The Impact of Developmental Stage of Osteoblasts and Collagen Fibril Matrix Properties On Hematopoietic Stem Cell Function.

Abstract 3638

Poster Board III-574

Previously we reported the impact of hematopoietic niche (HN) elements, calvaria-derived osteoblasts (OB) and bone marrow derived stromal cells (SC), on murine hematopoietic stem cells (HSC, defined as Lin-sca1+c-kit+ or LSK) cultured in a conventional two-dimensional (2D) format. Interestingly, we found that OB maintained the functional properties of LSK cells including their long-term marrow repopulating potential significantly better than SC thus corroborating the importance of OB in the overall competence of the HN. We also reported that this stem cell function-enhancing activity is induced by the up regulation of Notch-mediated signaling between HSC and OB. However, which specific lineage or developmental stage of OB enhances HSC function is still unknown. In the present study, we evaluated the interactions between HSC and OB at various stages of development. We also examined the impact of physical parameters of the extra cellular matrix, such as stiffness/storage modulus using a more physiologically relevant 3D context consisting of collagen-fibril matrices in which the fibril density and shear storage modulus was systematically varied. OB were harvested from murine calvaria and used directly (fresh OB) or were cultured for 1, 2, or 3 weeks in basic or complete medium (supplemented with ascorbic acid and β-glycerophosphate). A total of 1000 LSK cells were co-cultured for 1 week with either fresh OB or OB cultured for 1, 2, or 3 weeks. Cultures were examined for hematopoietic cell number increase, colony forming unit (CFU) expansion, LSK phenotype, and plating efficiency. In addition, OB function was analyzed by measuring alkaline phosphatase activity and calcium deposition/mineralization. Surprisingly, total hematopoietic cell number on day7 was significantly higher in cultures containing fresh OB (34.5±3.3×10⁵) compared to OB cultured for 1 week (12.4±4.0 ×10⁵, p=0.05), 2 weeks (9.8±4.6 ×10⁵, p=0.03), or 3 weeks (7.8±3.4 ×10⁵, p=0.02). Total CFU fold expansion was also significantly elevated in fresh OB co-cultures (149.6±45.7) vs OB cultured for 1 week (62.4±18.7, p=0.04), 2 weeks (51.0±14.5, p=0.01), or 3 weeks (52.4±23, p=0.03). The percentage of Lin-Sca1+ cells was also significantly higher in co-cultures of fresh OB (29.5±3.6%) vs OB cultured for 1 week (5.3±0.9, p=0.02), 2 weeks (0.9±0.2, p=0.008), or 3 weeks (1.0±0.2, p=0.008). Interestingly, alkaline phosphatase activity and calcium deposition were inversely associated with all hematopoietic properties examined. In order to examine the significance of 3D spatial and physical properties of the extra cellular matrix microenvironment, which is absent in traditional culture systems, LSK cells were cultured in both the presence and absence of calvariae-derived OB within 3D engineered collagen type I matrix constructs in an effort to recreate the in vivo niche conditions. Matrix parameters, including collagen fibril density (9-20%) and shear storage modulus (50-800 Pa, G') were systematically varied and quantified. LSK proliferated most rapidly when co-cultured with OB in low fibril density/G' (50 Pa) collagen type I matrices (836.8±74 fold-increase) compared to 200Pa (172.3±26, p=0.007) or 800Pa (128.4±27, p=0.006). Proliferation of LSK cells in matrices not containing OB was minimal. Interestingly, the clonogenicity of cells harvested from 800Pa matrices was nearly 3-fold and 1.5-fold higher than that for cells from 50Pa and 200Pa matrices, respectively. Furthermore, 800Pa matrices maintained the highest percentage of Lin-Sca1+ cells for 7days (15.9±7.3) compared to 200Pa (9.3±3.5) and 50Pa (6.9±1.5) matrices. These data illustrate that collagen matrices with relatively high fibril density/G' suppressed rapid proliferation and expansion and maintained progenitor cell function. Alternatively, low stiffness matrices promoted cell expansion at the apparent expense of differentiation and loss of clonogenic potential of progenitor cells. Collectively these results demonstrate that early stage OB maintains HSC function significantly better than later stage OB. Furthermore, physical properties such as fibril density and shear storage modulus (stiffness) of the surrounding collagen fibril matrix also play a critical role in HSC proliferation and maintenance of stem cell pool.