Cytoskeleton-based forecasting of stem cell lineage fates

Abstract

Stem cells that adopt distinct lineages cannot be distinguished based on traditional cell shape. This study reports that higher-order variations in cell shape and cytoskeletal organization that occur within hours of stimulation forecast the lineage commitment fates of human mesenchymal stem cells (hMSCs). The unique approach captures numerous early (24 h), quantitative features of actin fluororeporter shapes, intensities, textures, and spatial distributions (collectively termed morphometric descriptors). The large number of descriptors are reduced into "combinations" through which distinct subpopulations of cells featuring unique combinations are identified. We demonstrate that hMSCs cultured on fibronectin-treated glass substrates under environments permissive to bone lineage induction could be readily discerned within the first 24 h from those cultured in basal-or fat-inductive conditions by such cytoskeletal feature groupings. We extend the utility of this approach to forecast osteogenic stem cell lineage fates across a series of synthetic polymeric materials of diverse physicochemical properties. Within the first 24 h following stem cell seeding, we could successfully "profile" the substrate responsiveness prospectively in terms of the degree of bone versus nonbone predisposition. The morphometric methodology also provided insights into how substrates may modulate the pace of osteogenic lineage specification. Cells on glass substrates deficient in fibronectin showed a similar divergence of lineage fates, but delayed beyond 48 h. In summary, this high-content imaging and single cell modeling approach offers a framework to elucidate and manipulate determinants of stem cell behaviors, as well as to screen stem cell lineage modulating materials and environments.biomaterials | differentiation | imaging and modeling | stem cells | actin organization

Actin stress in cell reprogramming

“…Other mechanical cues correlated with fate commitment of mesenchymal stem cells are cell shape and the mean stress in the cytoskeleton (15). Because actin transfers physical cues between the cell exterior and interior, it likely plays a major role in stem cell linage commitment (14,25). However, there have been no measurements available on the forces in specific structural proteins in adult cell reprogramming and dedifferentiation.…”

Morphological features of IFN-γ–stimulated mesenchymal stromal cells predict overall immunosuppressive capacity

“…Using high-content imaging of MSCs following IFN-γ stimulation, morphological features were identified that significantly correlated with immunosuppressive capacity and predicted the immunosuppressive capacity of other MSC and non-MSC lines, as well as the effects of IFN-γ pretreatment on immunosuppressive capacity. These findings highlight MSC morphology as an attribute associated with immunosuppressive capacity and provide additional evidence that morphological profiling can be used to predict MSC in vitro function (12,(15)(16)(17). …”

“…Morphology has been identified in many studies as a predictive marker of osteogenic capacity (15,16); however, limited research exists that demonstrates a correlation of morphology with other MSC functions such as immunosuppression. Further improvement on the methods for morphological assessment and applicability across multiple donors is necessary to fully demonstrate its possible utility as a predictor of not only in vitro but also in vivo MSC immunosuppressive capacity.…”

Three-Dimensional In Vitro Tri-Culture Platform to Investigate Effects of Crosstalk Between Mesenchymal Stem Cells, Osteoblasts, and Adipocytes

“…5,6 Toward this end, use of biomaterials may provide a way to recreate these 3D environments, while allowing the study of complex cellular interactions. This includes the application of methods for high-throughput, multivariate analyses of highcontent data (e.g., from gene microarrays, suspension arrays, time-of-flight-mass spectrometry, and microscopy images) [7][8][9][10][11] that yield system-level information of complex cellular processes at or close to a single-cell level. However, innovative strategies that more closely mimic in vivo microenvironments need to be further coupled with the sophisticated methods outlined above.…”