Stratos Pistikopoulos [Closing Remarks]

Characterizing Human Embryonic Stem
Cells Function With Dielectrophoresis And Flow Cytometry

Tayloria Adams, Clarissa Ro, Shubha Tiwari, and Lisa

Neurology Department, University of California, Irvine

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 hNSPCÕs 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 process for cell
characterization is challenging due to a lack of biomarkers that provide an
adequate picture of hNSPCs specific functions. Therefore, weÕve implemented
dielectrophoresis (DEP), a label-free characterization technique that uses
nonuniform electric fields, to determine cellsÕ dielectric properties such as
membrane capacitance (Cmem). 
Additionally, hNSPCs were analyzed by flow cytometry to characterize
surface protein expression answering the scientific question Òare there correlations
between Cmem, measured by DEP, and protein expression?Ó

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, Cmem, 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 with Shef4-1 > Shef4-2 > Shef4-3. Similarly, there is
variability across lots in the DEP spectra, which is corroborated with Cmem.
At early and late passages between 0-100kHz the DEP spectra is shifted right
for Shef4-2 as compared to Shef4-1 and Shef4-3 (Figure 1A and 1B). The
corresponding Cmem at early and late passages, Figure 1C and 1D,
shows that each cell batch is different with statistical significance (one-way
ANOVA). Across
passages, Shef4-1 and Shef4-2 Cmem gradually decreases and Shef4-3 Cmem
fluctuates minimally. Alpha6
and alphaV integrin markers were assessed by flow cytometry for each batch of
cells, and the % protein expression was plotted with Cmem, Figure 1E
and 1F. For alpha6, there is a strong correlation to Shef4-2 Cmem (R2=0.882),
and essentially no correlation to Shef4-3 (R2=0.081). For alphaV,
there is a weak correlation for Shef4-2 (R2=0.244), and no
correlation for Shef4-3 (R2=0.082). Shef4-1 was not included because
alpha6 and alphaV expression was low (less than 14%).

Figure 1. hESC derived hNSPCs were characterized using DEP and
flow cytometry. Shef4-1, Shef4-2, and Shef4-3 DEP spectra at (A) early (p4) and (B) late (p6 and p7) passage. Average Cmem for (C) early and (D) late passage Shef4-1, -2, and -3 cells. (E) Alpha6 and (F) alphaV
integrin expression with the associating Cmem (passages are
matched). One-way ANOVA posthoc Tukey test conducted for multiple comparisons,
**p <0.01 and ****p < 0.0001.

Significant lot-to-lot variability exists
among hESC-derived hNSPCs, and DEP plus flow cytometry provide a good
quantitative assessment of cell phenotype variability. Shef4-2 Cmem correlates
well to alpha6 while no correlations were seen with alphaV. Alpha6 and alphaV
integrins are important for cell migration and differentiation. Linking
integrin expression with DEP measurements provides additional insights to the
biological meaning of Cmem to expand beyond the core definition,
which is the ability to store charge. Effectively discerning the functional
behavior of stem cells before their use in transplants is essential to advance
stem cell therapeutics.