(251x) Characterizing Human Stem Cell Function with Dielectrophoresis and Flow Cytometry

Human embryonic stem cells (hESCs) provide great opportunities in stem cell therapeutics because they can differentiate into the three germ layers: ectoderm, mesoderm, and endoderm. More specifically, hESCs can be directed toward human neural stem/progenitor cell (hNSPC) differentiation and used to treat neurological diseases and injuries. Sufficiently characterizing hNSPCâ??s functional behavior before using them in transplant therapy is essential to the development of reliable therapeutic treatment options. Having measures that will accurately reflect cell phenotype after transplantation is critical. Currently, the cell characterization process is challenging due to a lack of biomarkers that provide an adequate picture of the specific functions of hNSPCs. Therefore, weâ??ve implemented dielectrophoresis (DEP), a label-free characterization technique that uses nonuniform electric fields, to determine cell dielectric properties such as membrane capacitance (C_mem). Additionally, cells were analyzed by flow cytometry to characterize hNSPC surface protein expression to determine any correlations between characteristics measured by DEP and cell surface markers.

In this work three sets of hESC-derived hNSPCs (Shef4-1, Shef4-2, Shef4-3) were derived from passage 6 EZ spheres and established as monolayers before analysis. The Shef4-1, Shef4-2, and Shef4-3 nomenclature indicates that these cells were derived from the same set of EZ spheres but separately differentiated to form hNSPCs, as would be done to generate sufficient numbers of stem cells for therapeutic purposes. Once established as monolayer hNSPC cultures, their cell size, DEP spectra, C_mem, and cell surface protein expression were quantified at a variety of passage numbers to determine lot-to-lot variability or variability over passaging. Results show that cell diameter varies across lots, so Shef4-1 > Shef4-2 > Shef4-3. Similarly, there is variability across lots in the DEP spectra, which is corroborated with Cmem. At passage 7 between 0-100kHz the DEP spectra is shifted right for Shef4-2 (C_mem = 8.6mF/m²) as compared to Shef4-1 and Shef4-3 (C_mem = 16.5mF/m² and 16.4mF/m², respectively). Over passages, Shef4-2 C_mem decreases with passage number, Shef4-3 C_mem increases with passage number and Shef4-1 does not have a clear C_mem trend. Cell surface markers were assessed by flow cytometry resulting in alpha6 and alphaV integrin expression decreasing over passage for Shef4-1, increasing for Shef4-2, and decreasing for Shef4-3. The changes in alpha6 and alphaV integrin expression over passage correlate well with changes in C_mem over passage. Generally, these findings correspond well with DEP theory such that high alpha6 and alphaV expression indicates a complex cell surface, which translates to lower C_mem (Shef4-2 C_mem decreases and integrin expression increases over passage) with the opposite also being true (Shef4-3 C_mem increases and integrin expression decreases over passage).

Here weâ??ve shown that significant lot-to-lot variability exists among hESC-derived hNSPCs, and DEP plus flow cytometry provide a good quantitative assessment of cell phenotype variability. Additionally, effectively discerning the functional behavior of stem cells before their use in transplants is essential to advance stem cell therapeutics; alpha6 and alphaV integrin are important for cell migration and differentiation. Linking integrin protein expression with DEP measurements provides additional insights to the biological meaning of C_mem to expand beyond the core definition, which is the ability to store charge. Future work will compare transplantation efficiencies of different hESC-derived hNSPCs.