Identification of a Novel Murine CD45 Negative Bone Marrow-Derived Cell Population with In Vivo Marrow Repopulating Potential Following Hematopoietic Specification In Vitro.

Murine bone marrow-derived cells expressing Sca-1+c-kit+lin− (KSL), as well as subfractions of these cells, represent an enriched population of hematopoietic stem cells (HSC) capable of long-term reconstitution of lethally irradiated recipients. Commitment to the hematopoietic lineage is invariably associated with expression of the pan-leukocyte marker CD45 which is also expressed on KSL cells. Whether KSL cells are the most primitive population of HSC present in the bone marrow (BM) is not fully resolved. We hypothesized that putative HSC that are more primitive than KSL cells may not express CD45 or genetic elements that mark early hematopoietic specification and commitment, but may mature under appropriate conditions into CD45+ cells capable of hematopoietic differentiation in conditioned hosts. BM cells from 8 to 10-week old BoyJ mice were collected by flushing and erythrocytes were lysed. The remaining cells were stained and sorted to yield CD45+ Sca-1+ c-kit+ (CD45+HSC) and CD45− Sca-1+ c-kit− (CD45−) cells which represented approximately 0.02% of total cells analyzed. PCR analysis of both cell populations revealed that CD45+HSC expressed CD45 and SCL but not PU.1 while CD45− cells did not express any of these genes. Directly after sorting, CD45+HSC, but not CD45− cells contained clonogenic cells that gave rise to hematopoietic colonies in progenitor cell assays. Similarly, while fresh CD45+HSC were able to respond to exogenous hematopoietic cytokines including SCF, TPO, and FL in liquid suspension cultures as evidenced by expansion and differentiation, their CD45− counterparts failed to proliferate under these conditions and none survived beyond 7 days of culture. When transplanted competitively into lethally irradiated congenic recipients, only freshly isolated CD45+HSC sustained donor-derived hematopoiesis, whereas hematopoiesis in mice injected with freshly isolated CD45− cells was sustained long term by competitor cells and endogenous host-derived stem cells. Both groups of CD45+HSC and CD45− cells could be expanded on irradiated M210B4 stromal cells when supplemented with SCF, TPO, and FL, with CD45− cells giving rise to cobblestone foci of small, round translucent cells beginning on day 7 of culture. Cultured CD45+HSC continued to express CD45 and SCL and, depending on the length of culture, also expressed PU.1. Interestingly, after 15 days in culture, CD45− cells expressed CD45 by RT-PCR and FACS (in addition to Sca-1) and also expressed mRNA for SCL. Given the ability of CD45− cells to expand under these conditions and to acquire CD45 expression, we next compared the repopulating potential of fresh and cultured CD45+HSC and CD45− cells using lethally irradiated C57Bl/6 recipients. As expected, fresh CD45+HSC sustained donor-derived engraftment and culture of these cells over M210B4 for 15 days reduced their repopulating potential more than 7-fold. In contrast, CD45− cells maintained on M210B4 (the expansion equivalent of 750 cells seeded) contributed to hematopoietic engraftment, albeit at low levels (under 5% chimerism). These data demonstrate that CD45− Sca-1+ c-kit− cells may be marrow resident precursors of hematopoietic stem cells and suggest that early stages of the HSC hierarchy may include CD45− cells. Whether these CD45− cells also posses endothelial differentiation potential and can give rise to CD45+HSC in vivo is now under investigation.