This method can be used to answer key questions in the reproductive medicine field about regarding the underlying molecular mechanisms of decidualization, and understanding the roles of specific genes in endometrial stromal cell decidualization. This study provides a detailed protocol for the isolation of human endometrial stromal cells and subsequent artificial decidualization by the supplementation of hormones to the culturing media. The expression of hormonal change, such as prolactin and insulin-like growth factor binding protein one can be utilized to confirm and quantify the degree of decidualization.
Importantly, the viability of this protocol to conduct gene specific knockdown is also demonstrated. Transfer the biopsy with sterilized forceps into a 50 milliliter tube containing ten milliliters fresh HBSF, followed by centrifugation at 500x G for 90 seconds. Using a one mL pipette, remove the media and add three mL pre-warmed hES isolation media.
Decant the biopsy into a 10 centimeter dish containing five mL of hES isolation media, and begin chopping using Dumont 5 forceps and fine straight stitch scissors for about 20 minutes. Using one mL pipette, transfer the chopped biopsy into a new 50 mL tube, and add an additional seven mL of hES isolation media to the dish, to transfer the remaining pieces to the 50 mL tube. Then centrifuge the sample for two minutes at 800x G, and remove the supernatant using a one mL pipette.
Next filter the DNase-1 and collagenase digestion mix through a 0.2 micron filter attached to a 10 milliliter syringe directly onto the pellet. Then vortex the tube for 10 seconds to resuspend the pellet, and incubate for 90 minutes in a water bath. After every 10 minutes, vortex the tube for 10 seconds to make sure the tissue is properly digested.
After incubation, filter the suspension through a 40 micron cell strainer stacked over a new 50 milliliter tube to recover the stromal cells, then centrifuge the collected cells at 800x G for 2.5 minutes. Following centrifugation, gently remove the supernatant by using a one milliliter pipette, making sure not to disturb the cell pellet. Then resuspend the cell pellet in 10 milliliters of fresh hES isolation media, followed by centrifugation at 800x G for 2.5 minutes.
After centrifugation, aspirate the supernatant carefully, and resuspend the cells in six milliliters fresh hES media. Next, to remove blood cells, slowly layer resuspended cells onto three milliliters of density gradient media, and centrifuge the sample at 400x G for 30 minutes. Following centrifugation, collect the supernatant without disturbing the density gradient media.
Then add hES media to make the total volume up to 10 milliliters, and centrifuge the sample again at 800x G for 2.5 minutes. After centrifugation, remove the supernatant and resuspend the cells in six milliliters of hES media. Next plate the cells in a 75 centimeter flask by adding six milliliters of the cell resuspension and 10 milliliters of hES media to the flask, followed by gentle mixing through rocking the flask.
After plating, check the cell density under the microscope, then culture the cells in a 5%carbon dioxide incubator for three to five days, making sure to change the hES media every two days. After 48 hours, the cells should grow as a monolayer. Generally after five days, cells achieve 80 to 90%confluency in a 75 centimeter flask.
For siRNA transfection, begin by plating 800, 000 cells per well in a six well plate in triplicates for each condition, and incubate the cells in a 5%carbon dioxide incubator for two days to prepare for transfection. After two days, add 150 microliters of Opti-MEM serum free media in a five mL tube, followed by the addition of five microliters of transfection reagent. Mix well by inversion, and incubate for five minutes.
Next add 100 microliters of Opti-MEM serum free media in a new five mL tube, followed by the addition of 16 nanomoles of SRC-2 siRNA. Then add 100 microliters of Opti-MEM serum free media in a five mL tube, followed by the addition of 16 nanomoles control siRNA. After incubation, combine the diluted transfection reagent with the SRC-2 siRNA and mix well by pipetting forward and reverse.
Then combine the diluted transfection reagent with the control siRNA and mix well by pipetting forward and reverse. Then incubate the complexes for five minutes. During this incubation, aspirate out the hES media, and add one milliliter of change media to each well.
After five minutes of incubation, centrifuge the complexes at 500x G for two minutes to collect the solution. Then add 250 microliters of the siRNA transfection reagent complexes to each well containing cells and media, and mix by gentle rocking, followed by incubation in a 5%carbon dioxide incubator for five hours at 37 degrees Celsius. After five hours, aspirate the media and change to hES media, and continue incubation for two days in preparation for an in-vitro decidualization assay.
Post 48-hour transfection and prior to initiating the in vitro decidualization assay, collect the day zero control siRNA and SRC-2 siRNA transfected cells. First, aspirate the media and wash with one mL PBS per well. Then remove the PBS, and add 300 microliters of RNA lysis buffer per well and incubate for three minutes.
After three minutes of incubation, collect the cells in RNA lysis buffer into a 1.5 ml tube for later downstream analysis. Then prepare the EPC media by aliquoting the appropriate volume of decidualization media, and add 10 nanomolar estradiol, one micromolar medroxyprogesterone acetate, and 50 micromolar cyclic adenosine monophosphate in a 50 milliliter tube. For remaining day six wells, add two milliliters of EPC media into each well, and incubate the cells in a 5%carbon dioxide incubator at 37 degrees Celsius for six days, changing the EPC media every 48 hours.
After the sixth day, collect the day six control siRNA and day six SRC-2 siRNA transfected cell pellets in RNA lysis buffer for downstream analysis using real-time PCR. The induction of decidualization by estrogen medroxyprogesterone acetate and cyclic adenosine monophosphate, results in the cytoskeletal rearrangement of stromal cells from elongated fibroblastic cells to rounded epitheliod cells. Phalloidin staining of endometrial stromal cells further reveals the active fiber cytoskeleton reorganization characteristic of hES decidualization.
In vehicle treated hES, cells remain fibroblastic in appearance, whereas in EPC treated cells, hES appeared epithelioid like. To confirm the efficacy of the in-vitro decidualization assay, mRNA transcript levels of decidualization markers prolactin and insulin-like growth factor binding protein-1 were assessed over a 6 day period. Upon decidualization of human endometrial stromal cells, prolactin and IGFBP1 levels increased in a time-dependent fashion.
The time-dependent increase of secreted prolactin levels was also confirmed by examining the hES tissue culture media via ELISA method. Gene specific knockdown effects on the cellular and molecular changes of human endometrial stromal cell decidualization were assessed on the sixth day of culture post 48-hour siRNA transfection, with control or steroid receptor co-activator 2 siRNA. Control siRNA transfected hES demonstrated cellular and molecular changes, consistent with proper decidualization, including a cobblestone morphological change and increased transcript levels of decidualization markers PRL and IGFBP1.
In SRC-2 siRNA knockdown, hES remained fibroblastic in appearance as compared to the control siRNA transfected hES, and showed significantly decreased PRL and IGFBP1 transcript levels. SRC-2 transcript levels were significantly decreased in SRC-2 siRNA transfected hES when compared to control siRNA, indicating the viability of this method for gene silencing. After watching this video, you should have a good understanding of how to isolate primary stromal cells from endometrial biopsies for the purpose of in-vitro decidualization and knockdown a specific gene of interest using siRNA transfection.
