Poster BB1 - “Under pressure”: Understanding How Hydrostatic Pressure Influences the Stromal-Hematopoietic Stem Cell Interplay

Introduction: Hematopoietic stem cell (HSC) culture systems have become a highly desirable goal of biomedical research due to the globally urgent need for transplantable HSCs. While numerous biological/biochemical modulators of HSC activity have been identified to support the development of such systems, mechanical regulators of hematopoiesis remain poorly understood. Moreover, while systemic blood pressure is in the range of 110-140 mmHg, reports have indicated a unique hydrostatic pressure (HP) of approximately 30 mmHg within the bone marrow space. Therefore, our objective is to investigate the influence of HP as a mechanical regulator of cell proliferation and development, focusing on the stromal component in the absence and presence of HSC. Specifically, we aim to define how HP regulates mesenchymal cell behavior to promote the BM HSC niche compartment. We hypothesize that increased HP will influence mesenchymal stem cell (MSC) stemness/differentiation properties and HSC interplay in-vitro.

Materials and

Methods: A HP bioreactor was constructed through controlled air compression within a
sealed acrylic chamber. To investigate the influence of HP on MSC properties, P5 murine MSCs were monocultured in Lonza Ex Viva 15-based basal medium at 34x103 cells/cm2 or 8.5x103 cells/cm2 and subjected to 0 or 120 mmHg continuous pressure for 14 days on TCPS before enzymatic detachment. Subsequently, MSC were cocultured with murine hematopoietic progenitors, isolated from the bone marrow of 5–10-week-old B6 mice, (1 HSC: 10 MSC) in a similar medium supplemented with 100 ng/mL Flt3L, 100 ng/mL SCF, and 100 ng/mL TPO and subjected to 0 or 120 mmHg pressure for 14 days on TCPS before mechanical detachment to examine HSC expansion/stemness. Cell culture was performed in humidified incubators supplying 5% CO2, 2% O2, and 37°C.

Results, Conclusions, and Discussions: MSCs subjected to 120 mmHg pressure presented with circular cell morphology after 10 days in culture. At day 14, 34x103 cells/cm2 seeded MSCs subjected to 120 mmHg pressure exhibit elevated expression of stemness/chondrocyte marker CD44, while 8.5x103 cells/cm2 seeded MSCs showed decreased expression of stemness marker CD73. Altogether, these preliminary data suggest that HP cues differentiation of MSCs. MSC and MSC/HSC samples are now being assessed for differences in hematopoietic coupling factor expression and hematopoietic expansion/stemness, respectively.

Ultimately, the findings in this work provide valuable insights into the role of HP in regulating MSC properties, and potentially MSC-HSC interplay.

Acknowledgements (Optional): We would like to acknowledge the NIDCR Cytometry Core for cytometric resources, UMD Terrapin Works for machining services, and Drs. Kathryn Futrega/Pamela Robey for supplying the O2 controlled incubator. All graphics were created using BioRender.