Experimental BMT 101 – part 6: Considerations for serial bone marrow transplantation

by Alexey Bersenev on April 10, 2014 · 0 comments

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This is fresh issue of the series “Experimental bone marrow transplantation 101“. In this series we talk about basics of whole bone marrow and hematopoietic cells transplantation models to study hematopoietic stem cells and blood formation in normal conditions and malignancies.

Why serial BMT?
The purpose of serial bone marrow transplantation (BMT) is to test the self-renewal capability of HSC or/and their aging and functional exhaustion.

Recipients and experimental scheme
Recipient mice should be identical throughout all series of transplant. Conditioning (usually lethal irradiation) should be the same in each series. The optimal age for recipients is 8-12 weeks.
In BMT models, donor-derived cells are traceable throughout all rounds of serial transplantation if you use the same recipient mice.

Donors
Donor mice are previously transplanted (underwent at least primary transplant) and (usually) genetically different from recipients. Donors should be at the final readout time point, because bone marrow harvested from euthanized animals. Donor-recipient could be one-to-one or pooled. One-to-one transplant is more precise method, but may could require more animals for statistics. Try to harvest bone marrow from the same type of bones throughout experiment.
On the day of transplant, detection of donor HSC engraftment in donor’s BM is highly recommended. For example, you can harvest 3 leg bones for flow cytometry and one bone (femur) save in the media for transplant.

Cell dose and number of rounds
Your cell input in the primary transplant could be any that justify your experimental goals. For the second and subsequent rounds there are some recommended doses. For whole marrow transplantation you have to input a sufficient number of primary HSC, capable of self-renewing for at least 2-3 rounds with significant readable output. For normal wild type mouse, anything between 1 to 10 million (M) total BM cells will be good. Typically, 2M or 5M of unseparated BM cells are used. Usually 3-4 rounds is enough to see a difference between control and test groups. Many labs do only 2 rounds to see if cells are capable to self-renew at all.

Keep in mind that transferring 5 – 10M BM cells from normal wild type mice will require at least 4 rounds of serial transplant in order to see significant self-renewal decline and HSC exhaustion. So, you can adjust the cell dose, based on the timeframe of your experiment. For example, If each round takes 4-6 months, and you want to see a difference sooner than later, you may consider transferring 2M of unseparated BM cells each round of transplantation instead of 5M.

Time planning and readout
The conclusion about donor HSC performance should be based on positively repopulated primary recipients. At least 16 weeks post-transplant is recommended for valid readout of blood repopulation and bone marrow engraftment by donor HSC. Therefore, each round of serial transplant will take at least 16 weeks. However, there is no unified opinion on intervals between rounds, starting from the second. For example, you can transplant with an interval of 2 months between transplant from round 2 to 5, with valuable readouts (16 weeks) at 1st and 5th transplants.

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