I irradiated mice and and transferred new BM into them. After 3 weeks I checked their blood and I found that 80% of the T cells are host cells while only 20% are from the donor. Is it too early to evaluate the success of the transfer or I did not get rid of the host stem cells?
T cell depletion after irradiation is much faster. The most probably, the dose of irradiation was unsufficient and some radioresistant T cells remained in irradiated mice. These cells have advantage over transplanted cells during the restoration of immune system of recipient. This trouble is very frequent in making chimeras. To avoid this, try to use RAG KO mice as recipients.
As Dmitry said, the irradiation dose is probably the main factor causing your chimerism problem. In my opinion, an irradiation dose between 5 and 10 Gy following by a transplantation of 1X10^7 BM cells give good results. Moreover, in our lab we are now using chemotherapy for myeloablation which is very effective. See the reference.
Lampron, A., Lessard, M. & Rivest, S. Effects of Myeloablation, Peripheral Chimerism and Whole Body Irradiation on the Entry of Bone Marrow-Derived Cells Into the Brain. Cell Transplant (2011). doi:10.3727/096368911X593154
Check the cell count at different periods of time [here you have checked after 3 weeks] and plan your experiments with the irradiation dose etc. lalitha kabilan
As Peter, I use 10 Gy following by the transfer of 1X107 bone marrow cells, but you have to use T cell deficient recipients anyways to completely replace T cell compartment. T regulatory cells, for example, are radio resistant and are not completely eliminated after irradiation.
It also depends on the way you administrate the irradiation, according to the quaity of the source, dose by unit of time, upper and lower body dose delivery etc... Controls without any reconstitution should die by about 8-10 days if the irradiation is letal, insurring full depletion of recipient lymphoid cells by that time. Bone marrow transfer should aso be done 24 hours after irradiation.
The dose of irradiation is a factor that should be the key consideration. What was the age and the mouse strain that you are using?
You have 2 different parameters to take in consideration. Firstly, a full reconstitution of hematopoiesis will be achieved 6-8 weeks after transplantation. Secondly, as said by others you have to keep in mind that there are radioresistant T cells which are still there after a lethal irradiation. If you do not want to use RAG-/- mice, you can perform 2 sublethal irradiations (2x 6.5 Gy with 5h in between, instead of 1 dose of 10 Gy) and in these conditions, you should be able to eliminate these radioresistant T cells. Do not use hosts younger than 6 weeks.
We found waiting 24-48 hours after irradiation to transfer avoided most of these problems, including with tregs--
Our Lab have another problem, we used Rag-/- mice and give irradiation, after that we did bone marrow transplantation.And 3 weeks after the transplant, we tested that more than 90% of stem cells were replaced. But the mice who only have irradiation without bone marrow transplantation are well alive and gain body weight after recover from the surgery. We want to know that the bone marrow transplantation is successful or not ? if this kind of irradiation dose we can use in the future?
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology