Date Published: March 8, 2018
Publisher: Public Library of Science
Author(s): Tom Bongiorno, Jeremy Gura, Priyanka Talwar, Dwight Chambers, Katherine M. Young, Dalia Arafat, Gonghao Wang, Emily L. Jackson-Holmes, Peng Qiu, Todd C. McDevitt, Todd Sulchek, Austin John Cooney.
The highly proliferative and pluripotent characteristics of embryonic stem cells engender great promise for tissue engineering and regenerative medicine, but the rapid identification and isolation of target cell phenotypes remains challenging. Therefore, the objectives of this study were to characterize cell mechanics as a function of differentiation and to employ differences in cell stiffness to select population subsets with distinct mechanical, morphological, and biological properties. Biomechanical analysis with atomic force microscopy revealed that embryonic stem cells stiffened within one day of differentiation induced by leukemia inhibitory factor removal, with a lagging but pronounced change from spherical to spindle-shaped cell morphology. A microfluidic device was then employed to sort a differentially labeled mixture of pluripotent and differentiating cells based on stiffness, resulting in pluripotent cell enrichment in the soft device outlet. Furthermore, sorting an unlabeled population of partially differentiated cells produced a subset of “soft” cells that was enriched for the pluripotent phenotype, as assessed by post-sort characterization of cell mechanics, morphology, and gene expression. The results of this study indicate that intrinsic cell mechanical properties might serve as a basis for efficient, high-throughput, and label-free isolation of pluripotent stem cells, which will facilitate a greater biological understanding of pluripotency and advance the potential of pluripotent stem cell differentiated progeny as cell sources for tissue engineering and regenerative medicine.
Tissue-engineered organs and regenerative medicine therapies are estimated to require >107 cells of one or more prescribed cell types , which is difficult to achieve using autologous cell sources. Embryonic stem cells (ESCs) hold great potential as scalable, phenotype-specific “cell factories,” but progress is hampered by the two-fold challenge of directing cell fate commitment to specific lineages and controlling the maturity of a particular cell type (Fig 1A).
Characterizing ESCs with known days of differentiation revealed that the cells stiffen within 1 day of differentiation and, on average, remain at a similar stiffness level for at least 5 more days, while changes to the viscoelastic relaxation response of cells were minimal. An increase in Feret’s diameter and a concomitant decrease in circularity were also observed as differentiation progressed. After sorting cells by stiffness using a microfluidic device, pluripotent cells were enriched in the soft outlet and differentiated cells were enriched in the stiff outlet. Using a 3-outlet device to sort a mixed population of pluripotent and differentiated cells, the soft subset of cells was more characteristic of the known day 0 cell population than the middle or stiff subsets, as assessed by stiffness and morphology. An assessment of the gene expression levels of sorted cells revealed decreased Nanog in the stiff outlet, increased Pou5f1 in the soft outlet, and increased Actn1 in the middle and stiff outlets, which reflect the enrichment of pluripotent cells in the soft outlet of the device.
In the present study, pluripotent ESCs were enriched via mechanically-driven cell sorting, which highlights cell mechanics as a basis for efficient, high-throughput isolation of pluripotent ESCs. Further optimization of cell sorting parameters, such as flow rate, cell concentration, and device geometry, in addition to employing multiple sorts in series, will enable stiffness-based, microfluidic sorting to be used as a novel, label-free, and highly efficient method for the purification of pluripotent ESCs. The ability to generate pure populations of pluripotent ESCs will facilitate a greater understanding of pluripotency and serve as a step toward realizing the potential of ESCs as cell sources for various applications. Technologies that can select for or against pluripotent cells, such as stiffness-based microfluidic sorting, also hold great potential to be adapted for the enrichment of specific differentiated lineages, with applications to improving directed differentiation for regenerative medicine and tissue engineering.