ASH 1999 b



Cytomatrix 212 West Cummings Park Woburn, MA 01801 *Phone:* 781-939-0995		*Targeted Differentiation of CD34+ Progenitors in a 3-Dimensional Matrix*

		Naheed Banu, Michael Rosenzweig, Mark Pykett, Cytomatrix, Woburn, MA
		In this study, we describe the effects of culture conditions and serum on the growth factor dependent expansion of CD34⁺ cells into erythroid and megakaryocyte lineages. We have developed a long-term bone marrow bioreactor system that mimics the bone-marrow microenvironment in which CD34⁺ cells are cultured in an inert, three-dimensional porous biomatrix termed Cellfoam^TM (CF). Our previous study of short-term culture of CD34⁺ cells showed that culture in Cellfoam^TM improved both the maintenance and multipotency of hematopoietic stem cells compared to cells cultured on bone marrow stroma or fibronectin coated plastic dishes. Using variable combinations of lineage specific cytokines, we now demonstrate that the cell expansion and differentiation is higher in serum free media in Cellfoam^TM compared to controls. To achieve terminally differentiated progeny from CD34⁺ cells, we used a combination of EPO⁺IL^-3⁺SCF for erythroid differentiation and TPO⁺IL^-6 or TPO⁺IL^-3⁺SCF for megakaryocytic differentiation in both serum free media (Stemspan) and serum containing media (RPMI with 10%FCS) for 13 weeks of culture. After 13 weeks of culture, we observed a 5 fold increase in CD45⁺CD34⁺ cells in serum free cultures in Cellfoam^TM compared to serum containing cultures in the presence of TPO containing supplements, but not EPO containing supplements. A similar observation was made when comparing Cellfoam^TM to plastic cultures, where a cultures in serum free environment. The highest number of mature red cell progeny (CD45^-Cd34^±CD71⁺) cells were obtained form 6 to 13 weeks in Cellfoam^TM cultures in a serum free environment compared to serum containing cultures in Cellfoam^TM or serum free cultures in plastic. Cells cultured in serum free media both in Cellfoam^TM and plastic demonstrated erythroid colonies even after 13 weeks of cultures, whereas no erythroid colonies were seen in serum containing cultures after 6 weeks. Higher numbers of megakaryocyte cells (CD45⁺Cd34^±CD61⁺) cells were observed at 9 and 13 weeks in serum free Cellfoam^TM cultures supplemented with TPO⁺IL^-6 and TPO⁺IL^-3⁺SCF cytokine combinations. Cellfoam^TM cultures both in serum free and serum containing media supplemented with TPO⁺IL^-3⁺SCF cytokine combinations showed 8 fold higher number of CD45⁺CD34^±CD61⁺ cells than TPO⁺IL^-6 cytokine combinations at the corresponding weeks of cultures. Megakaryocyte colony assays were performed after 2 weeks of cultures and the high test number of CFU^-MK were obtained in serum free Cellfoam^TM cultures with the addition of TPO⁺IL^-3⁺SCF. The structure of Cellfoam^TM appears to augment the terminal maturation process of megakaryocytes by providing a three-dimensional matrix for their proplatelet formation. Our data, which demonstrated that serum free media enhanced megakaryocytopoiesis, is also in agreement with previous findings about the inhibitory effects of serum on the proliferation of megakaryocyte progenitors. All these findings indicate that the three dimensionality of Cellfoam^TM assists stem cell maintenance, as well as the terminal differentiation of progenitor cells with the addition of appropriate growth factors.