Ex Vivo Bone Marrow Model for Investigating Platelet Production in Physiological and Pathological Conditions

RNA-seq analysis of MSCs revealed that as the stiffness of the 3D silk model increased, the cells' behavior became more similar to that seen in traditional 2D cultures. In the stiffer 3D cultures, there was an upregulation of genes involved in the synthesis of collagen-containing extracellular matrix components (e.g., COL1A1, COL4A1, COL4A2, COL8A2) and collagen fibril formation (e.g., LOX, LOXL4, SPARC, PXDN). Additionally, genes typically associated with osteogenic processes (e.g., NPR3, XYLT1, RFLNA, ISG15) were also upregulated in these stiffer cultures. This suggests that stiffer environments promote an endosteal/osteoblastic phenotype, while the softer 3D silk microenvironment supports the maintenance of MSCs in their stromal role. This was further supported by enhanced differentiation into β1-tubulin+ CD42b+ megakaryocytes in the softer niches, where they produced highly branched proplatelets and platelets, as opposed to the smaller, more quiescent megakaryocytes observed in the stiffer niches. 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 (SMA)+ myofibroblasts. This transition was characterized by the upregulation of smooth muscle-related genes (e.g., TPM1, MYL99) 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. When cultured healthy human HSPCs in the 3D 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. Such insights are vital for elucidating the conditions associated with abnormal megakaryopoiesis and bone marrow fibrosis.

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, which are crucial for regulating cellular behavior. These physical and chemical characteristics of BM are critical considerations when developing three-dimensional (3D) models of the BM. Modeling dysfunction in human BM is particularly challenging, especially in disease contexts. BM fibrosis, marked by excessive and uncontrolled ECM deposition, leads to the formation of fibrous tissue. High levels of transforming growth factor-β1 (TGF-β1) play a key role in triggering this dysfunctional microenvironment. Elevated TGF-β1 drives MSCs to transform into myofibroblasts, which contribute to the buildup of ECM, disrupting the normal structure and function of the BM niche. Current methods for studying the BM niche often struggle to effectively incorporate and regulate the biophysical and biochemical factors, such as controlled ECM composition and cellular environments. Natural silk fibroin from Bombyx mori silkworm cocoons was used to fabricate 3D scaffolds designed to mimic the human BM niche. These scaffolds were engineered with adjustable properties, such as stiffness and bioactive ligand distribution. The elasticity (Young's modulus) of the silk scaffolds ranged from 34 to 200 kPa, reflecting the stiffness gradient of the native BM environment, which transitions from the rigid outer cortical bone to the softer interior trabecular bone, surrounded by extracellular matrix, blood vessels, and various cell types. This design facilitated optimal MSCs engraftment and enabled customizable ECM deposition based on the stiffness of the niche.