(805a) Biophysical and Biochemical Cues Regulate the Expansion and the Differentiation of Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are adult stem cells with the capacity to give rise to all blood and immune cells in the body. They reside in a specialized microenvironment in the bone marrow (BM) known as the stem cell niche, which presents cellular (niche cells), biochemical (cytokines, growth factors, signaling molecules), and mechanical signals believed to impact HSC fate decisions. Previous in vivo and in vitro studies have suggested potential HSC niche constituents and regulatory signals, but the exact mechanisms by which they work to modulate HSC fate remain elusive. In particular, little is known about how niche-mediated biophysical and biochemical cues (i.e., stiffness, ECM ligand presentation) directly impact early HSC fate decisions such as self-renewal, proliferation, and differentiation. Inspired by gradations in ECM protein content and mechanical properties across the bone marrow, we created an in vitro culture platform using mechanically-tunable polyacrylamide (PA) gels and decorated them with ECM ligands (type I collagen, fibronectin, laminin) to examine early HSC fate decisions as a function of the biophysical and biochemical environment.

As a first step, we created PA gels with varying stiffness (moduli: 3.7kPa, 44kPa, 227kPa) and uniformly coated the gel surface with type I collagen, fibronectin, or laminin. HSCs (LSK: Lin^-cKit⁺Sca1⁺) isolated from murine tibial and femoral BM via fluorescence-activated cell sorting (FACS) were cultured on top of these substrates for 24h, with changes in viability, morphology, proliferation, and functional capacity (via colony-forming unit (CFU) assays) compared to freshly isolated HSCs. Cultured HSCs showed significant changes in their spreading (size) and morphology (shape) with increasing substrate stiffness and/or ligand density (1). We subsequently examined changes in HSC phenotype and proliferation via flow cytometry. Although the majority of the cells were still Lin-, about half of the cells were no longer LSK cells and they were actively proliferating. Examining the lineage commitment state of the cultured HSCs, colony-forming unit (CFU) assays indicated significantly different colony-forming patterns compared to freshly isolated HSCs. Notably, overall increases (CFU-M, -E, -Mk) and decreases (CFU-G), were observed for some differentiated myeloid colonies. Most interestingly, a fibronectin-mediated, substrate stiffness-dependent increase in early myeloid progenitors (CFU-GEMM) was observed. Critically, inhibiting myosin II (via blebbistatin) or α5β1 integrin (via α5β1 antibody) negated this effect but did not impact other CFU assay results, suggesting HSC mechanotransduction impacts early fate decisions. Our findings suggest that stiffness and ligand presentation impact HSC morphology, viability, bioactivity in myosin-II and integrin dependent manner, suggesting design parameters for biomaterials targeting the expansion and differentiation of HSC ex vivo.  

To build on this PA gel culture platform, we fabricated polydimethylsiloxane (PDMS) stamps with circular posts (<=50μm in diameter) to microcontact print protein domains via EDC/NHS chemistry. Separately, we also created PA gels with a stiffness gradient (1-50kPa) using a UV photoiniatior. With these additional design features, we can better mimic the physiologically relevant mechanical and structural gradients in the bone marrow HSC niches. Currently, we are investigating the effect of substrate stiffness, protein concentration, and cell seeding density on the expansion and the differentiation of HSCs on each of these substrates. With further advancement, we plan to combine the different design parameters of the PA gels to construct an artificial HSC niche dictated by HSC-material interactions.

References: 1.           Choi JS & Harley BAC (2012). Biomaterials 33(18):4460-4468.