ASH 2000 a



Cytomatrix212 West Cummings Park Woburn, MA 01801 *Phone:* 781-939-0995		*CD 34^-Lin*^- Cells Cultured in Lineage Specific Cell Growth Conditions Can Give Rise to Hematopoietic and Non hematopoietic Cells

		Naheed Banu, Julie Reitter, Erika von Schild, Kristen Biber, Michael Rosenzweig, Mark Pykett. Cytomatrix, Woburn, MA.
		We present new evidence showing CD34^-Lin^- cells have the potential to give rise to hematopoietic and nonhematopoietic cells ex vivo. The relative engraftment capability in NOD/SCID mice of the cultured and noncultured CD34^-Lin^- cells compared to similarly treated CD34⁺Lin^- cells was investigated. CD34^-Lin^- and CD34⁺Lin^- cells were isolated from adult human bone marrow and cultured in three different cell growth media which were specific for human bone marrow and cultured in three different cell growth media which were specific for hematopoietic progenitors cells (HPC-media), mesenchymal stem cells (MSC-media), neural stem cells (NSC-media). FACS analysis showed that after 3 or 4 weeks of culture, CD34^-Lin^- and CD34⁺/Lin^- cells cultured in HPC media were found to contain CD45⁺CD33⁺ and CD45⁺CD38⁺ hematopoietic progenitor cells. However only CD34^-Lin^- cells cultured in MSC^- media gave rise to mesenchymal cells (CD45^-Vimentin⁺), epithelial cells (CD45^-Cytokeratin⁺) and endothelial cells (vwf⁺). Similarly only CD34^-Lin^- cells cultured in neural growth medium had cells that expressed neurofilament. CD34^-Lin^- and CD34⁺/Lin^- cells cultured in HPC-media were observed to form hematopoietic colonies in standard methylcellulose assays whereas cells cultured in MCS or NSC media did not. To extend this work, bone marrow CD34^-Lin^- and CD34⁺/Lin^- cells cultured for 4 weeks in NSC-media were injected into irradiated NOD/SCID mice. 3 weeks post transplantation, mice were sacrificed and cells from the brain, muscle, thymus, spleen, and bone marrow were collected and stained with antibodies specific to human CD45 and MHC Class-1. Human cells (CD45⁺ or MHC Class 1⁺) were detected in the brain of the mice injected with CD34^-Lin^- cells, but not in other mouse tissues. PCR for human globin was performed which again confirmed the presence of human cells only in the brain of the CD34^-Lin^- cells transplanted mice. Freshly isolated CD34^-Lin^- failed to engraft when injected into NOD/SCID mice which may have been consequence of cell number. CD34⁺Lin^- cells, either freshly isolated or cultured in HPC-media were found to engraft in the bone marrow and spleen of the irradiated NOD/SCID mice. Interestingly, CD34⁺Lin^- cells cultured in NSC-media engrafted only in the bone marrow of the NOD/SCID mice, not in the brain. These results, indicate the organ specific homing of the injected cells and confirm our hypothesis that a common precursor present in CD34^-Lin^- cell population of adult human bone marrow has both hematopoietic and nonhematopoietic potential.