Silk Bone Marrow Niches to Model Fibrosis and Altered Megakaryopoiesis in Myeloproliferative Neoplasms

The bone marrow (BM) niche is a complex microenvironment that supports hematopoietic stem and progenitor cells (HSPCs). Mesenchymal stem cells (MSCs) are essential components of this niche, maintaining the BM microenvironment, regulating HSPC functions and ensuring effective blood cell production. Both cell types are embedded within the extracellular matrix (ECM), which provides mechanical properties such as stiffness and elasticity, crucial for regulating cellular behavior. These physical and chemical characteristics of the BM are critical considerations when developing three-dimensional (3D) models of the BM. Modeling dysfunction in the human BM is particularly challenging, especially in the context of diseases. BM fibrosis, characterized by excessive and uncontrolled ECM deposition, leads to the formation of a fibrous tissue. Myeloproliferative Neoplasms (MPNs) are HSPC-driven cancers where myelofibrosis is triggered by elevated levels of transforming growth factor-β1 (TGF-β1) in the BM microenvironment. This elevation promotes the transition of MSCs to myofibroblasts, which contribute to the deposition of ECM components, thereby disrupting the niche's physiological architecture and function. Current methods for studying the BM niche often lack the ability to incorporate and regulate biophysical and biochemical cues, such as a controlled ECM and cellular environment. We used natural silk fibroin biomaterial from Bombyx mori silkworm cocoons to fabricate 3D scaffolds mimicking the human BM niche. The scaffolds were engineered with adjustable features including stiffness and bioactive ligand distribution. The elasticity (Young's modulus) of the 3D silk scaffold was set at less than 40 kPa, aligning with the native BM environment and significantly softer than the 1 GPa Young's modulus of standard 2D cell culture plastic plates. This configuration promoted optimal MSC engraftment and customizable ECM deposition. RNA-seq analysis of MSCs demonstrated that the more physiological 3D silk model, compared to 2D cultures, led to distinct cellular responses. In 2D cultures, there was an upregulation of genes associated with the synthesis of collagen-containing ECM components (e.g., COL1A1; COL4A1; COL4A2; COL8A2) and collagen fibril formation (e.g., LOX; LOXL4; SPARC; PXDN). Furthermore, 2D cultures also showed an upregulation of genes typically associated with pro-fibrotic processes (e.g., ACTA2, ACTG2, CCN2, TGFB1). This suggests that the 2D cultures induce a pro-fibrotic phenotype, while the softer 3D silk microenvironment prevents MSCs from transitioning into myofibroblasts. We then functionalized the 3D silk scaffolds with TGF-β1. The slow release of this cytokine over a 2-week culture period induced MSCs to transition into α-smooth muscle actin (α-SMA)+ myofibroblasts. This transition was characterized by the upregulation of smooth muscle-related genes (e.g., TPM1, CALD1, MYL9, MYH9) and ECM synthesis-related genes (e.g., collagens, elastic fibers, proteoglycans), as well as increased deposition of reticulin, and fibronectin extra domain A (EDA-FN). These fibrotic markers are similarly elevated in the bone marrow of MPN patients with overt fibrosis. BM fibrosis in MPN is associated with abnormal megakaryocytes. To determine if the 3D silk model could effectively simulate this condition, we cultured human HSPCs in the 3D silk scaffolds with thrombopoietin. This setup resulted in enhanced differentiation into β1-tubulin+ CD42b+ megakaryocytes, which could produce highly branched proplatelets and platelets. However, when healthy HSPCs were cultured in the modified niche, there was a marked impairment in thrombopoiesis. This impairment was characterized by the proliferation of small megakaryocytes that were unable to elongate proplatelets or undergo platelet shedding under ex vivo perfusion conditions. BM HSPCs from selected MPN patients were cultured in either ‘physiologic’ or ‘fibrotic’ silk scaffolds. The ‘fibrotic’ niche showed dysmorphic megakaryocytes and defective proplatelet formation, while the ‘physiologic’ silk scaffold demonstrated improved platelet production capacity. These findings underscore the crucial role of a well-regulated BM niche environment in understanding HSPC-niche interactions. Such insights are vital for elucidating the conditions associated with abnormal megakaryopoiesis and bone marrow fibrosis.