Functional Studies of Human Bone Marrow Derived Side Population Cells.

Rationale:

We have previously reported that staining of murine or human bone marrow with the vital dye, Hoechst 33342, identifies a rare population of dye-effluxing cells termed the “side population” (SP). In mice, competitive blood repopulation assays demonstrate that murine SP cells are highly enriched in hematopoietic stem cells (HSC). However, whether bone marrow SP cells in humans similarly possess functional stem cell characteristics is not currently known. We tested the stem cell potential of human SP cells in comparison to non-SP and AC133+ cells by transplantation of bulk or purified marrow sub-populations into irradiated NOD/SCID mice.

Methods:

Ficolled human bone marrow preparations were stained with Hoechst dye or a fluorescence-conjugated antibody against AC133. Flow cytometry was used to purify SP cells, non-SP cells designated as ‘MP”, AC133+ cells, or unfractionated mononuclear (MNC) cells. Several cell doses of each sub-population were intravenously transplanted into sub-lethally irradiated SCID/NOD recipient mice. Long-term bone marrow chimerism was analyzed 6–8 weeks post transplant to assess the SCID/NOD repopulating potential of each sub-population.

Results:

Transplantation of 1x10^6, 500, 000 and 100, 000 mononuclear cells resulted in detectable but low-level marrow chimerism 5.14%, 1.13%, 0.17%. Transplantation of same number of MP cells also resulted in low level of chemerism (3.88%, 1.20%, 0.67%) compared to SP cells. High level of chemerism was obtained with 40,000, 20,000, and 10,000 SP cells 7.09%, 1.4%, and 0.50% respectively. In comparison 40,000, 20,000, and 10,000 AC133+ cells resulted in 2.57%, 0.97, and 0.07% chimerism. To examine whether the ability of AC133+ cells to efflux Hoechst dye could better identify cells with HSC potential we transplanted 5,000 AC133+ SP cells vs. 5,000 AC133+ non-SP cells into SCID/NOD recipients and found that AC133+ cells that do not efflux Hoechst (non-SP) do not contribute to long-term marrow chimerism whereas AC133+ SP cells resulted in 0.63% chimerism.

Conclusion:

We show for the first time that human bone marrow SP cells possess functional hematopoietic stem cell characteristics. Similarly, all of the NOD/SCID repopulating activity of adult human bone-marrow AC133+ cells are found to be more enrich in the “SP”AC133+ fraction (0.1% SP) compare to total mononuclear, or “MP”AC133+ or total AC133+ cells.