The overall goal of this procedure is to turn primary differentiated cells into progenitor cells of the same lineage by transient expression of the transcription factor YAP. This reprogramming procedure offers the possibility to generate in vitro progenitor like or stem like cells of various tissues starting from their differentiated counterparts even when the endogenous stem cells are rare or difficult to identify. The possibility here outlying to expand somatic cells ex vivo has implications for regenerative medicine for understanding mechanisms of cell plasticity that is also relevant in cancer and for cell tissue and development of biology studies.
This procedure fills a black box in the current reprogramming arena as we're not aiming to make specialized cells through embryonic stem cells differentiation, but tissue specific stem cells from differentiated cells. After sacrificing 10 female mice according to the text protocol, dissect the mammary glands by making a y shaped incision along the abdominal skin. Then use dumont forceps to carefully separate the glands from the peritoneum by gently pulling.
Place the dissected glands in a non cell adhesive dish with 10 milliliters of ice cold HBSS+PS. Under a tissue culture hood use 10 milliliters of fresh HBSS+PS to wash each gland once and place them in an empty non cell adhesive dish. Then use two sterile scalpels to finely mince the glands into one cubic millimeter fragments.
Proper mincing of the glands is essential for efficient cell isolation. Pull 20 glands together and try to perform this step within three to five minutes for optimal cell viability. Using a 25 milliliter serological pipette and 10 milliliters of dissociation medium, recover the minced tissue from each dish and transfer the suspension in a 50 milliliter conical tube pipetting at least five times to disaggregate the tissue clumps.
Incubate the tissue at 37 degrees celsius for one hour with continuous vigorous shaking. Then spin down the digested tissue at 400 times g at room temperature for five minutes. After discarding the supernatant, use three milliliters of hemolytic solution to resuspend the tissue and incubate on ice for three minutes.
Next, with 10 milliliters of wash medium number one, wash the cells. After spinning the digested tissue and discarding the supernatant, resuspend the tissue pellet in 10 milliliters of wash medium number two and plate the cells in 10 centimeter tissue culture dishes. Then incubate the dishes at 37 degrees celsius for one hour.
Recover the cell suspension into a 50 milliliter conical tube and spin the tube at 400 times g for five minutes. Then use 10 milliliters of the calcium chelating solution to resuspend the pellet. Following centrifugation and after repeating the wash, resuspend the pellet in five milliliters of 0.25%trypsin-EDTA.
After incubating the sample, add five milliliters of dispase solution supplemented with one microgram per milliliter of DNase one on top of the trypsin solution. To disaggregate the clumps use a one milliliter pipette tip to pipette up and down at least five times and incubate the sample at 37 degrees celsius for 10 minutes shaking every three minutes. Add 10 milliliters of wash medium number two and filter the solution through a 40 micrometer cell strainer into a new 50 milliliter conical tube.
Then spin down the cell suspension. To carry out cell purification by FACS, use 200 microliters of wash medium number one to resuspend each pellet. Then add 44.5 microliters of antibody mix, pipette thoroughly, and incubate the sample in the dark on ice for 30 minutes.
Dillute the cell suspension in 10 milliliters of wash medium number one and spin down the sample. Resuspend the cell pellet in two milliliters of sorting solution and filter through a cap strainer FACS tube before proceeding to FACS separation of the cell populations. Use wash solution number one to wash the cells recovered from FACS.
After removing the supernatant, use mammary 2D culture medium to resuspend the cell pellet and plate 500 microliters per well on a collagen treated plate. Seven days after doxycycline induction of lentiviral infected primary LD cells, add 150 microliters per well of 05%trypsin-EDTA to the cells and incubate them at 37 degrees celsius for 10 minutes. Once the cells have been washed and counted, use memory colony medium, supplemented with two micrograms per milliliter of doxycycline to resuspend them.
Then seed 1, 000 cells per well in 24 well ultra low attachment plates for culturing YAP expressing mask like colonies. To subculture y masks, collect each sample and add a 10 to one volume of ice cold HBSS, then incubate the samples on ice for one hour in order to solubilize the basement membrane matrix. After spinning down the colonies, use ice cold HBSS to wash the cells three times.
Then add 05%trypsin-EDTA to the colonies and incubate them at 37 degrees celsius for 10 minutes. Pass the colonies through a P1000 pipette tip 10 times to obtain a single cell suspension. Then count and reseed the cells in mammary colony medium without doxycycline in 24 well ultra low attachment plates at a colonogenic density of 1, 000 cells per well.
Prior to the third passage, transfer y mask colonies in organoid culture conditions by recovering the colonies from the mammary colony medium and use 100%growth factor reduced basement membrane matrix to resuspend them. Following incubation when the basement membrane matrix has solidified overlay the gels with 500 microliters of the mammary organoid medium. Passage the cultures by collecting each organoid sample and incubating them in an excess volume of HBSS on ice for one hour.
Then use ice cold HBSS to wash the organoids three times. Trypsinize the organoids as demonstrated earlier in this video and reseed single cell suspensions in a drop of 100%growth factor reduced basement membrane matrix, then use 500 microliters of mammary organoid medium to overlay the gels. After washing digested mouse pancreatic tissue according to the text protocol, use six milliliters of acinar recovery medium to carefully resuspend the digested tissue and split it between two wells of a six well tissue culture plate.
Under a stereomicroscope carefully assess the quality of the acinar isolation and remove any big tissue clumps eventually present. To seed primary pancreatic acini, after incubating the clusters for two hours for cell recovery, collect the acinar cell suspension in a conical tube and centrifuge the samples at 100 times g and 18 degrees celsius for five minutes. After resuspending acini in acinar culture medium dilute the suspension with an equal volume of neutralized rat tail collagen one solution, keeping the tubes on ice.
Mix carefully and quickly and seed the cell suspension in 16 wells on top of the collagen cushion. For optimal seeding of pancreatic acini it is crucial to keep all tubes in ice before plating and proceed with speed but at the same time careful pipetting to avoid air bubble formation is essential to create a homogenous collagen matrix. Incubate the cells at 37 degrees celsius for one hour to allow a hydrogel to form.
Then to induce pancreatic organoids, overlay the collagen hydrogels with 500 microliters of acinar culture medium supplemented with doxycycline. To subculture the organoids, discard the culture medium, carefully extract the hydrogels, and transfer them to conical tubes containing four milliliters of collagenase I solution B then incubate the tubes at 37 degrees celsius with vigorous shaking for 30 minutes before spinning down the cells and removing the supernatant. After trypsinizing the cells and removing the PBS supernatant, use ice cold basement membrane matrix to resuspend the cell pellet and seed the suspension in ultra low attachment plates.
Let the basement membrane matrix hydrogel solidify by incubating the plates at 37 degrees celsius for 40 minutes and then overlay with pancreatic organoid medium. yDucts will grow as cyst like organoids in seven to 10 days. Careful gating of primary mammary LD epithelial cells into the three subpopulations shown here is essential to isolate a pure preparation of LD cells that are fully differentiated and completely growth arrested when seeded in mammary gland colony forming conditions.
Conversely, when induced to express exogenous YAP, LD cells start proliferating to form easily recognizable dense epithelial colonies in 5%basement membrane matrix suspension cultures. Under basement membrane matrix organoid culture conditions, reprogrammed luminal cells self organize into complex organoid like structures that develop around multiple lumens and self renew even in the absence of doxycycline. Within the five to seven days of culture in 3D collagen I based hydrogel in the presence of doxycycline, differentiated panrcreatic acini readily turn into duct like clusters or yDucts composed of a thin monolayer of epithelial cells that proliferate around an expanding central cavity.
The reprogramming efficiency can be easily measured by scoring the number of duct like clusters over the total number of seeded acini. Reprogrammed yDucts can then be passaged at the single cell level into matrigel based organoid culture conditions displaying remarkable self renewal ability even in the absence of endogenous YAP expression. We have detailed two procedures.
One allowed reprogramming of FACS purified cells through lentiviral vectors and a second one that avoids viral infection and takes advantage of transgenic YAP expression. While attempting reprogramming of mammary cells it is important to avoid the use of excessive viral titer as this can be detrimental for reprogramming efficiency. The fully transgenic strategy is appropriate for primary pancreatic acini as these are scarcely amenable to lentiviral infection.
It also offers the same advantage of doxycycline dependent lentiviral vectors for the type control of gene expression. In addition to the exampes demonstrated here, this YAP reprogramming protocol has proven successful for converting differentiated neurons into proliferating neurons themselves. After watching this video you should have a good understanding on how to perform a YAP dependent reprogramming to convert differentiated cell types from different adult tissues into their corresponding tissue specific stem cells.
This procedure expands the current reprogramming strategies by providing a means to generate somatic stem cells that may have relevance for regenerative medicine for the study of somatic stemness and for expansion of somatic stem cells in vitro. Please consider that handling of lentiviral preparation can be extremely hazardous and by safety level two measures should be always adopted while performing this procedure.
