iPSC generation
Fibroblasts from skin biopsies
of the three PD donors, one MSA
donor, and two age-matched nondiseased control individuals were
reprogrammed to iPSCs using the
CytoTune®-iPS Sendai Reprogramming
Kit. Our workflow is summarized
in Figure 1. After plating in normal
growth medium, fibroblasts were
simultaneously transduced with the
four reprogramming vectors included
in the kit: hOct3/4, hSox2, hKlf4, and
hc-Myc. Seven days after transduction,
cells were harvested and plated onto
MEF culture dishes. The medium was
changed to iPSC medium (KnockOut™
DMEM/F-12, 20% KnockOut™
Serum Replacement (KSR), 10 mM
non-essential amino acids, 2 mM
GlutaMAX ™-1, and 10 ng/mL bFGF)
and monitored for formation of iPSC
colonies consisting of tightly packed
groups of cells with large nuclei
and defined edges. Representative
phasecontrast images of cells at
various stages of reprogramming are
shown in Figure 2.
Day 7 Plating
on MEFs
Day 20-26 Picking
colonies
1. Images of cells were taken before
harvesting.
1. Plates were observed every day for
the emergence of colonies.
2. Cells harvested using TrypLE™
Select at room temp.
2. Starting Day 20, colonies were
picked and transferred to 12-well
MEF plates.
Fibroblast culture
Day 1 Transduction
1. 1.25 x 105 cells/well were plated on
6-well plates coated with
Attachment Factor.
1. Protocol described in the User
Guide for the CytoTune®
reprogramming kit was followed.
2. Cells were cultured in a 37˚C, 5%
CO2 incubator for two days.
Additional cells were cultured as a
cell number control for accurate
MOI determination, and for
karyotype analysis and cell line ID.
2. The additionally plated cells were
harvested to determine the cell
number for accurate MOI
calculation.
3. CytoTune® virus was added at
MOI of 3.
3. Remaining cells were stored in
RNAlater® for RNA extraction.
4. Day 8, medium changed to iPSC
medium and replaced with fresh
iPSC medium every day thereafter.
4. Images of cells were taken before
the transduction and then every
other day after media change.
5. Remaining cells were stored in
RNAlater® for RNA extraction.
3. Cells plated at multiple seeding
densities onto 10 cm MEF dishes.
Figure 1. Workflow for iPSC generation and characterization.
11 Life Technologies | Parkinson's cell model
3. Colonies were picked based on
morphology, live staining, or both.
On average, 20 colonies were
picked for each line.
CULTURE
ENGINEERING
DIFFERENTIATION
CHARACTERIZATION
Colony expansion
iPSC characterization
1. 6 clones for each line were
further expanded onto 6-well
MEF plates and later
cryopreserved.
1. Karyotype analysis
2. 3 of the 6 clones were further
expanded, cryopreserved and
characterized.
2. Cell ID
3. Immunocytochemistry for SSEA4,
Oct4, Tra-1-60 and Tra-1-81
4. FACS analysis for Nanog and
SSEA4
5. Pluripotent gene expression panel
6. Embryoid body formation and
staining for 3 germ layer markers
7. TaqMan® Sendai detection to
make sure the cells are
transgene-free
