Mechanical plasticity of notch1 and α4β1 integrin ligand functionalized interpenetrating network hydrogels regulates T-cell lineage specification and expansion from human hematopoietic stem cells

Introduction:: Therapeutic T-cell engineering in vitro from human primary hematopoietic stem cells (HSCs) has relied on recapitulating critical molecular features of the thymus using either feeder stromal cell-based systems that ectopically express notch1 ligand delta-like ligand-4 (DLL-4) or surface immobilized DLL-4 and vascular adhesion protein-1 (VCAM-1). Current in vitro systems do not account for the importance of biophysical cues in guiding T-cell development, as T-cells are subjected to mechanical cues (e.g., stiffness) during their development, activation, and function. We report mechanically tunable interpenetrating network (IPN)-hydrogels functionalized with DLL-4 and VCAM-1 to promote T-cell differentiation from HSCs in 3-dimensional, mechanically confined cell culture. We additionally report a computational model of notch1/α4β1 integrin mechanical proofreading to explore in silico the effects of hydrogel mechanics on the production of transcription factors critical to T-cell development.

Materials and Methods:: Tz-polymer synthesis: Tz-VLVG alginates were synthesized using EDC/NHS carbodiimide coupling and subsequently dialyzed with a decreasing salt gradient followed by freezing and lyophilization for long-term storage at -20C. Preparation of DLL-4/VCAM-1 functionalized IPN-hydrogels: TCO-PEG5-DLL-4 and TCO-PEG5-VCAM-1 (5:1 mass ratio) was added to 1.0% -1.5% w/v of Tz-VLVG alginate with either TCO-PEG5k-methoxy, no covalent crosslinker, or bis-functional Nb-PEG4-Nb to fabricate viscous, intermediate relaxing, or elastic IPN-hydrogels. Pre-gels were mixed with collagen I (2 mg/mL), calcium carbonate nanoparticles, and GDL (1:4 molar ratio) to form IPN-hydrogels. Cell culture: CD34+ cord blood-derived HSCs were cultured in 3D IPN-hydrogels at a density of 2.5e6 cells/mL in a T cell differentiation media containing penn/strep, BIT 9500, b-ME, L-aa, SCF, Flt3L, IL-3, IL-7, TNF-a, and CXCL12 over the course of 14 days. Cells were characterized by flow cytometry. Modeling: Performed with MATLAB.

Results, Conclusions, and Discussions:: Herein, we report mechanically tunable interpenetrating network (IPN)-hydrogels functionalized with DLL-4 and VCAM-1 for HSC encapsulation in 3-dimensional, mechanically confined cell culture, and a computational model of notch1/α4β1 integrin mechanical proofreading to define key mechanical features critical to promoting T-cell differentiation in vitro. In silico, we find that the production of notch1 intracellular domain (NICD) transcription factors critical to T-cell differentiation and their nuclear import has a biphasic dependency on mechanical parameters such as modulus, stress relaxation, and viscosity. In vitro, we observe that stiff, elastic, IPN-hydrogels inhibit thymocyte differentiation, while softer, viscous, IPN-hydrogels promote thymocyte differentiation and proliferation. Our results suggest a critical role for biophysical cues in T-cell development in vitro and the importance of manipulating notch1 and α4β1 integrin mechanotransduction in 3-dimensional cell culture.

Acknowledgements (Optional): : We thank Dr. Bryan A. Nerger (Harvard University), Dr. John T. Ngo (Boston University), Dr. Nathaniel Huebsch (Washington University in St. Louis), Dr. Tetsuhiro Harimoto (Wyss Institute), Dr. Yoav Binenbaum (Wyss Institute), and Mason Dacus (Harvard University) for scientific discussions. We thank Maurice Perez, Eric Zigon, and Michael Carr of the Wyss Institute for technical staff support. We thank the Harvard John A. Paulson School of Engineering and Applied Sciences Active Learning Laboratory and Makerspace for technical staff support. We thank the Center for Macromolecular Interactions at Harvard Medical School for technical staff support. We thank the Center for Nanoscale Systems at Harvard John A. Paulson School of Engineering and Applied Sciences for technical staff support. We thank the Dana-Farber Cancer Institute’s Pasquarello Tissue Bank for providing cord blood samples for HSC isolation. We especially hold great appreciation for the patients who donated these aforementioned samples. We acknowledge funding from the NSF (MRSEC), Wellcome Leap Fund (HOPE), and NIH/NCI (i3 center).

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