Human progenitor T-cell differentiation regulated by the mechanical resistance of thymus-mimetic extracellular matrices

Nicholas Jeffreys(Harvard University Press), Kyle T Ruark(Harvard University Press), Joshua Price(Harvard University Press), Ella M Serrano-Wu(Harvard University Press), Blake F. Hanan(Harvard University Press), Andrew Khalil(Harvard University Press), Wei‐Hung Jung(Harvard University Press), Nuria Lafuente‐Gómez(Harvard University Press), Joshua M. Brockman(Harvard University Press), Andrew Lu(Harvard University Press), Izabela Zmirska(Harvard University Press), Kyle H. Vining(Harvard University Press), Junzhe Lou(Harvard University Press), Kwasi Adu‐Berchie(Harvard University Press), Siyoon Kwon(Harvard University Press), Hamza Ijaz(Harvard University Press), Azeem Sharda(Harvard University Press), David T. Scadden(Harvard University Press), David Mooney(Harvard University Press)
bioRxiv (Cold Spring Harbor Laboratory)
August 27, 2025
10.1101/2025.08.27.671384
Cited by 0Open Access
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Abstract

Abstract Therapeutic T-cell engineering ex vivo from human hematopoietic stem cells (HSCs) focuses on recapitulating notch1-signaling and α4β1-integrin-mediated adhesion within the thymic niche with supportive stromal cell feeder-layers or surface-immobilized recombinant protein-based engineered thymic niches (ETNs). The relevant Notch1-DLL-4 and α4β1-integrin-VCAM-1 interactions are known to respond to mechanical forces that regulate their bond dissociation behaviors and downstream signal transduction, yet manipulating the mechanosensitive features of these key receptor-ligand interactions in thymopoiesis has been largely ignored in current ETN designs. Here, we demonstrate that human T-cell development from cord blood-derived CD34 + HSCs is regulated via molecular cooperativity in notch1 and integrin-mediated mechanotransduction. Mechanically confining interpenetrating network (IPN) hydrogel-based 3D cell culture comprised of collagen type I and alginate polysaccharides functionalized with DLL-4 and VCAM-1 is used as a model viscoelastic 3D ETN to manipulate human progenitor (pro)T-cell differentiation. This ETN enables orthogonal control of the mechanical and biomolecular features of the thymic niche, including thymopoietic ligand density, modulus, and viscoelastic properties (e.g., stress relaxation kinetics). We identify that soft, viscous matrices that enhance activation of the notch1-pathway, and subsequently notch1 intracellular domain (NICD) nuclear import sustain the T-cell development gene regulatory network during proT-cell differentiation. Conversely, stiff, elastic matrices inhibit HSC commitment to the T-lineage, and rather promotes Myeloid-cell differentiation. Our observations indicate mechanical reciprocity in signaling pathways indispensable to thymopoiesis, and highlights extracellular matrix mechanics as a variable in controlling hematopoietic stem cell fate decisions.

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