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Novel hydrogel system eliminates subculturing and improves retention of nonsenescent mesenchymal stem cell populations.

ABSTRACT: Aim: To compare the physiological behavior of mesenchymal stem/stromal cells (MSCs) within an expandable tissue-mimetic 3D system relative to in vitro expansion in a traditional 2D system. Methods: Adipose-derived MSCs (ASCs) were continuously cultured for 6 weeks on either 2D culture plastic or in a 3D hydrogel system that eliminated subculturing. ASCs were assessed for senescence, 'stem-like' MSC markers, and ability for their secretome to augment a secondary cell population. Results: The 3D hydrogel system resulted in an enhanced retention of more regenerative, nonsenescent ASC populations that exhibited increased expression of 'stem-like' MSC surface markers. Conclusion: This study introduces a proof-of-concept design for a novel modular 3D system that can improve in vitro expansion of stem-like cell populations for future regenerative therapies.

SUBMITTER: Hodge JG

PROVIDER: S-EPMC9732917 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Novel hydrogel system eliminates subculturing and improves retention of nonsenescent mesenchymal stem cell populations.

Hodge Jacob G JG Robinson Jennifer L JL Mellott Adam J AJ

Regenerative medicine 20221012 1

Aim: To compare the physiological behavior of mesenchymal stem/stromal cells (MSCs) within an expandable tissue-mimetic 3D system relative to in vitro expansion in a traditional 2D system. Methods: Adipose-derived MSCs (ASCs) were continuously cultured for 6 weeks on either 2D culture plastic or in a 3D hydrogel system that eliminated subculturing. ASCs were assessed for senescence, 'stem-like' MSC markers, and ability for their secretome to augment a secondary cell populati ...[more]

PMID: 36222003

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Project description:The host immune system (T-lymphocytes and their pro-inflammatory cytokines) has been shown to compromise bone regeneration ability of mesenchymal stem cells (MSCs). We have recently shown that hydrogel, used as an encapsulating biomaterial affects the cross-talk among host immune cells and MSCs. However, the role of hydrogel elasticity and porosity in regulation of cross-talk between dental-derived MSCs and immune cells is unclear. In this study, we demonstrate that the modulus of elasticity and porosity of the scaffold influence T-lymphocyte-dental MSC interplay by regulating the penetration of inflammatory T cells and their cytokines. Moreover, we demonstrated that alginate hydrogels with different elasticity and microporous structure can regulate the viability and determine the fate of the encapsulated MSCs through modulation of NF-kB pathway. Our in vivo data show that alginate hydrogels with smaller pores and higher elasticity could prevent pro-inflammatory cytokine-induced MSC apoptosis by down-regulating the Caspase-3- and 8- associated proapoptotic cascades, leading to higher amounts of ectopic bone regeneration. Additionally, dental-derived MSCs encapsulated in hydrogel with higher elasticity exhibited lower expression levels of NF-kB p65 and Cox-2 in vivo. Taken together, our findings demonstrate that the mechanical characteristics and microarchitecture of the microenvironment encapsulating MSCs, in addition to presence of T-lymphocytes and their pro-inflammatory cytokines, affect the fate of encapsulated dental-derived MSCs.Statement of significanceIn this study, we demonstrate that alginate hydrogel regulates the viability and the fate of the encapsulated dental-derived MSCs through modulation of NF-kB pathway. Alginate hydrogels with smaller pores and higher elasticity prevent pro-inflammatory cytokine-induced MSC apoptosis by down-regulating the Caspase-3- and 8- associated proapoptotic cascade, leading to higher amounts of ectopic bone regeneration. MSCs encapsulated in hydrogel with higher elasticity exhibited lower expression levels of NF-kB p65 and Cox-2 in vivo. These findings confirm that the fate of encapsulated MSCs are affected by the stiffness and microarchitecture of the encapsulating hydrogel biomaterial, as well as presence of T-lymphocytes/pro-inflammatory cytokines providing evidence concerning material science, stem cell biology, the molecular mechanism of dental-derived MSC-associated therapies, and the potential clinical therapeutic impact of MSCs.

Project description:UnlabelledMesenchymal stem cells (MSC) hold promise in promoting vascular regeneration of ischemic tissue in conditions like critical limb ischemia of the leg. However, this approach has been limited in part by poor cell retention and survival after delivery. New biomaterials offer an opportunity to localize cells to the desired tissue after delivery, but also to improve cell survival after delivery. Here we characterize the mechanical and microstructural properties of a novel hydrogel composed of pooled human platelet lysate (PL) and test its ability to promote MSC angiogenic activity using clinically relevant in vitro and in vivo models. This PL hydrogel had comparable storage and loss modulus and behaved as a viscoelastic solid similar to fibrin hydrogels despite having 1/4-1/10th the fibrin content of standard fibrin gels. Additionally, PL hydrogels enabled sustained release of endogenous PDGF-BB for up to 20days and were resistant to protease degradation. PL hydrogel stimulated pro-angiogenic activity by promoting human MSC growth and invasion in a 3D environment, and enhancing endothelial cell sprouting alone and in co-culture with MSCs. When delivered in vivo, the combination of PL and human MSCs improved local tissue perfusion after 8days compared to controls when assessed with laser Doppler perfusion imaging in a murine model of hind limb ischemia. These results support the use of a PL hydrogel as a scaffold for MSC delivery to promote vascular regeneration.Statement of significanceInnovative strategies for improved retention and viability of mesenchymal stem cells (MSCs) are needed for cellular therapies. Human platelet lysate is a potent serum supplement that improves the expansion of MSCs. Here we characterize our novel PL hydrogel's desirable structural and biologic properties for human MSCs and endothelial cells. PL hydrogel can localize cells for retention in the desired tissue, improves cell viability, and augments MSCs' angiogenic activity. As a result of these unique traits, PL hydrogel is ideally suited to serve as a cell delivery vehicle for MSCs injected into ischemic tissues to promote vascular regeneration, as demonstrated here in a murine model of hindlimb ischemia.

Project description:BackgroundFactors such as poor engraftment, retention, and survival of the transplanted stem cells are deemed to limit their therapeutic efficacy for wound regeneration. Hence, it is necessary to explore these issues in order to resolve them. In this study, we aim to investigate the role of Pluronic F-127 (PF-127) hydrogel plus antioxidant sodium ascorbyl phosphate (SAP) in enhancing Wharton's jelly mesenchymal stem cell (WJMSC)-mediated effectiveness on full-thickness skin wound healing in mice.MethodsFirst, the cytotoxicity of PF-127 and the biological effect of SAP on the survival of WJMSCs were tested in vitro using cell viability and proliferation assays. Next, a cell suspension containing WJMSCs, PF-127, and SAP was topically administered onto an 8-mm diameter excisional full-thickness wound bed. Eight days after transplantation, the mice were sacrificed and the skin tissue was excised for histological and immunohistochemical analysis. Finally, in vivo distribution of transplanted WJMSCs was traced to investigate cell engraftment and the potential therapeutic mechanism.ResultsPF-127 was found to be cytotoxic to WJMSCs while SAP significantly improved the survival of PF-127-embedded WJMSCs. When this combination was topically transplanted onto the wound bed, wound healing was facilitated and dermis regeneration was achieved on the 8th day after surgery, as evidenced by an increase in dermal thickness, newly developed hair follicles, and collagen fiber deposition accompanied by a reduction in scar width. Further, immunohistochemical analysis demonstrated a higher number of anti-inflammatory M2 macrophages, proliferating cells, and newly formed blood vessels in the WJMSCs/PF-127/SAP group relative to all other groups. In addition, in vivo tracking results revealed a highly enhanced engraftment of WJMSCs accumulated in the dermis in the WJMSCs/PF-127/SAP group.ConclusionsSAP significantly improves the survival of WJMSCs in PF-127 encapsulation. Further, PF-127 plus SAP is an effective combination that enhances WJMSC engraftment in the dermis, which then promotes full-thickness wound healing through potential M2 macrophage formation and angiogenesis.

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Novel hydrogel system eliminates subculturing and improves retention of nonsenescent mesenchymal stem cell populations.

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Novel hydrogel system eliminates subculturing and improves retention of nonsenescent mesenchymal stem cell populations.

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