Development of Peptide-Based Hydrogel Scaffolds for the Extended Maintenance of Mesenchymal Stem Cell Phenotype In Vitro

Mesenchymal stem cells (MSCs) have been widely studied during the last thirty years for their potential applications in tissue engineering and cell therapy. They are multipotential cells that produce bioactive factors for the proliferation of tissue specific stem cells, inhibition of apoptosis, angiogenesis signaling, and modulation of the immune response. However, there are still unmet challenges for their translation to the clinic, such as the gradual osteogenic differentiation <i>in vitro</i> caused by the stiffness of tissue culture plastic, and the low cell survival and engraftment <i>in vivo </i>caused by the lack of vascularization into scaffolds, ischemia, and poor removal of cell metabolic waste. A way to address these challenges is by developing scaffolds with suitable chemical and mechanical properties that both mimic the stem cell niche and promote extended survival of MSCs. In this study, we propose the utilization of multidomain peptides (MDPs) as a base for such scaffolds, evaluating different chemical functionalities and their effect on stem cell phenotype preservation, survival, maintenance of immunomodulatory properties, and stem cell proliferation and differentiation.<br/>MDPs are self-assembling peptides with an ABA motif, where B represents an amphiphilic domain of alternating hydrophilic and hydrophobic amino acids, and A represents the terminal domains that control peptide self-assembly. In aqueous solutions, MDPs self-assemble into nanofibers to form compliant hydrogels with storage moduli between 20 and 1200 Pa. In the present study, MSCs were 3D encapsulated in O₅(SL)₆O₅ (a neutral MDP), K₂(SL)₆K₂ (a cationic MDP), and D₂(SL)₆D₂ (an anionic MDP) hydrogels, to evaluate the cell viability and cell density between one and five days post-encapsulation. While K₂(SL)₆K₂ proved to be highly cytotoxic to MSCs and D₂(SL)₆D₂ showed high cell viability with moderate proliferation, the results obtained from O₅(SL)₆O₅ show a high cell viability percentage with low cell proliferation rate throughout the experiment. This is indicative of cells that have entered quiescence, a metabolic state also known as cell cycle arrest that is known to enhance the survival of MCSs in ischemic environments and improving their engraftment in vivo. Stem cell phenotype and quiescence induction is being further characterized by quantifying the expression of CCNB2 (expected in proliferating cells) and CDNK1 (expected in quiescent cells), and the retention of differentiation potential by inducing osteogenesis and adipogenesis on MSCs pre and post encapsulation. The maintenance of immunomodulatory properties was evaluated via IL-6 secretion measurements using ELISA, done by encapsulating cells in MDP hydrogels for 1, 5, 10, 15 and 20 days before retrieving them, stimulating them with TNFα and IFNγ for 24 hours, and recovering the supernatant to measure IL-6 concentration. From these experiments, neither O₅(SL)₆O₅ and D₂(SL)₆D₂ showed a decrease in IL-6 production, indicating the maintenance of MSCs immunomodulatory properties.<br/>Overall, our current data suggest that by encapsulating MSCs in some MDP hydrogels, cells enter a quiescent state during encapsulation that could enhance their stem cell phenotype and preserve their immunomodulatory properties. Such scaffolds would have potential as delivery vehicles for enhanced survival and engraftment of MSCs <i>in vivo</i> and have broad applications for facilitating the use of stem cells in medical research and the clinic.