Aligned electrospun scaffolds are promising tools for engineering fibrous musculoskeletal tissues, as they reproduce the mechanical anisotropy of these tissues and can direct ordered neo-tissue formation. However, these scaffolds suffer from a slow cellular infiltration rate, likely due in part to their dense fiber packing. We hypothesized that cell ingress could be expedited in scaffolds by increasing porosity, while at the same time preserving overall scaffold anisotropy. To test this hypothesis, poly(epsilon-caprolactone) (a slow-degrading polyester) and poly(ethylene oxide) (a water-soluble polymer) were co-electrospun from two separate spinnerets to form dual-polymer composite fiber-aligned scaffolds. Adjusting fabrication parameters produced aligned scaffolds with a full range of sacrificial (PEO) fiber contents. Tensile properties of scaffolds were functions of the ratio of PCL to PEO in the composite scaffolds, and were altered in a predictable fashion with removal of the PEO component. When seeded with mesenchymal stem cells (MSCs), increases in the starting sacrificial fraction (and porosity) improved cell infiltration and distribution after three weeks in culture. In pure PCL scaffolds, cells lined the scaffold periphery, while scaffolds containing >50% sacrificial PEO content had cells present throughout the scaffold. These findings indicate that cell infiltration can be expedited in dense fibrous assemblies with the removal of sacrificial fibers. This strategy may enhance in vitro and in vivo formation and maturation of functional constructs for fibrous tissue engineering.

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Human mesenchymal stromal cell (MSC) lines can vary significantly in their functional characteristics, and the effectiveness of MSCbased therapeutics may be realized by finding predictive features associated with MSC function. To identify features associated with immunosuppressive capacity in MSCs, we developed a robust in vitro assay that uses principal-component analysis to integrate multidimensional flow cytometry data into a single measurement of MSC-mediated inhibition of T-cell activation. We used this assay to correlate single-cell morphological data with overall immunosuppressive capacity in a cohort of MSC lines derived from different donors and manufacturing conditions. MSC morphology after IFN-γ stimulation significantly correlated with immunosuppressive capacity and accurately predicted the immunosuppressive capacity of MSC lines in a validation cohort. IFN-γ enhanced the immunosuppressive capacity of all MSC lines, and morphology predicted the magnitude of IFN-γ-enhanced immunosuppressive activity. Together, these data identify MSC morphology as a predictive feature of MSC immunosuppressive function.H uman mesenchymal stromal cells (MSCs) can potently suppress immune responses in vitro and in animal models of human disease (1, 2), but to date MSC-based therapies have produced mixed results in clinical trials for treatment of inflammatory and autoimmune diseases (3, 4). A major challenge in the development of consistently effective MSC-based immunosuppressive therapies is that MSC lines derived from different donors and manufacturing processes (i.e., cell expansion) can possess markedly dissimilar immunosuppressive function (3,5,6). Although methods exist to assess MSC immunosuppression in vitro, they are often based on only a few measured outcomes, assay culture conditions, and donor MSC samples (5, 7-9). To improve upon these methods, we developed an experimental and analytical approach to quantify MSC-mediated immune suppression using principal-component analysis (PCA) to integrate multiple measurements of T-cell activation assessed at a range of MSC densities. This approach allowed us to determine a single value for immunosuppressive capacity for MSC lines derived from two different manufacturing processes and 13 independent donors.Another major challenge associated with MSC-based immune therapies is the lack of well-defined predictive markers to identify MSC lines with therapeutically relevant biological activities or manufacturing processes that produce more effective MSCbased products. Efforts have been made to identify MSC quality attributes associated with immunosuppression (6, 7), but the majority of clinical studies (10) rely upon the surface markers described by Dominici et al. (11). Having previously shown that morphology can predict MSC mineralization capacity (12), we hypothesized that morphological features associated with immunosuppression in MSCs could be identified and used to predict their performance in our quantitative immunosuppression assay.Using our quantitative method for as...

Advances in our understanding of stem cell interactions with their environment are leading to the development of new materials-based approaches to control stem cell behavior toward cellular culture and tissue regeneration applications. Materials can provide cues based on chemistry, mechanics, structure, and molecule delivery that control stem cell fate decisions and matrix formation. These approaches are helping to advance clinical translation of a range of stem cell types through better expansion techniques and scaffolding for use in tissue engineering approaches for the regeneration of many tissues. With this in mind, this progress report covers basic concepts and recent advances in the use of materials for manipulating stem cells.