(251e) Biomaterial Systems to Assess the Influence of Cell-Cell and Cell-Matrix Interactions On Hematopoietic Stem Cell Biology

Hematopoietic stem and progenitor cells (HSPCs) are an ideal platform for studying extrinsic cues on stem cell behavior and for developing material systems than can act as stem cell niche analogs. Regulation of HSPC fate decisions (quiescence, self-renewal, differentiation) is thought to be partially regulated via extrinsic (cell-cell, cell-matrix, soluble regulator) cues presented by distinct microenvironments, termed niches, in the bone marrow. Here we describe development of biomaterial systems that enable independent assessment of the effects of cell-matrix and cell-cell cues on HSPC biophysical parameters and early fate decisions (proliferation, viability, differentiation). For these experiments we isolated HSPCs as Lin^-cKit⁺Sca1⁺ (LSK) cells from murine tibial and femoral bone marrow via flow cytometry.

We created type I collagen coated (100μg/ml), two-dimensional polyacrylamide (PA) gels and three-dimensional type I collagen hydrogels (1.45, 2.9 mg/ml) to assess the effect of matrix stiffness and dimensionality on HSPC cytoskeletal (CSK) organization. Two collagen hydrogel densities (1.45, 2.9 mg/ml) were used with elastic moduli of 24.3 ± 6.6 and 41.7 ± 12.5 Pa as characterized via rheology. Three PA gels were created with elastic moduli ranging from 150 Pa to 11kPa to 2 MPa, as determined via AFM. HSPCs were cultured within or on top of the 3D collagen hydrogels as well as on top of the 2D PA gels for up to 32 hours, at which point HSPC CSK organization was quantified via fluorescence microscopy. No effect of substrate elasticity or dimensionality was observed on HSPC CSK organization; as expected, significant differences in cell spreading and CSK organization was observed for MC3T3-E1 osteoblasts cultured on the identical substrates. Throughout, HSPCs maintained an amorphous CSK organization; these results parallel recent findings in the literature regarding embryonic stem cell CSK organization. Ongoing work is characterizing HSPC viability, mitosis rates, surface antigen expression profiles (Lin, Flk2), as well as changes in HSPC stiffness/softness in response to these defined 2D and 3D matrix environments.

To quantify the significance of HSPC interactions with a putative niche cell, osteoblasts, we have developed a mould capable of creating collagen hydrogels with uniform and linear gradient, both single and multiple, opposing gradients, properties. Cells within these hydrogels can be imaged via conventional fluorescence microscopy (Fig. 1). We have cultured MC3T3-E1 osteoblasts and primary HSPCs (LSK cells) for up to 24 hours within 2mg/ml collagen hydrogels while maintaining >50% viability; using spectral deconvolution (Zeiss LSM 710 Multiphoton Confocal Microscope) we can assess cKit and Sca1 surface antigen expression over time using the original flow cytometry antibodies (Fig. 1). After extended culture, distinct regions of the gradient hydrogels can be isolated, allowing quantitative analysis of differential rates of HSPC viability and mitosis. Ongoing work is quantifying frequency differences in HSPC viability proliferation, and surface antigen expression profiles (cKit, Sca1, Lin, Flk2), in collagen hydrogels with opposing linear gradients of HSPCs and osteoblasts, thereby creating defined ratios of HSPCs:osteoblasts in distinct gel regions.

Fig. 1. (top) Mould capable of creating gradient populations of HSPCs, niche cells, and hydrogel matrix environments. (bottom) Spectral deconvolution allows confocal imaging of primary HSPCs via flow cytometry antibodies within the collagen hydrogel mould: Lin^-, cKit⁺ (APC, red), Sca1⁺ (PE, yellow).