(453f) Hydrogel Systems to Examine Diffusion-Mediated Paracrine Signaling On Hematopoietic Stem Cell Fate

                                           Bhushan Mahadik^*, Dr. Sara Pedron^*, Dr. Paul Kenis^*,#, Brendan Harley^*,#

^*Dept. of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801

^#Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801

Introduction: The stem cell niche provides extrinsic signals that regulate stem cell fate. 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. We have developed a microfluidic platform to create microgels containing counter gradients of cell and matrix environments to determine differences in primary mHSC fate decisions in response to niche cell co-cultures using monolithic and counter-gradient 3D hydrogel environments designed to alter diffusion-mediated paracrine signaling.

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, gelatin, and HA hydrogel cultures; hydrogel density as well as both HSC density and the HSPC:niche cell ratio (1:10, 1:100, 1:500) were varied to alter cell-cell communication. Cell-laden microgel constructs were polymerized thermally or via UV exposure and cultured over a period of 7 days with liquid co-culture used as a non-limited diffusion control. Hydrogel mechanical and diffusive properties were determined via compression and FRAP assays, respectively. HSPC proliferation was tracked via a fluorescent cell division assay while post culture HSPC fate was analyzed via FACS and gene expression analysis. A previously described microfluidic mixing device was used to create counter-gradient hydrogels comprised of HSPCs as well as multiple niche cells. Systematically varying HSPC:niche cell ratio as well as hydrogel type and density provides a rich landscape for examining the functional role of paracrine signaling on HSC fate.

Results and Discussion:Cell viability was not acutely affected by hydrogel gelation; higher HSPC viability and expansion (~50%) was observed for smaller gel volumes (50µL vs. 100µL). After 2 days of culture, 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. These results suggest a strong influence of niche cell interactions on HSPC fate.

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).