The overall goal of the following experiment is to position a population of healthy stem cells adjacent to stem cell ablated tissue within a parian in order to analyze stem cell and progeny behaviors in vivo during regeneration. This is achieved by anesthetizing and immobilizing either an un irradiated or a Leafly irradiated parian for further manipulation. Next, the immobilized un irradiated parian is partially shielded with lead and then x-ray irradiated, which produces an uninjured parian with stem cell ablated tissue juxtaposed to the shielded tissue that remains stem cell laden.
Alternatively, a tissue graft taken from a non irradiated donor may be transplanted into a previously irradiated host in order to produce a parian with an isolated population of stem cells. Fally present within an otherwise stem cell. Ablated host results are obtained that show isolated stem cell populations adjacent to the stem cell ablated tissues by whole mount RNA in situ two hybridization for stem cell marker genes.
So the methods described here aim to better understand regeneration planarians by studying the behavior of stem cells in both transplantation paradigm and a partial irradiation paradigm, which allows us to test whether or not the surrounding tissues and the local environments in which these stem cells reside, affect or affect the behavior of these neo blasts or stem cells. During the process of regeneration, One to two months prior to the experiment, feed the plin according to the accompanying manuscript. In order to obtain the desired size in this demonstration, plin between one to two centimeters in length and wider than two millimeters are selected.
Then they are starved three to seven days prior to use. Next, prepare the Chloro tone solution a mild local anesthetic by dissolving 0.1 to 0.2%weight per volume cleone in the parian water. Then chill the solution on ice for tissue transplantation with a bunsen burner, bend a capillary tube with 0.75 millimeter interior diameter at one to two centimeters from the end to a 90 degree angle for cutting the graft tissue.
Then bend another capillary tube with 0.7 millimeter exterior diameter at one to two centimeters from the end to a 90 degree angle for creating a hole in the host to receive the graft. Next, cut the black filter paper watman. Number three, filter paper Kim wipe and cigarette rolling paper according to the sizes indicated in the accompanying manuscript.
After that, prepare the modified Holt Fers solution and Caine saturated Holt Fretter solution. Chill both of them to four degrees Celsius. Then attach a folded Kim wipe to a square of Paraform.
Place it on the Peltier cooler plate or other cooling device under a dissecting microscope. Subsequently saturate the Kim wipe with chilled Holt Fretter solution. Then place two black filter paper rectangles on the Kim wipe.
Now place a piece of Watman number two filter paper in a Petri dish. Moisten the filter paper with Holt Fretter solution. Then chill the dish on ice for partial irradiation.
Place a larger paper liner in a larger dish similar to tissue transplantation. Moisten the filter paper with Holt Fretter solution. Then chill the dish on ice.
In this procedure, fill a Petri dish with chilled cleone solution. Pipette the Planaria into the dish for transplantation. Only anesthetize one host and one donor at a time.
For partial irradiation, many animals can be anesthetized at once. Then allow the paria to soak in the cleone solution for five to 10 minutes until they become motionless. Rinse the paria by pipetting them into a dish filled with chilled Holt Fretter solution.
After that, immobilize the plin area by pipetting them onto the black filter papers saturated with chilled Holt fretter solution. Then orient them ventral side down with a pair of forceps. Now place a chilled Petri dish prepared earlier in an ice bucket that will fit inside a top source X-ray irradiator.
Arrange the anesthetized plin area in a Petri dish by moving the black filter papers. Then transfer them to the top source X-ray irradiator situate the ice bucket such that the distance from the cathode tube to the paria is minimized. Thus maximizing the effective dose rate.
Then position the lead shields between the paria and the cathode tube for the dose of x-ray. If complete stem cell ablation from non shielded regions is desired, deliver 30 grays or greater. Immediately after the x-ray emission, transfer the parian into chilled parian water.
After that, allow the parian water to warm to room temperature and the animals to dislodge themselves from the black filter paper. The partial irradiation procedure is now complete. To begin this procedure under the dissecting microscope, arrange the anesthetized host and donor plin area on separate rectangular black filters on the Kim wipe, which has already been cooled on the peltier or cooler plate.
Use a 0.75 millimeter inner diameter capillary tube to cut out the graft plug from the donor. Then use a pair of forceps to place it on an out of the way portion of the host. Then use a 0.7 millimeter outer diameter capillary tube to remove a plug from the host.
Subsequently position the graft in the hole. Transfer the transplanted host on the rectangular black filter paper to the Petri dish prepared earlier. Wet a piece of rolling paper with Caine saturated Holt Fretter solution.
Then place it on top of the transplanted host. Next, soak four pieces of filter paper encasing saturated Holter solution, and in case the transplanted host sub subsequently soak four wads of cut, Kim wipe encasing saturated Holt Fretter solution. Lay them over the filter papers.
After that, close the lid. Place the Petri dish on ice. Then transfer the donor parian to the parian water for recovery and regeneration.
When all the transplants are completed, place the transplanted parian in a 10 degree Celsius incubator overnight. The next morning, transfer it to a Petri dish filled with parian water. Allow the parian to either dislodge itself from the filter paper or gently remove it with forceps and change the parian water once every two to three days afterward.
Hole mount in situ hybridization in the irradiated, but fully shielded control parian fixed three days following irradiation shows a stem cell distribution that is indistinguishable from that of the wild type parian. On the other hand, for the parian that was only partially shielded, leaving the anterior and posterior parts exposed stem cells appear to be ablated from the non shielded regions. Here shows the dorsal and ventral views of a successfully transplanted parian.
Three days after transplantation, the graft is clearly visible on both dorsal and ventral surfaces, and is surrounded by characteristic un pigmented tissue at the graft host interface. However, the unsuccessful transplantation displays no visible graft tissue at the transplantation site. Whole mount in situ hybridization revealed the presence of stem cells only in or around the graft location, which indicates the success of transplantation Once mastered.
Transplantations can be done in as little as five minutes each. With success rates approaching 90 to 100%and partial irradiation can be greatly scaled up. Assuming the field size of your irradiator is sufficiently large.
