Cancer Research Foundation Women's Board; S. Kregel is supported by an HHMI: Med-into-Grad Fellowship (56006772) and a Cancer Biology Training Grant (T32-CA09594). Finally, we would like to thank Robert Clark, Dr. VenkateshKrishnan, and Nathan Stadick for their critical evaluation of the manuscript.
|Hank’s Balanced Salt Solution (HBSS)||GIBCO||14170|
|R1881||Sigma||965-93-5||Mix to 1 ug/ml in Ethanol (1,000x stock)|
|Pen-Strep Solution||GIBCO||15070||100x stock|
|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|
|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|
|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|
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