Engineering the MSC Secretome: A Hydrogel Focused Approach

Abstract

The therapeutic benefits of exogenously delivered mesenchymal stromal/stem cells (MSCs) have been largely attributed to their secretory properties. However, clinical translation of MSC-based therapies is hindered due to loss of MSC regenerative properties during large-scale expansion and low survival/retention post-delivery. These limitations might be overcome by designing hydrogel culture platforms to modulate the MSC microenvironment. Hydrogel systems could be engineered to i) promote MSC proliferation and maintain regenerative properties (i.e., stemness and secretion) during ex vivo expansion, ii) improve MSC survival, retention, and engraftment in vivo, and/or iii) direct the MSC secretory profile using tailored biochemical and biophysical cues. Herein, it is reviewed how hydrogel material properties (i.e., matrix modulus, viscoelasticity, dimensionality, cell adhesion, and porosity) influence MSC secretion, mediated through cell-matrix and cell-cell interactions. In addition, it is highlighted how biochemical cues (i.e., small molecules, peptides, and proteins) can improve and direct the MSC secretory profile. Last, the authors' perspective is provided on future work toward the understanding of how microenvironmental cues influence the MSC secretome, and designing the next generation of biomaterials, with optimized biophysical and biochemical cues, to direct the MSC secretory profile for improved clinical translation outcomes.

Keywords: biomaterials; cell therapy; hydrogels; mesenchymal stromal cell; secretome.

Figure 1.. Mesenchymal stem cell-based clinical trials in the United States between 2010–2020.

a) MSC clinical trials based on the organ system affected by the disease. b) Source of MSCs used in clinical trials. c) Number of clinical trials using MSCs over time. d) MSC clinical trials categorized by phase. e) Methods of delivering MSCs in clinical trials. Local refers to direct injection into the targeted tissue; systemic refers to intravenous infusion; and biomaterial refers to its combination with MSC delivery. These data were obtained from clinicaltrials.gov and searching for “Mesenchymal Stem Cell” in the “Other” category, limited to the United States. Data was collected on October 11, 2020.

Figure 2.. Culture systems to expand and deliver MSCs.

A variety of material platforms are used to expand and deliver MSCs, including tissue culture polystyrene (TCPS), hydrogels (either on 2D surfaces or 3D encapsulation), microspheres, porous scaffolds, and or multi-cellular spheroids. Each of these systems has the ability to influence MSC proliferation, secretory properties, and survival upon delivery. Strategies that encapsulate cells (e.g., hydrogels, microspheres) lead to higher levels of cell-matrix interactions compared to 2D surfaces. Porous scaffolds and multi-cellular spheroids lead to more cell-cell interactions. As a qualitative assessment, minus sign (−) indicates a system that does not improve the corresponding property, while the single up arrow (↑) indicates a slight improvement and the double arrow (↑↑) indicates a higher level of improvement.

Figure 3.. Matrix composition and physical properties influence MSC secretion.

a) To promote cell adhesion, bioactive cell adhesion molecules are often incorporated in hydrogel formulations. Integrins, present on the cell surface, bind to amino acid sequences found in ECM adhesion proteins. For example, RGD and GFOGER peptides, fibronectin and collagen mimics respectively, have been shown to differentially influence MSC secretory profiles. Similarly, peptides derived from N-cadherins (e.g., HAVDI) can mimic cell-cell interactions and influence MSC secretory properties. b) Bulk hydrogel properties (e.g., stiffness, viscoelasticity) influence MSC interactions and global secretory properties. Porosity and degradation properties can direct MSC clustering and promote secretion through increased cell-cell contacts.

Figure 4.. Biochemical microenvironmental modifications to influence MSC secretion.

a) Biochemical compounds such as small molecules, peptides, or proteins, can be incorporated into hydrogels by a variety of methods focused on either bulk adsorption or immobilization to the matrix. b) Release profiles of biochemical compounds will vary depending on how the compound is incorporated into the hydrogel and can be tailored for immediate or prolonged release and cell exposure over time. c) Immobilization of biochemical compounds to hydrogel matrices enables slower release profiles to be obtained, compared to bulk adsorption, which results in the need for lower biochemical doses. Bulk adsorption of biochemical factors is the simplest method for incorporation; however, burst release profiles often result, necessitating higher concentrations of the bioactive factor.

Figure 5.. Design considerations for the next generation of hydrogels to direct the MSC secretome.

Prior to hydrogel use, MSC donor characteristics, such as, gender, age, health, and genetics, must be evaluated. A hydrogel platform with specific material properties can then be utilized for MSC culture. Material characteristics should be chosen based on whether the hydrogel will be injected or implanted, in addition to how these properties will affect the MSC secretome. Furthermore, various biochemical compounds can be incorporated into the material system. Specific release profiles of bioactive factors can be obtained depending on how the molecules are incorporated into the hydrogel system.

Figure 6.. Proteomic approaches used to detect and quantify MSC secretory components.

Shot-gun based proteomics (primarily mass spectrometry based) are used for the detection of unknown or unique proteins. Once these proteins are identified, publicly available databases and bioinformatics can be used for pathway analyses to determine specific protein roles (extensive analyses). Immunological-based assays are used for testing a broad range of known proteins. These assays are often user-friendly involving minimal sample preparation and analysis methods resulting in quantitative or semi-quantitative outcomes.