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.

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
<ol>
	<li>Euthanize a timed-pregnant mouse (day 17.5) or rat (day 18.5) by inhalation of CO2 for 5 min. Death is confirmed by cessation of vital signs in the rat. Then break its neck humanely. The rat is euthanized at this point. Spray 70% ethanol to sterilize the abdominal skin and flanks. Cut the abdominal skin and muscle layers and then cut the uterus, separate the rat embryos from the amniotic sac, and cut the umbilical cord. Transfer the embryos into a 50 ml Falcon tube containing ice-cold Hank's Balanced Salt Solution (HBSS) and keep on ice. Fetuses are euthanized by decapitation with surgical scissors.</li>
	<li>Place the embryo in 100 mm Petri dish. Bisect the embryo with scissors at the level of the umbilicus (Figure 1A). Remove the stomach and small intestine. Reveal the ovaries in the female embryo and testis in the male embryo (Figure 1B and 1C). (The ovaries are located at the caudal poles of the kidneys. The testes are approximately at the level of the bladder). At this developmental stage, UGSM isolated from both male and female embryos are capable of inducing a prostatic epithelial lineage in the presence of host testosterone.4 After this developmental time point, however, prostatic buds begin forming into the UGSM, and isolation of pure UGSM is very challenging.4</li>
	<li>Under the dissection microscope, cut the abdominal wall in the middle line with Vannas scissors to expose the bladder and posterior wall of the abdomen. Free the upper genital tract from attachments (in male embryos, cut the ureter, Wolffian duct and M&uuml;llerian duct; in female embryos, cut the ureter and M&uuml;llerian duct). Free the bladder from the umbilical cord. Cut pubic bone and bluntly separate from the anterior wall of the urogenital sinus with Dumont fine tip forceps. Finally, separate the posterior wall of the urogenital sinus from the rectum. Cut the urogenital sinus with the bladder at the lower end (from the urethra) and store the urogenital sinus en bloc (with the bladder) in ice cold HBSS.</li>
</ol>
2. Separation of the Urogenital Sinus Mesenchyme
<ol>
	<li>Remove the bladder from the urogenital sinus under the dissection microscope (Figure 1D).</li>
	<li>Transfer the urogenital sinus to a falcon tube containing 1% trypsin in Calcium (Ca2+) and Magnesium (Mg2+) free HBSS. Place the tube in a refrigerator at 4 &deg;C for 75 min.</li>
	<li>Remove the trypsin and neutralize trypsinization with 10% Fetal Bovin Serum (FBS) in DMEM/F12 medium.</li>
	<li>Carefully tease the sticky mesenchymal sleeve from the epithelial tube with a needle or Dumont fine tip forceps (maintaining the epithelial tube intact reduces the chances of epithelial cell contamination of the mesenchyme; this can result in rodent glands forming along with your stem cell-derived human glandular epithelium) (Figure 1D).12</li>
	<li>Transfer UGSM into a new Falcon tube containing DMEM/F12 medium + 10% FBS + 1% Penicillin/Streptomycin (Pen/Strep; 0.5 mg/ml) + 1% Non-Essential Amino Acids (NEAA) plus 1nM R1881. Place the tube in an incubator at 37 &deg;C with 5% CO2 overnight.</li>
	<li>Centrifuge the tissue at 200 x g and discard the supernatant. Wash with Phosphate Buffer Saline (PBS) (do not disturb the tissue pellet).</li>
	<li>Add 0.2% collagenase (in DMEM/F12) to re-suspend the tissue. Incubate in 37 &deg;C with gentle rocking for 1 hr.</li>
	<li>Vigorously vortex the digestion tissue three times until it becomes homogenous single-cell mixture and add DMEM/F12 medium + 10% FBS + 1% P/S + 1% NEAA + 1nM R1881 to neutralize the collagenase.</li>
	<li>Centrifuge at 200 x g and then wash by re-suspending the UGSM cells in HBSS, and repeat three times to fully wash the UGSM. Finally suspend mesenchymal cells in DMEM/F12 and count the cells using a hemocytometer or cell counting device.</li>
	<li>Dissociate hES cells cultured using feeder-free protocol with mTeSR1 media (Stem Cell Technologies; Vancouver, B.C.) using an Accutase digestion (Millipore; Billerica, MA). Wash the hES cells during the log phase of their growth in warm DMEM/F12 media, then add warm 1x Accutase solution, and incubate at 37 &deg;C for 15-20 min until single cells are uniformly dissociated from colonies. Add an equal amount of 1x defined Trypsin Inhibitor (Invitrogen; Grand Island, NY) to neutralize the Accutase and gently pipette up and down to mix and break up cell clumps. Pipette cells into a centrifuge tube and centrifuge for 3 min at 200 x g, and re-suspend cell pellet in DMEM/F12 media. Centrifuge tube for 3 min at 200 x g again and remove the supernatant, then re-suspend in cold HBSS or DMEM/F12 at desired volume.</li>
	<li>Add hES cells with 1:2.5 ratio of hES/mesenchymal cells.3 This will be a cellular composition of 1E5 UGSM cells with 4E4 hESCs per 10 ml injection (a single rat UGS typically yields about 2E6 mesenchymal cells). Centrifuge at 200 x g for 5 min and then discard the supernatant. Re-suspend with 75% Matrigel (BD Biosciences, San Jose, CA; mixed with 25% cold HBSS for easier handling) and place the tube in ice for sub-renal capsule injection.</li>
</ol>
3. Transplantation of Tissue Recombinant Underneath the Renal Capsule
<ol>
	<li>Prepare a sterile surgical area.</li>
	<li>Anesthetize a male athymic nude mouse with ketamine/xylazine i.p.; use a fresh mix containing a 1:1:8 ratio of ketamine:xylazine:saline. Dosing will be 100 mg/kg Ketamine and 2.5 mg/kg Xylazine. Animals unresponsive to a toe-pinch should be fully under for 20-30 min.</li>
	<li>Surgically prepare the mouse by shaving the surgical site (if not using a nude mouse), cleansing with three alternating wipes of 70% alcohol followed by betadine solution and applying a surgical drape.</li>
	<li>Put eye moisture salve (Altalube eye ointment) to the eyes of the mouse to prevent drying during anesthesia.</li>
	<li>During surgery, respiration rate and core body temperature of the mice are monitored. The respiration rates should be steady and maintained within 40-90 breaths/min. Core body temperature is monitored with observation of the color of the mucous membranes and the pink soles of the feet. </li>
	<li>Make a 1.0 cm long longitudinal skin incision on the back. Continue to cut the abdominal wall in a 0.5 cm longitudinal incision.</li>
	<li>Gently squeeze the kidney out of the abdomen and keep the kidney surface moist using drops of sterile saline.</li>
	<li>Gently puncture the renal capsule using an empty 27-gauge syringe needle. A very shallow puncture is sufficient. This will be where you insert your blunt needle to inject your cellular mixture.</li>
	<li>Fill a 28-gauge blunt syringe needle with 10 &mu;l of your cell mixture. Slowly insert the puncture site and penetrate the kidney parenchyma to reach an area underneath the renal capsule. Inject 10 &mu;l of the cellular mixture to form a bolus on the kidney underneath the renal capsule. Withdrawal the needle.</li>
	<li>Return the kidney to the abdominal cavity. Secure the muscular layer of the peritoneum with 4-0 nylon sutures.</li>
	<li>Close the skin with either suture or staple.</li>
	<li>Clean blood from the skin using sterile saline.</li>
</ol>
4. Post-operative Analgesia and Monitoring
<ol>
	<li>Inject the animal with buprenorphine (0.1 mg/kg every 12 hr) or ketoprofen (3-5 mg/kg in saline every 12 hr) to manage post-operative pain, and 1 ml of 37 &deg;C saline i.p. to keep the animal warm and prevent dehydration.</li>
	<li>Put the animal in a clean cage and place it on a mild heating pad.</li>
	<li>Send the animals back to animal room once they regain consciousness and are moving within the cage.</li>
	<li>Observe the animal daily for unexpected signs of illness and infection for the following three days. The mice are monitored with observation of activities in cages including accessing food and water 1 day post-operatively. If the mice display fewer activities, Ketoprofen is intraperitoneally administered again once a day.</li>
	<li>Remove the skin staples or sutures 2 weeks post-surgery.</li>
	<li>Xenograft tissues can be harvested as early as 8 weeks post-surgery. Xenografts can be imaged in vivo using small-animal ultrasound (Figure 2A), and fixed tissues can be sectioned and stained for various proteins using immunohistochemistry (Figure 3).</li>
</ol>

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The authors have no conflict of interest with the work presented.

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...

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

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

formation of human prostate epithelium using tissue recombination of rodent urogenital sinus mesenchyme and human stem cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve 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).

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...

Watch this Scientific Journal Video about Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells at JoVE.com

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

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.

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

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Research

JoVE Journal

Medicine

13.6K Views.  University of Chicago. 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.

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

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

Cancer Research

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Bioengineering

Handling and Assessment of Human Primary Prostate Organoid Culture

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process involves seeding of epithelial cells sparsely in a 3D matrix gel on a 96-well microplate with media changes to cultivate expansion into organoids. Morphology is then assessed by whole-well capturing of z-stack images. Compression of z-stacks creates a single in-focus image from which organoids are measured to quantify a variety of outputs, including circularity, roundness, and area.DNA, RNA, and protein can be collected from organoids recovered from the matrix gel. Cell populations of interest can be assessed by organoid dissociation and flow cytometry. Formalin-fixation-paraffin-embedding (FFPE) followed by sectioning is used for the histological assessment and antibody staining. Whole-mount immunofluorescent staining preserves organoid morphology and facilitates observation of protein localization in organoids in situ. Commercial assays that are traditionally used for 2D monolayer cells can be modified for 3D organoids. Used together, the techniques in this protocol provide a robust toolbox to quantify prostate organoid growth, morphologic characteristics, and expression of differentiation markers.

Here, we present a protocol to guide human primary prostate organoid handling then suggest endpoints to assess phenotype. Seeding, culture maintenance, recovery from matrix gel, morphologic quantification, embedding and sectioning, FFPE sectioning, whole-mount staining, and application of commercial assays are described.

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process ...

Murine Prostate Micro-dissection and Surgical Castration

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has been used as a surrogate for discoveries in human prostate development and disease. Prostate micro-dissection allows consistent study of lobe-specific prostate anatomy, histology, and cellular characteristics in the absence of contamination of other tissues. Testosterone affects prostate development and disease. Androgen deprivation therapy is a common treatment for prostate cancer patients, but many prostate tumors become castration-resistant. Surgical castration of mouse models allows for the study of castration resistance and other facets of hormonal biology on the prostate. This procedure can be coupled with testosterone reintroduction, or hormonal regeneration of the prostate, a powerful method to study stem cell lineages in the prostate. Together, prostate micro-dissection and surgical castration opens up a multitude of opportunities for robust and consistent research of prostate development and disease. This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse.

This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse. We also depict representative results produced by these protocols. Finally, we discuss the advantages and utilization of these protocols.

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has ...

Accessing Collagen Scaffold for Single-Stage Urinary Diversion Surgery in Pigs

Surgical Model for Single-Staged Tissue-Engineered Urothelial Tubes in Minipigs

Reconstructive surgeries are often challenged by a lack of grafting tissue. In the treatment of urogenital malformations, the conventional solution has been harvesting gastrointestinal tissue for non-orthotopic reconstruction due to its abundance to reestablish normal function in the patient. The clinical outcomes after rearranging native tissues within the body are often associated with significant morbidity; thus, tissue engineering holds specific potential within this field of surgery. Despite substantial advances, tissue-engineered scaffolds have not yet been established as a valid surgical treatment alternative, mainly due to the costly and complex requirements of materials, production, and implantation. In this protocol, we present a simple and accessible collagen-based tubular scaffold embedded with autologous organ-specific tissue particles, designed as a conduit for urinary diversion. The scaffold is constructed during the primary surgical procedure, comprises commonly available surgical materials, and requires conventional surgical skills. Secondly, the protocol describes an animal model designed to evaluate the short-term in vivo outcomes post-implantation, with the possibility of additional variations to the procedure. This publication aims to demonstrate the procedure step-by-step, with special attention to the use of autologous tissue and a tubular form.

Tissue-engineered implants for reconstructive surgery rarely progress beyond preclinical trials due to laborious ex vivo culturing, which includes complex and expensive scaffold components. Here, we present a single-staged procedure designed for urinary diversion with an accessible collagen-based tubular scaffold containing autologous micrografts.

Reconstructive surgeries are often challenged by a lack of grafting tissue. In the treatment of urogenital malformations, the conventional solution has ...

Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular mechanisms of disease, development, and evolution. To make primate induced pluripotent stem cells (iPSCs) more accessible, this paper presents a non-invasive method to generate human and non-human primate iPSCs from urine-derived cells, and their maintenance using a feeder-free culturing method.
The urine can be sampled from a non-sterile environment (e.g., the cage of the animal) and treated with a broad-spectrum antibiotic cocktail during primary cell culture to reduce contamination efficiently. After propagation of the urine-derived cells, iPSCs are generated by a modified transduction method of a commercially available Sendai virus vector system. First&#160;iPSC colonies may already be visible after 5 days, and can be picked after 10 days at the earliest. Routine clump passaging with enzyme-free dissociation buffer supports pluripotency of the generated iPSCs for more than 50 passages.

The present protocol describes a method to isolate, expand, and reprogram human and non-human primate urine-derived cells to induced pluripotent stem cells (iPSCs), as well as instructions for feeder-free maintenance of the newly generated iPSCs.

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular ...

Biology

Extracting Prostate from Mouse Urogenital System: A Technique to Separate Prostate Gland from Other Urinary and Genital System Organs in Male Mouse

Source: Nath, D. et al. <a href="https://www.jove.com/video/58397" target="_blank">Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models.</a> J. Vis. Exp. (2018)
In this video, we describe the procedure for the dissection of the urogenital system or UGS from a male mouse model. This UGS is further dissected ex vivo to isolate the prostate gland and study its role in prostate cancer development.

Source: Nath, D. et al. Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models. J. ...