Hematopoietic Stem Cell Potential within the Primary Sorted Lineage Positive Population: What Are We Losing with Hematopoietic Stem Cell Purification?

Lineage positive cells within murine whole bone marrow (WBM) are classically considered to be devoid of stem cell capacity. As such, lineage depletion prior to further selection for stem cell marker positive, lineage negative cells dominates virtually all hematopoietic stem cell (HSC) purification protocols. Here, we present data showing a significant population of long-term multi-lineage hematopoietic stem cells within the primary sorted lineage positive (Lin+) murine marrow population. Using limiting dilution competitive bone marrow transplantation assays, we found significant long-term multi-lineage engraftment capacity within the total primary sorted Lin+ population (cells positive for erythroid (TER119), myeloid (GR1, CD11b) or lymphoid (B220, CD4, CD8) markers). When 0.25 x 10⁶ Lin+ cells were competed against 1 x 10⁶ competitor WBM cells, average percent donor chimerism in recipient mice at 6 months post-transplant was 7.2% ± 2.9%. Donor chimerism was multi-lineage. There was still 1.9% ± 0.5% donor chimerism when only 62,500 primary sorted Lin+ cells were competed against 1 x 10⁶ competitor WBM cells at 3 months post transplant, and further limiting dilution assays are ongoing to quantitate the stem cell compartment within the primary sorted Lin+ population. These levels of engraftment were similar to that found within un-separated donor WBM at the same dilutions, and multi-lineage engraftment capacity within the primary sorted Lin+ population persisted in secondary transplant. When further sub-fractionated into erythroid, myeloid or lymphoid sub-populations, every primary sorted individual sub-population contained long-term multi-lineage engraftment capacity at 6 months post transplant. Primary sorts were between 96-97.5% pure. When primary Lin+ sub-sets (B220+ cells, GR1+ cells, or CD11b+ cells) were double sorted, there was loss of engraftment potential within the double sorted persistently positive Lin+ subsets, but surprisingly high engraftment within those cells negative for only the single Lin+ marker on double sort (31%-59% donor chimerism at 6mo post-transplant; 1 x 10⁵ donor cells + 3 x 10⁵ competitor WBM cells). Immunophenotype analysis showed that these populations were not enriched in c-Kit+/Sca-1+/CD150+ cells. The high engraftment capacity of cells within the primary sorted Lin+ population, further isolated solely based on their being negative for a single Lin+ marker on the double sort prompted us to directly compare their engraftment capacity to that of purified Lineage negative (Lin-) cells. Interestingly, although isolated solely based on the presence or absence of GR1 on double sort, we found that the average percent donor chimerism at 6 months post-transplant was equal between purified Lin- cells (58.3% ± 17%) and the population of GR1 positive cells that became negative for GR1 on double sort (58.3% ± 26%; 1 x 10⁵ donor cells + 3 x 10⁵ competitor WBM cells). Using tritiated thymidine suicide, we found that the stem cell activity within these populations isolated from the primary sorted Lin+ fraction was due to actively cycling cells. These data further indicate that the engraftment capacity within the primary sorted Lin+ population is not simply due to conventionally purified quiescent HSCs contaminating our primary sort. In sum, there is a cycling population of stem cells within the primary sorted Lin+ fraction that appears to be distinct from quiescent highly purified HSCs. As primary sorted Lin+ cells, routinely discarded in most HSC studies, comprise up to 98% of WBM, engraftment capacity within this population represents a potentially large and understudied stem cell population within WBM worthy of further characterization. Work is currently underway to further quantitate and define this population, and delineate how cell cycle-related changes influence the phenotype and function of this cycling murine HSC population.