Hematopoietic Stem Cell Purification Leads to Loss of a Stem Cell Population within the Lineage Positive Cellular Fraction

Background: Hematopoietic stem cells (HSCs) have tremendous self-renewal and differentiation capacity. The majority of murine hematopoietic stem cell studies have focused on rare purified populations of HSCs, conventionally described as negative for lineage-specific markers and positive for particular cell surface epitope profiles, including c-Kit, Sca-1, and CD150. However, our data indicate that such purifications lead to the loss of a significant population of actively cycling marrow cells with long-term multi-lineage stem cell potential. In the studies presented here, we tested the hypothesis that this discarded stem cell population lies, in part, within the lineage positive (Lin+) fraction of marrow.

Methods: We flushed whole bone marrow (WBM) from B6.SJL mice and incubated it with allophycocyanin-tagged antibodies against erythroid (TER119), myeloid (CD11b, GR1), B-lymphoid (B220), or T-lymphoid (CD3, CD4, CD8) markers. Different doses of each specific Lin+ subset isolated by fluorescence activated cell sorting were competitively engrafted into lethally irradiated C57BL/6 host mice. At 1,3, and 6 months post-transplant, peripheral blood was analyzed for donor contribution to chimerism and lineage specificity.

Results: Although typically considered to be without stem cell activity, we found that all Lin+ sub-fractions upon single sorting were able to contribute to marrow repopulation in competitive bone marrow transplants. For example, when lethally irradiated recipient mice received 3x10⁵ C57BL/6J competitive whole bone marrow cells in combination with single-sorted GR1⁺ ± CD11b⁺ cells (2x10⁶ cells/mouse), peripheral blood showed 15% donor chimerism at 6 months. Similarly, if single sorted CD3⁺ ±CD4⁺ ±CD8⁺ cells (70,000 cells/mouse), B220⁺ cells (1x10⁶ cells/mouse), or Ter119⁺ cells (1x10⁶ cells/mouse) were competitively engrafted with 3x10⁵ C57BL/6 WBM cells, the donor Lin+ sub-fractions contributed to 2%, 15%, and 35% peripheral blood chimerism at 6 months post-transplant, respectively. This contribution was multi-lineage in all cases. When we performed double sorting of the Lin+ subsets, there was a dramatically reduced engraftment capacity between 1-6% donor chimerism for all subgroups. However, we do not think the loss of stem cell capacity with double sorting seen in these studies is due merely to the loss of classical hematopoietic stem cells (Lineage-/stem cell marker+). In our earlier studies, we showed that the total Lin+ population contains long-term multi-lineage engraftment capacity due almost entirely to actively cycling cells. Therefore, if the engraftment capacity within the single sorted Lin+ sub-fractions was due solely to the presence of classical HSCs lost with double sorting, the engraftment capacity found within the Lin+ compartment should be due only to quiescent cells in keeping with the cell cycle status of engrafting highly purified stem cells.

Conclusions: Based on these data, we predict that a cycling population of stem cells exists within this single sorted, Lin+ enriched fraction discarded with conventional HSC purification. Future studies are ongoing to further characterize the subsets of Lin+ cells that both remain Lin+ and are found to be Lin- upon double sorting. We will analyze these populations for engraftment capacity, concomitant stem cell marker expression and cell cycle status, in order to fully characterize the total stem cell potential within whole bone marrow that is not included in the purified HSC populations.