This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.
Decidualisering är en progesteronberoende differentieringsprocessen av endometrial stromaceller och är en förutsättning för framgångsrik embryoimplantation. Även om många ansträngningar har gjorts för att avslöja den underliggande mekanismerna för decidualisering, förblir den exakta signaleringen mellan epitelcellerna som är i kontakt med embryot och de underliggande stromaceller dåligt kända. Därför studerar decidualisering på ett sätt som tar både epiteliala och stromala celler hänsyn skulle kunna förbättra vår kunskap om de molekylära detaljerna i decidualisering. För detta ändamål in vivo-modeller av konstgjord decidualisering är fysiologiskt de mest relevanta; emellertid är begränsad manipulation av intercellulär kommunikation. Närvarande, in vitro-kulturer av endometrial stromaceller som används för att undersöka modulering av decidualisering av flera signalmolekyler. Konventionellt, människa eller mus endometrial stromaceller ärBegagnade. Emellertid är tillgången på mänskliga prover ofta begränsad. Vidare är användningen av murina vävnader åtföljs med variation i metoden enligt odling. Denna studie presenterar en validerad och standardiserad metod för att få ren Endometrial epitelceller (EEG) och Stromacells- (ESC) kulturer på vuxna intakta möss som behandlats med östrogen under tre dagar i följd. Protokollet är optimerad för att förbättra utbytet, livskraft, och renheten hos cellerna och utvidgas ytterligare för att studera decidualisering i en samodling av EEG och ESC. Denna modell kan vara lämpligt att utnyttja vikten av att båda celltyper i decidualisering och utvärdera bidraget av betydande signalmolekyler som utsöndras av EEG eller ESC under kommunikationen mellan.
Den mänskliga livmoderslemhinnan är den inre slemhinnan i livmodern och genomgår månatliga cykler av nedbrytning och reparation som en förberedelse för möjliga graviditeter. Den består av ett enda skikt av epitelceller som bekläder livmodern lumen och underliggande stroma som varierar i tjocklek beroende på variationer i äggstockshormoner östrogen och progesteron. Under lutealfas kommer postovulatory progesteron surge inducera differentiering av endometrial stromaceller till större, runda decidualcell, en process som kallas decidualisering 1, 2. I människa, sker detta fenomen spontant för att bilda predecidua, men blir mer uttalad vid embryo implantation. En funktionell konsekvens av decidualisering är att livmodern övergående blir mottaglig för embryo implantation. Detta intervall är märkt som "fönster implantation". Under den tidiga perioden av embryo implantation, den decidualized endometrial stromaceller som omger implantera embryot kommer att tillhandahålla en barriär för att förhindra passage av skadliga ämnen till embryot 3. Under graviditeten, kommer detta decidua ge näring för det växande fostret innan placentation har skett, och kommer senare att bilda moderns del av moderkakan 4.
Etiska och praktiska överväganden begränsar utformningen av studier på människa för att undersöka processen för decidualisering och har föranlett användningen av djurmodeller. Att vara medlem i hemochorial placentation gruppen, kommer livmoderslemhinnan hos gnagare också genomgå decidualisering före placentation 5. Hos gnagare, men inledningen av decidualisering processen beror på närvaron av blastocyster i livmoder lumen, vilket indikerar att en extra stimulans är nödvändig för att utlösa decidual svar 6. Detta innebär ett intrikat kommunikation mellan the endometriala epitelceller som har direkt kontakt med blastocysten, och underliggande stromala celler. Ändå kan decidualisering vara artificiellt med hjälp av stimuli som mekanisk repor eller instillation av oljan i en hormon primas livmoder. Även in vivo-studier med hjälp av konstgjorda decidualisering modeller har tillåtit oss att förbättra våra insikter i den komplexa processen av decidualisering signalering, mekaniska fördjupade studier, t.ex. undersökning av oumbärlig kommunikation mellan epiteliala och stromala celler, är begränsade. Därför kan decidualisering in vitro-studier kan användas för att manipulera viktiga vägar och studera grundläggande processer. För detta ändamål kan primära endometrial stromal cellkulturer sättas upp från människa eller gnagare endometrium. Anställa gnagare samtycker användningen av genetiskt modifierade djur för att undersöka betydelsen av ett specifikt protein av intresse under decidualisering processen. Emellertid omogna, prepuberty-möss används ofta i syfte att undvika komplikationer av Östrogencykeln, medan i andra fall uteri samlas in från alla faser av Östrogencykeln, som resulterar i en heterogenitet i kulturerna. Dessutom har processen för decidualisering studerats i olika protokoll, till exempel, genom att (i) kompletterar hormoner (östrogen, progesteron eller cykliskt adenosinmonofosfat (cAMP)) till odlingsmediet, eller genom (ii) isolering av decidualcell från möss vid dag 4,5 av (pseudo) graviditet, som kräver ytterligare behov av plug kontroll och vasektomiserade hanar. Dessa begränsningar påvisa behovet av en standardiserad metod för att studera in vitro decidualisering. I denna studie, att en fysiologisk modell undersöka in vitro decidualisering början från vuxen, kommer icke (pseudo) dräktiga möss presenteras. I denna metod, kommer daglig injektion av 17β-östradiol (E2) inducera proliferation av endometriala celler, vilket resulterar i ett ökat utbyte av homogen och pure cellpopulationer. Vidare har användningen av en samodling system tillåter studiet av den epiteliala-stromal överhörning och förmågan att manipulera processen för decidualisering i olika nivåer.
Decidualization is the progesterone-dependent differentiation of endometrial stromal cells into round secreting decidual cells. In human, this process occurs spontaneously during the luteal phase of the menstrual cycle and is initiated in the stromal cells surrounding the vascular cells to form the predecidua. However, in rodents, the presence of a blastocyst is imperative to induce decidualization. The fact that decidualization only occurs after contact with the endometrial epithelial cells implies that important factors are secreted by the epithelial cells to induce decidualization in the underlying stroma. Although this difference in the initiation of the decidualization process should be acknowledged, the fact that human decidualization becomes more robust upon embryo implantation suggests that similar mechanisms could be involved. In vitro cell cultures are often used to study the effect of specific molecules during the process of decidualization. Nevertheless, the availability and amount of human tissue that can be collected is often limited and accompanied by variations, e.g., cycle phase, number of pregnancies and presence of gynecological diseases like endometriosis or adenomyosis. Many of these issues can be prevented by the use of murine tissue that is easily accessible and cycle phase independent. Another strong advantage of using murine tissue is the opportunity to use genetically modified rodents and the possibility to setup a large number of experiments. At the moment, many different isolation protocols have been described in literature with high variability in the age of the animals and the period in the estrus phase at the moment of use.
This study presents a new method for primary endometrial co-cultures of stromal and epithelial cells in which advantage was taken of a standardized estrous cycle by a daily injection of estrogen prior the isolation. Furthermore, estrogen is known to induce proliferation in epithelial and stromal cells which further increases the yield per isolation. The isolation protocol described in this paper was based on Grant et al.7, however, further optimization steps were included to increase the yield, purity, and viability of the different cell cultures. First, HBSS was always supplemented with antibiotics to avoid contamination of the primary culture. During the isolation of MEEC, a temperature adaptation was done (15 min at 37 °C) to increase the harvest of the epithelial cells during the first digestion. Next, an additional step was included to temper enzymatic activity after trypsin-digestion by adding medium containing FBS to inhibit its activity. Furthermore, MEECs were passed through a filter of which the mesh was larger than what is commonly described (100 µm) as they often come in clusters and cell sheets. Moreover, contamination by stromal cells was reduced by performing an additional step in which MEECs and MESCs were separated based on gravity sedimentation. Finally, MEECs were seeded on collagen-coated coverslips to increase the attachment of cells to glass coverslips, as it became clear that attachment to uncoated or PLL-coated glass coverslips was insufficient. During the isolation of the MESCs, collagenase (1 mg/mL) was supplemented to improve digestion. Furthermore, this digestion step was performed in duplicate to avoid contamination of remaining MEECs. All these additional steps resulted in pure and viable cultures of homogenous cell populations.
The purity of the cell population was evaluated with immunostaining and qRT-PCR using vimentin as a stromal marker and cytokeratin as an epithelial marker. Clearly, an opposite expression pattern of vimentin and cytokeratin was observed in MEEC and MESC. However, it can be noticed that the relative mRNA expression of vimentin and cytokeratin is similar in the MEEC cultures. Similar results are published by other research groups, in which epithelial cells in culture acquire vimentin expression9. More important is the difference in protein expression between vimentin and cytokeratin as was observed in the immunohistological stainings of the different cell populations. Double immunostaining showed that EECs were positive for cytokeratin and ESC were positive for vimentin. The use of these markers in immunostaining is an established method and standardly used to indicate the cell types10.
Although a lot of research has been performed on the decidualization of MESC, it remains possible that the presence of epithelial cells in this model may alter the results of decidualization. Firstly, it has been shown that if MEEC grow to a monolayer in the presence of MESC-conditioned medium or in a co-culture setting, the monolayer has an improved epithelial cell transepithelial electrical resistance (TEER), resulting from factors secreted by MESC7. Moreover, Pierro et al.11 provided evidence that epithelial proliferation, influenced by 17β-estradiol, might be mediated by factors secreted from the stromal cells, and alternatively, epithelial cells can release factors that modulate stromal decidualization12,13. Overall, it is clear that the epithelial-stromal co-culture environment provides a more physiological situation that allows for paracrine interaction and communication. The method of culturing two different cell types using an insert co-culture system was described earlier14,15 and was adapted for the use of murine primary cells. By the detection of prolactin mRNA levels as a read-out for decidualization, the described technique has a very reproducible read-out for decidualization available and allows thereby the study of the epithelial-stromal relationship during decidualization. Paracrine signals mediated from MEEC might alter the extent of decidualization in the stromal cells. Moreover, the model allows for specific modulation of the epithelial side without directly affecting the stromal cells. Therefore, this co-culture of MEEC and MESC offers a perfect tool to evaluate the effect of hormones, cytokines, etc. on epithelial cells with a read-out on stromal decidualization. Another advantage of this co-culture system is that it allows researchers to investigate the involvement of a specific protein in the decidualization-process in either the epithelial or stromal compartment by using cells isolated from transgenic animals. Furthermore, this technique will be very helpful to investigate blastocyst adhesion to evaluate whether paracrine factors secreted by epithelial cells in the presence of a blastocyst are sufficient to induce decidualization of the underlying stromal cells. An important step in trophoblast invasion correlated to extracellular matrix degradation, which is mediated by the action of proteolytic enzymes. The proposed technique can be applied as an in vitro model to investigate the cross-talk among the blastocyst, the epithelial cells and the supporting stroma.
However, at the moment little is known about the origin of the epithelial cells which can be as well luminal as glandular. Therefore, further characterization of the genetic and molecular profile of the epithelial cells is required. In addition, the described method is limited in the available knowledge about the polarization of the epithelial cells. At the moment, the polarization of epithelial cells was not taken into account. However, differences in expression of membrane proteins are described in apical and basal membranes. Inappropriate polarization of the epithelial layer could have an impact on the paracrine interaction between epithelial and stromal cells. However, methods to obtain polarized epithelial cells has been described and could be included in the described technique15,16.
Altogether, the described protocol proposes a technique to successfully establish primary cell cultures of mouse endometrial epithelial and stromal cells. This technique results in pure cell cultures as confirmed by qRT-PCR and immunostaining of vimentin and cytokeratin. Ultimately, an epithelial and stromal co-culture was introduced that allows for the investigation of paracrine signals mediated by either MEEC and/or MESC in the decidualization process.
The authors have nothing to disclose.
This work was supported by grants from the Research Foundation-Flanders (FWO G.0856.13N) and the Research Council of the KU Leuven (OT/13/113). K.D.C. is funded by the FWO Belgium. A.H. is funded by OT/13/113. We would like to thank the Cell Imaging Core facility of the KU Leuven (http://gbiomed.kuleuven.be/english/corefacilities/microscopy/cic/cic.htm) for the use of the confocal microscope.
ThinCert Tissue culture insert for 24 well plates; 1 µm pore size; transparant | Greiner Bio-one | 662610 | Inserts are also provided for other multiwell plates and/or other pore sizes. Other suppliers: Merck Millipore. https://shop.gbo.com/en/row/articles/catalogue/article/0110_0110_0090_0030/13408/ |
Nunc Cell culture treated 24 well plates | Thermo Scientific | 142475 | http://www.thermofisher.com/order/catalog/product/142475 |
8-Bromoadenosine 3′,5′-cyclic monophosphate sodium salt | Sigma Aldrich | B7880 | Prepare a stock solution of 100 mM in Milli Q water. Store aliquots at -20 °C. http://www.sigmaaldrich.com/catalog/product/sigma/b7880?lang=en®ion=BE |
Medroxyprogesterone 17-acetate | Sigma Aldrich | M1629 | Prepare a stock solution of 250 µM in ethanol. Store aliquots at -20 °C. http://www.sigmaaldrich.com/catalog/product/sigma/m1629?lang=en®ion=BE |
Arachis oil | Supermarket | Standard cooking arachis oil that can be found in every supermarket is used |
|
PrestoBlue cell viability reagent | Thermo Scientific | A13261 | https://www.thermofisher.com/order/catalog/product/A13261?ICID=cvc-prestoblue-c1t1 |
HBSS 1x, no calcium, no magnesium, no phenol red | Gibco | 14175-046 | Store in fridge as long as possible. Use cold HBSS+ during the protocol. https://www.thermofisher.com/order/catalog/product/14175046 |
17-β Estradiol | Sigma Aldrich | E8875 | Prepare a stock solution of 1mg/ml in EtOH before diluting in arachis oil (1:1000). http://www.sigmaaldrich.com/catalog/product/sigma/e8875?lang=en®ion=BE |
Cell medium filter Stericup, 0.22 µm, polyethersulfone, 500 mL, radio-sterilized | Merck Millipore | SCGPU05RE | http://www.merckmillipore.com/BE/en/product/Stericup-GP%2C-0.22%C2%A0%C2%B5m%2C-polyethersulfone%2C-500%C2%A0mL%2C-radio-sterilized,MM_NF-SCGPU05RE |
Penicillin-Streptomycin (5,000 U/mL) | Gibco | 15070-063 | https://www.thermofisher.com/order/catalog/product/15070063?ICID=search-15070-063 |
DMEM/F12, phenol red, L-glutamine, HEPES | Gibco | 11330-032 | https://www.thermofisher.com/order/catalog/product/11330057?ICID=search-11330-057 |
Fetal Bovine Serum, qualified, heat inactivated, E.U.-approved, South America Origin | Gibco | 10500-056 | https://www.thermofisher.com/order/catalog/product/10500064?ICID=search-10500-064 |
Amphotericin B | Gibco | 15290-018 | https://www.thermofisher.com/order/catalog/product/15290018?ICID=search-15290-018 |
Gentamicin (50mg/ml) | Gibco | 15750-037 | https://www.thermofisher.com/order/catalog/product/15750037?ICID=search-15750-037 |
DMEM, low glucose, pyruvate, no glutamine, no phenol red | Sigma Aldrich | D5921 | http://www.sigmaaldrich.com/catalog/product/sigma/d5921?lang=en®ion=BE |
MCDB-105 | Sigma Aldrich | M6395 | Dilute in Milli Q water, adjust pH to 7 with NaOH and filter before use. Store in the dark. http://www.sigmaaldrich.com/catalog/product/sigma/m6395?lang=en®ion=BE |
Insulin from bovine pancreas | Sigma Aldrich | I1882 | http://www.sigmaaldrich.com/catalog/product/sigma/i1882?lang=en®ion=BE |
Coverslips, glass, 18 mm Ø | Glaswarenfabrik Karl Hecht | 1001/18 | http://www.hecht-assistent.de/187/?L=1&tx_rmproducts_pi1[g]=937&tx_rmproducts_pi1[p]=3504&tx_rmproducts_pi1[v]=20088&cHash=abd12065683fe74b00eaa5e562824d06 |
Poly-L-lysine | Sigma Aldrich | P2636 | http://www.sigmaaldrich.com/catalog/product/sigma/p2636?lang=en®ion=BE |
Collagenase type IA from Clostridium histolyticum | Sigma Aldrich | C2674 | http://www.sigmaaldrich.com/catalog/product/sigma/c2674?lang=en®ion=BE |
DPBS, no calcium, no magnesium | Gibco | 14190-094 | https://www.thermofisher.com/order/catalog/product/14190094?ICID=search-14190-094 |
Trypsin from bovine pancreas | Sigma Aldrich | T7409 | http://www.sigmaaldrich.com/catalog/product/sigma/t7409?lang=en®ion=BE |
Trypsin-EDTA (0.05%), phenol red | Gibco | 25300-054 | Warm to room temperature before use. https://www.thermofisher.com/order/catalog/product/25300054?ICID=search-25300-054 |
Cell strainer, 40 µm mesh, disposable | BD Falcon | 352340 | https://www.fishersci.com/shop/products/falcon-cell-strainers-4/p-48680 |
Cell strainer, 100 µm mesh, disposable | BD Falcon | 352360 | https://www.fishersci.com/shop/products/falcon-cell-strainers-4/p-48680 |
Millex-GP Syringe Filter Unit, 0.22 µm, polyethersulfone, 33 mm, gamma sterilized | Merck Millipore | SLGP033RS | https://www.merckmillipore.com/BE/en/product/Millex-GP-Syringe-Filter-Unit%2C-0.22%C2%A0%C2%B5m%2C-polyethersulfone%2C-33%C2%A0mm%2C-gamma-sterilized,MM_NF-SLGP033RS?bd=1 |
Acetic acid (glacial) 100% | Merck Millipore | 1.00063 | https://www.merckmillipore.com/BE/en/product/Acetic-acid-%28glacial%29-100%25,MDA_CHEM-100063 |
Collagen I | Corning | 354236 | From rat tail. https://catalog2.corning.com/LifeSciences/en-BR/Shopping/ProductDetails.aspx?categoryname=&productid=354236%28Lifesciences%29# |
Pancreatin from porcine pancreas | Sigma Aldrich | P3292 | http://www.sigmaaldrich.com/catalog/product/sigma/p3292?lang=en®ion=BE |
35 mm Tissue Culture Dishes, Polystyrene, Sterile | BD Falcon | 353001 | https://us.vwr.com/store/catalog/product.jsp?product_id=4675630 |
Ethanol absolute AnalaR NORMAPUR ACS, Reag. Ph. Eur. analytical reagent | VWR Chemicals | 20821296 | https://uk.vwr.com/store/catalog/product.jsp?catalog_number=20821.310DP |
monoclonal rabbit anti-human vimentin (D21H3) | Cell Signalling Tech | #5741 | use 1/500. http://www.cellsignal.com/products/primary-antibodies/vimentin-d21h3-xp-rabbit-mab/5741 |
monoclonal mouse anti-human pan cytokeratin | Sigma Aldrich | C2562 | use 1/1000. http://www.sigmaaldrich.com/catalog/product/sigma/c2562?lang=en®ion=BE |
Paraformaldehyde Solution, 4% in PBS | Affymetrix | 19943 | http://www.affymetrix.com/catalog/130621/USB/Paraformaldehyde+Solution+4+percent+in+PBS#1_1 |
Triton X-100 | Sigma Aldrich | X100 | http://www.sigmaaldrich.com/catalog/product/SIAL/X100?lang=en®ion=BE&gclid=Cj0KEQjwhN-6BRCJsePgxru9iIwBEiQAI8rq879lESeuIU4N4AEQRLNEXj4f8z65KkD-q8Xr35sQDRgaAp-k8P8HAQ |
Goat serum | Sigma Aldrich | G9023 | http://www.sigmaaldrich.com/catalog/product/sigma/g9023?lang=en®ion=BE |
Goat anti-rabbit Alexa Fluor 488 | Thermo Scientific | A-11034 | http://www.thermofisher.com/order/genome-database/searchResults?query=488&resultPage=1&resultsPerPage=15&autocomplete=&searchMode=keyword&productTypeSelect=antibody&targetTypeSelect=antibody_secondary&species=ltechall&keyword=488#filters=genericsort2:%22Capra%20hircus%22;;generic6:^%22Oryctolagus%20cuniculus%22$;;conjugates:^%22Alexa%20Fluor®%20488%22$;; |
Goat anti-mouse Alexa Fluor 594 | Thermo Scientific | A-11032 | http://www.thermofisher.com/order/genome-database/searchResults?query=594&resultPage=1&resultsPerPage=15&autocomplete=&priorSearchTerms=&searchMode=keyword&productTypeSelect=antibody&targetTypeSelect=antibody_secondary&species=ltechall&keyword=594#filters=genericsort2:%22Capra%20hircus%22;;generic6:^%22Mus%20musculus%22$;; |
VECTASHIELD mounting medium with DAPI | Vector Laboratories | H-1200 | http://vectorlabs.com/vectashield-mounting-medium-with-dapi.html |
DPBS, with calcium and magnesium | Gibco | 14040174 | https://www.thermofisher.com/order/catalog/product/14040133 |