(236g) Hydrogel Systems to Examine Diffusion-Mediated Paracrine Signaling On Hematopoietic Stem Cell Fate

Introduction: The hematopoietic stem cell (HSC) niche in the bone marrow (BM) contains cells, the extracellular matrix (ECM), and ECM-bound or soluble biomolecules that present chemotactic, durotactic, and paratactic cues as well paracrine and juxtacrine signals to drive HSC fate. This niche regulates HSC fate decisions and hematopoiesis, the physiological process responsible for generating the body’s blood and immune cells. While multiple cell types have been proposed as key regulators of HSC fate, the direct influence of heterotypic cell interactions via paracrine and juxtacrine signaling remain relatively unknown. Here we investigate the influence of local environment on HSC differentiation and proliferation through feeder cell populations at varying ratios in 2D and 3D cultures. Culture analysis provides key information regarding the regulatory mechanics of stem cells under different environments as well as the driving forces determining stem cell differentiation or maintenance. Using a microfluidic platform we have also created microgels containing counter gradients of cell, matrix and biomolecules. These 3D gradient environments will be used to investigate roles crucial biomolecules play in HSC maintenance and mobilization, mimicking in vivoconditions of the active and quiescent BM niches.

Materials and Methods: Murine hematopoietic stem/progenitor cells (HSPCs) were isolated via FACS as Lin^-ckit⁺Sca1⁺ (LSK) or LSKCD34^- BM cells. HSPCs were co-cultured with Lin⁺BM cells under liquid as well as collagen and gelatin hydrogel cultures. Hydrogel density as well as both HSC density and the HSPC:niche cell ratio were varied to alter cell-cell communication. Using liquid co-culture as a non-limited diffusion control, the cell-laden microgels were cultured up to 7 days. HSPC proliferation was tracked via a fluorescent cell division assay while post culture HSPC fate was analyzed via FACS and colony forming unit (CFU) assays. A previously described microfluidic mixing device was used to create counter-gradient hydrogels of SCF-functionalized Gelatin with HSPCs. The effect of mobilized vs. immobilized SCF, a critical stem cell regulator, was analyzed. Systematically varying SCF dosage and introducing Mesenchymal stem cells (MSCs) as extrinsic cell regulators provides a rich landscape for examining the functional role of paracrine signaling on HSC fate.

Results and Discussion:FACS analysis indicates significant difference in HSPCs quiescence/maintenance (LSK) vs. myeloid lineage (CMP) differentiation due to both gel environment and niche cell co-culture. HSPCs showed increased specification towards myeloid lineages with increasing Lin+ niche cell co-culture. Significant differences were also observed between liquid and hydrogel conditions, with reduced niche cell effects observed in hydrogel environments that limited biomolecule diffusion. Cell viability was not acutely affected by hydrogel polymerization while higher HSPC viability and expansion (~50%) was observed for smaller gel volumes (50µL vs. 100µL). These results suggest a strong influence of niche cell interactions on HSPC fate. Early results indicate that SCF-functionalized gelatin maintained bioactivity up to 7 days. HSPC proliferation was noted to be higher when cultured in a SCF-functionalized gelatin as opposed to a soluble SCF environment.

Conclusions:We have established a biomaterial platform to explore the influence of niche cell mediated paracrine signaling on HSPC fate decisions. Ongoing experiments are examining longer culture periods and incorporating increasingly primitive niche cell populations (vascular endothelial cells, mMSCs) to alter the balance between lineage specification and quiescence. Counter gradients of quiescent and active niche populations are being incorporated into a single heterotypic microgel environment as an ectopic BM culture mimic to examine the dynamics of HSC fate decisions.

Acknowledgements: American Cancer Society, Illinois Division, Inc. (#160673, #189782), NSF #1254738