Analysis using the TaqMan® hPSC Scorecard™ Panel
To determine pluripotency of the iPSCs and any inherent
lineage bias of EBs, the samples were analyzed using the
TaqMan® hPSC Scorecard™ Panel. This panel evaluates
pluripotency and detects germ layer differentiation bias via
qPCR assays and intuitive data analysis software, which
compares all samples to an internal standard.
After cDNA synthesis, the samples were run on the
QuantStudio™ 12K Flex Real-Time PCR System and analyzed
using the hPSC Scorecard™ Analysis Software. Cells from
all six donor lines tested positive for pluripotency (data
not shown). Late-passage (>p25) iPSCs from the MSA and
Ctrl-2 samples tested positive for pluripotency, and their
corresponding EBs show potential for all three lineages,
suggesting both are good general-purpose pluripotent cell
lines. The evaluation of the samples is summarized in the
software (Figure 10).
MSA
MSA EBs
Ctrl2
Ctrl2 EBs
+
+++
+
+++
Pluri
Pluripotency markers
expressed. Test differentiated
cells to determine utility.
Ecto Endo Meso
Good general-purpose
pluripoint cell line.
Pluri
Pluripotency markers
expressed. Test differentiated
cells to determine utility.
Ecto Endo Meso
Good general-purpose
pluripoint cell line.
Figure 10. Gene expression results for pluripotency and lineage markers are
summarized after analysis with the TaqMan® hPSC Scorecard™ Panel.
How to Use the TaqMan® hPSC
Scorecard™ Panel
Instrument highlight—QuantStudio™ 12K Flex Real-Time PCR System
The QuantStudio™ 12K Flex Real-Time PCR System was used for gene expression analysis of the
iPSCs in three different characterization experiments:
• The first was a broad-based gene
expression analysis of the iPSCs with 609
genes in the the TaqMan® OpenArray®
Human Stem Cell Panel. Pluripotency
genes were shown to be up-regulated in
all iPSCs lines, and fibroblasts were easily
distinguished from iPSCs after results
were plotted in a heat map.
• The second experiment was a detection
of Sendai virus transgenes remaining
in the iPSCs after reprogramming. The
22 Life Technologies | Parkinson's cell model
TaqMan® iPSC Sendai Detection Kit was
used to detect the presence and level of
expression of exogenous transcription
factor genes (OCT3/4, SOX2, KLF4, and
c-MYC) delivered to cells, as well as the
presence of Sendai virus RNA. Clearance
was observed as early as passage 10 in
one clone and as late as passage 15 in
another.
• The third experiment was gene expression
analysis for pluripotency and lineage
bias of EBs using
the TaqMan® hPSC
Scorecard™ Panel.
Passage 25 iPSCs
from the MSA and
Ctrl-2 samples
tested positive for pluripotency, and their
corresponding EBs showed potential for all
three lineages, suggesting both are good
general-purpose pluripotent cell lines.
