Defining Engraftment Potential within the Lineage Positive Population in Murine Marrow

The study of highly purified hematopoietic stem cells (HSCs) has dominated the field of hematopoietic stem cell biology. It is widely believed that the true stem cell population lies within the Lineage negative (Lin-) population, further sub-fractionated using positive and negative selection for surface markers such as c-Kit, Sca-1, CD150, CD41, CD48, and CD34. It is research on these highly purified subsets of HSCs that forms the foundation for almost all our knowledge of HSCs, and has led to the dogma that marrow stem cells are quiescent with a stable phenotype and therefore can be purified to near homogeneity. In contrast, we have shown that a large percentage of long-term multi-lineage marrow repopulating cells in whole bone marrow (WBM) are actively cycling, that these cycling stem cells are lost during conventional HSC isolation, and that they can be found, in part, within the discarded Lineage positive (Lin+) population. Here we present data further characterizing the stem cell potential in the Lin+ fraction. We incubated WBM from B6.SJL mice with fluorescently tagged antibodies directed against TER119, B220, or T-cell markers (CD3, CD4, CD8), isolated the distinct Lin+ subsets by FACS, and then competitively engrafted each Lin+ subset into lethally irradiated C57BL/6 host mice. Donor chimerism and lineage specificity of donor cells in peripheral blood were analyzed by flow cytometry at 3 months. Although classically considered devoid of stem cell activity, we found that, when competed against equal numbers of C57BL/6 WBM, the TER119+ and B220+ B6.SJL donor cells contributed to 33% and 13% of the peripheral blood chimerism, respectively. In both cases, the engraftment was multi-lineage. When 70,000 T cell marker+ donor cells were competed with 300,000 C57BL/6 WBM, the donor cells contributed up to 1.6% of the peripheral blood multi-lineage chimerism. Given the size of the Lin+ fraction in WBM, such chimerism indicates a significant stem cell potential within this typically discarded population. Further time-points, secondary transplants and limited dilution studies are in progress to further define the prevalence and potency of this stem cell population.

We have been testing mechanisms governing the loss of this stem cell population during HSC purification. First, we have previously shown that bulk Lin+ engraftment potential is due to cycling stem cells. We hypothesize that fluctuations in surface epitope expression with cell cycle transit render this population difficult to isolate with antibody-mediated strategies that rely on stable epitope expression. To begin testing this, we tracked the fluctuation of stem cell markers on Lin- cells in vitro. We isolated Lin- cells that were also negative for the stem cell markers c-Kit and Sca-1, placed them in liquid culture and, 18 hours later, re-assessed for stem cell marker expression by flow cytometry. We found that, although initially stem cell marker negative, up to 6%, 14%, and 2% of the Lin-/stem cell marker negative cells became positive for c-Kit alone, Sca-1 alone, or both c-Kit and Sca-1 expression, respectively. We are currently testing this population for a correlation between gain of c-Kit- and Sca-1 expression and stem cell function. Second, it is possible that there is a distinct subset of HSCs that are positive for both Lin+ markers and stem cell markers with stable stem cell capacity and that these distinct stem cells are thrown out in the process of lineage depletion. To begin testing this hypothesis, we have simultaneously stained WBM with antibodies directed against the Lin+ markers and conventional stem cell markers. Our preliminary data indicate that each Lin+ fraction tested to date has a subpopulation that is also positive for c-Kit and Sca-1. For example, 21% of CD3+ cells, 6.2% of CD4+ cells, 2.26% of CD8+ cells, 0.5% of B220+, and 0.45% of TER119+ cells express both c-Kit and Sca-1. We suspect these two populations have distinct functional phenotypes and experiments characterizing the molecular phenotype and engraftment capacity of these subpopulations are ongoing. In sum, our data indicate that stem cell purification skews isolation towards a small population of quiescent stem cells, underrepresenting a potentially large pool of actively cycling HSCs that are found within the Lin+ fraction. These data underscore the need to re-evaluate the total hematopoietic stem cell potential in marrow on a population level.