Exploiting material softness to regulate mesenchymal stem cells secretome production for "cell-free" regenerative medicine

• Main Author: Yang, Haibo
• Other Authors: Dalton Tay Chor Yong
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2020
• Subjects: Engineering::Materials::Biomaterials
• Online Access: https://hdl.handle.net/10356/139672
• Institution: Nanyang Technological University

Mesenchymal stem cells (MSCs) are multipotent stem cells derived from various adult tissues. They have been widely used in regenerative medicine because of their capability to differentiate into a variety of cell types. However, the paradigm of MSCs therapy shifts from differentiation to the secreted trophic factors of MSCs. MSCs secretome is a collective term for growth factors, chemokines, and cytokines and a variety of biologically active molecules secreted by MSCs. The therapeutic value of MSCs secretome has been proven in the treatment of cardiovascular diseases, liver diseases, and brain diseases, etc. To activate MSCs and engineer MSCs secretome for regenerative medicine, various preconditioning methods, including gene modification, drug treatment, hypoxia culture, etc., are adopted. Considering the sensitivity of MSCs to their microenvironment, biomaterials of different properties could be a potent regulator of MSCs secretome. Hydrogel systems with tunable stiffness have been adopted to study the response of cells to substrates of different stiffnesses. In this thesis, polyacrylamide (PAAm) hydrogel was chosen due to its highly feasibility in tuning the stiffness mimicking to that of the human organs. Two different substrate stiffnesses, namely 0.15-1.5 kPa (brain mimicking) and 10-17.7 kPa (heart mimicking), were achieved. After conjugation of fibronectin (Fn) through sulfo-SANPAH chemistry, soft material for MSCs culture was prepared. With this PAAm hydrogel system, it was firstly found that primary MSCs are sensitive to substrate stiffness in aspects of cell spreading, cytoskeleton development, and intracellular ROS level even in the serum free condition. In addition, among the 42 cytokines screened with protein array, 15 of them were defined as “mechanosensitive” secretome since that their secretions were modulated by substrate stiffness. As a common mediator in MSCs preconditioning, reactive oxygen species (ROS) was found to regulate MSCs secretome production under the mechanical cue of substrate stiffness. Furthermore, compared with the CS of GPa stiffness, this “mechano-condition” of MSCs with soft PAAm hydrogel of kPa stiffness further improved the therapeutic efficacy of MSCs secretome in both in vitro scratch assay and in vivo full-thickness excisional splinted wound healing mouse model with higher wound healing index. In short, soft material can be utilized for preconditioning of primary MSCs to promote “cell-free” regenerative medicine. Secondly, to address the senescence issue that bothers primary MSCs, the feasibility of “mechano-condition” on immortalized MSCs was tested. Immortalized MSCs were first confirmed to maintain differentiation potential, especially at the late passage (P10). Meanwhile, they still secreted trophic factors that promoting migration of human dermal fibroblasts and keratinocytes. These results suggest that immortalized MSCs are also an ideal cell source for tissue repair. Furthermore, the immortalization procedure was found to have no impact on the mechanosensitivity of MSCs. The 0.15 kPa PAAm hydrogel also limits spreading, hinders cytoskeleton maturation, induces intracellular ROS expression of immortalized MSCs. The expressions of VEGF and bFGF by immortalized MSCs were stimulated by the 0.15 kPa PAAm hydrogel at both transcription and protein levels, with the involvement of redox signaling. This mechano-conditioned MSCs secretome promoted the therapeutic angiogenesis because of a higher amount of proangiogenic factors. In the end, by focusing on the angiogenesis stage of tissue repair, the mechanism for MSCs redox stress induced by the 0.15 kPa PAAm hydrogel to guide proangiogenic secretion production was studied. Substrate stiffness was proven to be the main controller of the intracellular ROS level, regardless of serum availability. Nrf2 antioxidant response was triggered by excess intracellular ROS in immortalized MSCs on the 0.15 kPa PAAm hydrogel. Furthermore, the interplay between Nrf2 signaling and HIF1 signaling causes a non-canonical activation of this important regulatory factor for angiogenesis (i.e., HIF1) in immortalized MSCs under normoxic condition. ROS inhibition assay consolidates the activating role of upregulated intracellular ROS on HIF1 signaling. Overall, this thesis shows that material softness can be utilized to modulate MSCs secretome for regenerative medicine, with the redox status of MSCs as a key mediator.