Name: formation of human prostate epithelium using tissue recombination of rodent urogenital sinus mesenchyme and human stem cells
Uploaded: 2013-06-22T14:00:00
Description: 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

Vi ønsker at anerkende den støtte fra University Of Chicago Section of Urology ledet af Dr. Arieh Shalhav, og direktøren for urologiske Research Dr. Carrie Rinker-Schaeffer. Vi vil også gerne anerkende den støtte fra University of Chicago Comprehensive Cancer Center (UCCCC), ledet af Dr. Michelle Le Beau. Vi er også med til at takke ekspert teknisk assistance af Human Tissue Resource Center core facilitet ledet af Dr. Mark Lingen, og bistand fra Leslie Martin og Mary Jo Fekete. Vi takker også Immunohistokemi Core Facility drives af Terri Li. Dette arbejde blev finansieret af University of Chicago Kirurgisk afdeling, Afdeling for Urologi, en American Cancer Society Institutional Research Grant (ACS-IRG, # IRG-58-004), en Cancer Center Support Grant (P30 CA14599) The Brinson Foundation, den Alvin Baum Family Fund, The University of Chicago Cancer Research Foundation Kvinders bestyrelse, S. Kregel er understøttet af en HHMI: Med-ind-Grad Fellowshofte (56006772) og et Cancer Biology Training Grant (T32-CA09594). Endelig vil vi gerne takke Robert Clark, Dr. VenkateshKrishnan og Nathan Stadick for deres kritiske evaluering af manuskriptet.

Denne undersøgelse blev udført i nøje overensstemmelse med anbefalingerne i Guide til Pleje og anvendelse af forsøgsdyr af National Institutes of Health. Protokollen blev godkendt af University of Chicago Institutional Animal Care og brug Udvalg (IACUC, protokol nummer 72.066 og 72231). Alle operation blev udført under anæstesi, og alle bestræbelser blev gjort for at minimere lidelse. Den humane embryonale stamceller linje WA01 (H1, NIH-registrering # 0043) blev erhvervet fra WiCell (Madison, WI) og kultiverede b...

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Tissue rekombination hjælp UGSM er en utrolig nyttig teknik til at undersøge udviklingen af ​​prostata og de molekylære begivenheder, der fører til prostatakræft initiering. Den induktive potentiale UGSM har været anvendt til mange applikationer i prostata forskning, disse omfatter øget tumor tage af prostata cellelinjer og tumorer, studerer stromal-epiteliale interaktioner, og danner tværs af arterne prostata rekombinanter 7,17-20 Korrekt forberedelse af UGSM. imidlertid er afgørende for eksperi...

Der er et enormt behov for bedre menneskelige modelsystemer prostatakræft. Navnlig vil de relevante humane modelsystemer for normale, ikke-maligne prostata væv, som kan være genetisk manipuleret til at direkte skelne rollen af ​​specifikke gener i indledningen af ​​prostatacancer være utroligt informativt. Fremkomsten af ​​den genomiske æra har identificeret en række gener, som kan have en rolle i dannelsen af ​​kræft. En mangel på eksperimentelle humane modelsystemer dog alvorligt svækker vores evne til funktionelt teste og karakterisere kandidat prostatakræft modtagelighed gener. En ideel model system vil lette en hurtig og hurtigere funktionelle analyser af kræft modtagelighed gener i kombination med passende transgene gnavere modelsystemer. Desuden ville en sådan menneskelig modelsystem muliggøre molekylær karakterisering af signalering mekanismer prostata carcinogenese mod opdagelsen og validering af nye behandlingsformer tilforebygge prostatakræft formation. Humane embryonale stamceller (hESCs) kan danne humane prostatavæv som xenotransplantater. I 2006 Taylor, et al. Rapporterede, at hESCs kan induceres til at danne prostata epiteler in vivo, når re-kombineret med gnavere urogenital sinus mesenchyme (UGSM) inden for en periode på 8-12 uger. 1. Disse undersøgelser var baseret på tidligere arbejde med Cunha lab der viser, at gnaver embryonale UGSM kan fremme prostata differentiering af stamceller og embryonale epitelcelletyper befolkninger in vivo. 2,3 Prostata udvikler sig fra en embryonale anlagen betegnes urogenitale sinus (UGS), og forud for embryonale dag 17 (mus E17 , dag E18 i rotten) UGS kan fjernes og fysisk opdelt i epitel (UGSE) og mesenkym (UGSM) 4 Dette væv rekombination tilgang har væsentligt forbedret vores forståelse af prostata udvikling og carcinogenese, især.ly vækstfaktor og hormonelle signalveje og de ​​molekylære forhold mellem prostata stroma og epithel. 5-8 Denne fremgangsmåde involverer ex vivo kombination af UGSM med stam-eller epitelceller fra samme eller adskilte arter, og disse cellulære / væv rekombinanter implanteres og dyrkes og xenotransplantater inden mus vært. 4,9 Efter en periode med in vivo vækst, indeholder implantatet prostata epiteliale kirtelstrukturer indlejret i stromavæv. Yderligere farvning kan udføres for at afgøre, om sådanne strukturer er virkelig prostata-og human oprindelse. 10,11

<table border="1">
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 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Hank'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

Fremskridt i prostatakræft forskning er stærkt begrænset af tilgængeligheden af ​​humant afledte og hormon-naive modelsystemer, som begrænser vores evne til at forstå genetiske og molekylære begivenheder underliggende prostata sygdom indvielse. Mod at udvikle bedre modelsystemer til at studere human prostata carcinogenese, har vi og andre draget fordel af den unikke pro-prostata-induktive potentiale af embryonale gnaver prostata stroma, betegnet urogenital sinus mesenkym (UGSM). Når rekombineret med visse pluripotente cellepopulationer såsom fosterstamceller, UGSM inducerer dannelsen af ​​normale humane prostata epitheler i en testosteron-afhængig måde. Sådan en menneskelig model kan anvendes til at undersøge og eksperimentelt teste evnen af ​​kandidat prostatakræft modtagelighed gener på en accelereret tempo i forhold til typiske gnavere transgene undersøgelser. Da humane embryonale stamceller (hESCs) kan genetisk modificeret i kultur using inducerbar genekspression eller siRNA knock-down vektorer før væv rekombination, en sådan model letter teste de funktionelle konsekvenser af gener eller kombinationer af gener, der menes at fremme eller forhindre carcinogenese. Teknikken med at isolere rene populationer af UGSM celler imidlertid udfordrende og læring kræver ofte en person med tidligere ekspertise til personligt undervise. Desuden kan podning af celleblandinger under nyrekapslen af ​​en immunkompromitteret vært være teknisk udfordrende. Her har vi skitsere og illustrere ordentlig isolering af UGSM fra gnavere embryoner og renal kapsel implantation af væv blandinger at danne menneskelige prostata epitel. En sådan fremgangsmåde, på dens nuværende stadium kræver in vivo xenografting af embryonale stamceller, fremtidige applikationer potentielt kunne omfatte in vitro-kirtel dannelse eller brug af induceret pluripotente stamceller populationer (iPSCs).

Med udgangspunkt i den spændende beretning af Taylor et al. Har vores laboratorium udviklet en engraftment protokol ved hjælp af almindeligt anvendte H1 (NIH-udpegede WA01, genetisk mandlige) humane embryonale stamceller linje. 1. Denne linje er blevet grundigt testet for kvalitetskontrol og er karyotypisk normal 13 Når dyrkes korrekt, kan hES celler opretholdes, udvidet, og kryokonserveres i en udifferentieret og pluripotente tilstand vha. et feeder-fri dyrkningsmetode (feeder-fri syst...

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

At optrævle de tidligste molekylære mekanismer bag prostatakræft initiering, nye og innovative menneskelige modelsystemer og tilgange er et desperat behov. Potentialet af præ-prostata urogenital sinus mesenkym (UGSM) at inducere pluripotente stamceller populationer at danne human prostata epitel er et stærkt eksperimentelt redskab i prostata forskning.

Dannelse af humane prostata Epitel Brug Tissue rekombination af Rodent Urogenital Sinus mesenkymet og menneskelige stamceller

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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The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Berigelse for kemoterapi kræft i æggestokkene stamceller fra humane cellelinier

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Evaluering af Stem Cell Properties i Human ovariekarcinomceller Brug Multi og enkelt celle-baserede Spheres Analyser

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

Laser-capture mikrodissektion for Human prostata epitel til RNA analyse

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

Prækliniske ortotopisk Murine Model of Human Prostata Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

Generering af en humaniseret Mouse Lever Brug humane lever Stamceller

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...