A bioprinted silk marrow niche reveals mechanical regulation of human megakaryopoiesis under genotoxic stress

Hematopoietic stem and progenitor cells (HSPCs) reside in a mechanically distinct bone marrow niche, yet how niche biomechanics shape genome stability and stress responses has been difficult to test because conventional two-dimensional (2D) culture lacks marrow viscoelasticity and uses surfaces that activate platelets, confounding hematopoietic readouts. Here, we show that this methodological gap has masked a basic principle: the marrow niche actively constrains genotoxic stress signaling in HSPCs, and 2D culture systematically overstates DNA damage and impairs differentiation in vitro. We engineered silk fibroin, a biologically inert biomaterial that does not activate platelets and recapitulates marrow viscoelasticity, into SilkInk, a 3D-bioprintable bioink, and used it to reconstruct a biomimetic marrow microenvironment. HSPCs encapsulated in SilkInk preserved clonogenic potential and multilineage differentiation, whereas 2D-cultured HSPCs activated cytoskeletal-tension and genome-surveillance programs characteristic of chronic stress, including pathways related to replication stress, DNA damage response, and redox stress. Cell phenotyping and single-cell RNA sequencing during megakaryopoiesis revealed that SilkInk supported coordinated endomitotic progression and terminal maturation, with progression from CD34+CD61-CD41-CD42b- progenitors to CD34-CD61+CD41+CD42b+ megakaryocytes, including increased 8N and >16N populations, whereas 2D culture and conventional 3D hydrogels sustained DNA damage signaling and impaired thrombopoiesis. The same hierarchy held under cytotoxic challenge, as 5-fluorouracil amplified DNA damage and crippled platelet output in 2D, whereas SilkInk-encapsulated HSPCs maintained differentiation, mirroring native marrow resilience. These findings reposition niche mechanics as an active determinant of hematopoietic genome stability and establish SilkInk as a physiologically faithful platform for studying hematopoiesis and predicting marrow responses to chemotherapy.