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Cytomatrix |
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Differentiation of Hematopoietic Progenitor Cells Into Erythrocytes or Megakaryocytes After Maintenance in Serum Free Culture in a Novel 3-Dimensional Structure With or Without Low Levels of Cytokines |
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Naheed Banu, Erika von Schild*, Michael Rosenzweig*, Mark Pykett*.
Cytomatrix, Woburn, MA. |
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Described is the development of a novel three-dimensional culture system capable of producing lineage-committed progenitors and mature cells subsequent to maintenance in serum free media. Hematopoietic progenitor cells (CD34+) were maintained in either serum free cultures with or without low concentrations of SCF (500pg/ml) or TPO (500 pg/ml). All cultures were maintained in a three-dimensional porous biomatrix termed CellfoamTM. At 2 week intervals, non-adherent (NA) or total cell populations (TC) were isolated separately from parallel CellfoamTM cultures. Each cell population was induced to differentiate along the erythrocyte or megakaryocytic lineages in media supplemented with 1% autologous plasma and lineage specific growth factors. After 2 weeks in differentiation media, cultures were harvested and phenotypic analysis of the cells was done by flow cytometry. TC cultures maintained in SCF supplemented serum free media for two weeks followed by two weeks of direct differentiation for erythrocyte exhibited a 375 fold expansion compared to maintenance control cultures. Similarly, after 4 weeks of maintenance followed by two weeks differentiation we observed a 555 fold expansion in erythrocytes progeny compared to the 4 week maintenance alone control cultures. TC cultures maintained in SCF supplemented serum free media for two weeks followed by two weeks of differentiation for megakaryocytes exhibited a 13 fold expansion of megakaryocyte progeny compared to 2 weeks maintenance control culture and also at 4 weeks, the SCF supplemented maintenance cultures showed 13 fold expansion compared to its maintenance control culture. Interestingly, maintenance cultures supplemented with TPO showed a higher number of megakaryocytes progenitors compared to 4 week of any other differentiation cultures. In megakaryocyte colony assays of the same cell populations we observed a similar enrichment of megakaryocytes. These study shows that the multipotency of the hematopoietic progenitor cells can be maintained in serum free media in this novel three dimensional matrix for extended periods of time and furthermore can be subjected to differentiation along a number of different pathways.   |
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