Probing hematopoietic stem cell function using serial transplantation: Seeding characteristics and the impact of stem cell purification

Appropriate regulation of hematopoietic stem cell (HSC) numbers and function is a requisite for life-long blood cell replenishment. Knowledge of factors that regulate HSC activity is derived largely from murine model systems, with serial transplantation often considered a “gold standard” to assess longevity and self-renewal of HSCs. In the literature, we noted inconsistencies in how serial transplantations are conducted and decided to assess a set of parameters at play in such experiments. We found that HSCs distribute and expand unevenly among individual bones following transplantation, suggesting that isolation of a limited number of bone marrow cells for serial transplantation and/or analysis can influence experimental outcomes. Comparing donor cell output from transplanted unfractionated bone marrow cells, as opposed to fluorescence-activated cell-sorted HSCs, revealed distinct differences in the output of mature blood cells. Specifically, we found that long-lived progenitor and/or mature co-transplanted cells can severely affect the interpretation of ongoing HSC activity in secondary hosts. The implications of these data for the design and execution of serial transplantation experiments are discussed.