23
Life Technologies
™
| lifetechnologies.com/parkinsons
A B
Figure 11. Characterization of TAL-edited MSA lines. (A) Cytogenetic analysis results of edited MSA clone
21 (SNCA
+/–
) and clone 32 (SNCA
–/–
). No clonal abnormality was detected at the G-banded level of resolution.
(B) Feeder-free iPSCs (MSA clone 21 and MSA clone 32) in Essential 8
™
medium on Geltrex
®
matrix–coated
plates were stained as indicated for Oct 4 (red), SSEA4 (green), Tra-1-60 (red), and Sox2 (green) using the PSC
4-Marker Immunocytochemistry Kit with DAPI (blue) nuclear DNA counterstaining. (C) Pluripotency analysis
using TaqMan
®
hPSC Scorecard Panel. Feeder-free iPSCs were lysed directly on the culture plates by adding
TRI Reagent
®
and incubating for 5 minutes at room temperature. RNA was isolated from the lysate using the
RiboPure
™
RNA Purification Kit. RNA was reverse transcribed into cDNA using SuperScript
®
VILO
™
Master Mix.
cDNA samples were combined with TaqMan
®
Gene Expression Master Mix and Nuclease-Free Water before
adding to the TaqMan
®
hPSC Scorecard
™
Panel and run on a QuantStudio
®
12K Flex Real-Time PCR System.
MSA iPSC
Self-
renew Ecto Meso Endo
MSA EBs
Self-
renew Ecto Meso Endo
MSA.21 iPSCs
Self-
renew Ecto Meso Endo
MSA.21 EBs
Self-
renew Ecto Meso Endo
MSA.32 iPSCs
Self-
renew Ecto Meso Endo
MSA.32 EBs
Self-
renew Ecto Meso Endo
C
Prior to further downstream work, fully characterized banks of the SNCA
+/–
and SNCA
–/–
lines were generated. Each line was
shown to be karyotypically normal (Figure 11A), and expressed pluripotency markers such as SSEA4, Tra-1-81, Tra-1-60, and
Oct4 (Figure 11B). Analysis using the TaqMan
®
hPSC Scorecard
™
Panel demonstrated that the edited clones were pluripotent,
and the 7-day embryoid bodies (EB) derived from these clones showed markers for all three lineages (Figure 11C).
Clone 21 (SNCA
+/-
) Clone 32 (SNCA
-/-
)
Tra1-81 SSEA4
Tra1-60
Oct4
21 (SNCA
+/-
) Clone 32 (SNCA
-/-
)
SSEA4
Oct4
Clone 21 (SNCA
+/-
) Clone 21 (SNCA
+/-
)—Tra-1-60 Sox2 DAPI Clone 21 (SNCA
+/-
)—Oct4 SSEA4 DAPI
Clone 32 (SNCA
-/-
)—Tra-1-60 Sox2 DAPI Clone 32 (SNCA
-/-
)—Oct4 SSEA4 DAPI
Clone 32 (SNCA
-/-
)
Results
Learn more about the TaqMan
®
hPSC Scorecard
™
Assay for quantitative
analysis of trilineage differentiation potential.
