Excessive stem cell competition in the competitive long-term repopulation assay - development of a solution

The competitive long-term repopulating stem cell assay is widely used as the standard method for detecting murine hematopoietic stem cells. An integral part of this assay is the competition between the test stem cells and those in the irradiated recipient, as well as the carrier cell population which ensures the short-term survival of the animal. Recently, we and others have observed that some populations of primitive stem cells repopulate the hematopoietic system poorly, if at all using the standard assay. We have determined that the explanation for this inability is that the competition from the recipient and carrier stem cells is too great, resulting in inadequate and inconsistent representation of the test cells in the reconstituted animal. This is particularly a problem when transplanting stem cells which are proliferatively quiescent and/or low in number. We have overcome this problem by developing a quantitative assay in which there is minimal stem cell competition. This has been achieved by firstly removing all stem cells in the recipient by significantly increasing the radiation dose to 1400 cGy. In addition, the ratio of stem cells to radioprotective cells in the carrier cell population has been decreased by compromising the stem cells prior to transplantation using a soluble, injectable form of Busulfan (Busulfex™). This is a chemical whose toxic effect is related to the stage of hematopoietic development, the primitive stem cells being affected more than later progenitors. In addition to its improved sensitivity the resulting REpopulating Stem Cell Unit (RESCU) assay has the added advantage of been much faster. The increased pressure to repopulate the recipient animal as a result of decreased competition enabling the presence of repopulating stem cells to be consistently determined and quantitated as early as 1-2 months after transplantation.