Dielectrophoresis based discrimination of human embryonic stem cells from differentiating derivatives

Abstract

Assessment of the dielectrophoresis (DEP) cross-over frequency (f xo), cell diameter, and derivative membrane capacitance (C m) values for a group of undifferentiated human embryonic stem cell (hESC) lines (H1, H9, RCM1, RH1), and for a transgenic subclone of H1 (T8) revealed that hESC lines could not be discriminated on their mean f xo and C m values, the latter of which ranged from 14 to 20 mF/m(2). Differentiation of H1 and H9 to a mesenchymal stem cell-like phenotype resulted in similar significant increases in mean C m values to 41-49 mF/m(2) in both lines (p < 0.0001). BMP4-induced differentiation of RCM1 to a trophoblast cell-like phenotype also resulted in a distinct and significant increase in mean C m value to 28 mF/m(2) (p < 0.0001). The progressive transition to a higher membrane capacitance was also evident after each passage of cell culture as H9 cells transitioned to a mesenchymal stem cell-like state induced by growth on a substrate of hyaluronan. These findings confirm the existence of distinctive parameters between undifferentiated and differentiating cells on which future application of dielectrophoresis in the context of hESC manufacturing can be based.

Combined hydrogels that switch human pluripotent stem cells from self-renewal to differentiation

“…Therefore, Matrigel and PVA were included in subsequent hydrogel formulations. Microscopy of cells within optimized composite hydrogels showed a rounded morphology with an average cell diameter of 10 ± 0.32 μm, consistent with published sizes for undifferentiated cells (25) (Fig. S7C).…”

A microfluidic method for the selection of undifferentiated human embryonic stem cells and in situ analysis

“…Wang et al (2011b) used optical tweezers within a microfluidic device to sort OCT4-GFP + hESCs. Dielectrophoretic methods could also be used to sort undifferentiated and differentiated hESCs based on differences in plasma membrane capacitance (Velugotla et al 2012) and have been used to sort neuronal stem cells (Flanagan et al 2008); however, subsequent immunostaining or other methods are necessary to determine hESC subsets (e.g., SSEA4 + cells). More recently, Singh et al (2013) employed extracellular-matrixcoated microfluidic channels to sort undifferentiated and differentiated hESCs based on adhesion strength.…”

A scalable label-free approach to separate human pluripotent cells from differentiated derivatives

“…Human dermal fibroblasts (HDFs) from neonatal foreskin (Cascade Biologics) were cultured on plastic (Corning), in M106 medium (Invitrogen) supplemented with low serum growth supplement (LSGS, 50Â), at 37 C, 5% CO 2 in air. H1-hESCs were differentiated to a mesenchymal progenitor phenotype (designated hESC-MP) as reflected by flow cytometric determination of negativity for CD14 and CD34 and detection of CD105, CD90, CD73, and Stro1, using a method previously developed and validated by ourselves and others ((De Sousa, 2006;Harkness et al, 2011;and Velugotla et al, 2012). The method involves plating hESCs (5 Â 10 5 cells/9.6 cm 2 ) on hyaluronan (HA; also known as hyaluronic acid or hyaluronate) (1200 kD; Calbiochem Cat Nr 385908) in a medium that would otherwise support hESC self-renewal (human dermal fibroblast conditioned medium (HDF-CM) composed of KO-DMEM, 20% Knockout serum replacement (KOSR, Invitrogen), 1 mM glutamine, 0.1 mM b-mercaptoethanol, and 4 ng/ml bFGF).…”