Methods
Preparation of Cell Culture Media
Media for Human Fibroblasts
We culture human fibroblasts in IMDM with 10% FBS, 1× NEAA, 1× l-Glutamine, and
1×-Antibiotic/antimycotics, 20-50ug/ml of Ascorbic Acid, 4ng/ml bFGF. For propagating
fibroblasts, the split ratios were 1:4 or 1:6 with cells passaged about every 4-days. Cells
were frozen down at each passage (>5 vials/passage). Cell are frozen down in 50%
IMDM complete, 40% FBS, 10% DMSO.
Media and Feeder Cells for hES and iPS Cells
Pre-inducible pluripotent and inducible pluripotent cells (iPS) cells were maintained in
HES media: DMEM/F12 containing 2mM of glutamine, 20% Knockout Serum
Replacement (KO), 1X NEAA, 0.1mM -mercaptoethanol (0.1mM), and 10ng/ml of
bFGF (Roche, Indianapolis, IN, www.roche.com) on 0.1% gelatin (Sigma, Inc.) coated
plates with irradiated mouse embryonic fibroblast (irMEFs).
Lentivirus production and infection
Lentiviral production and infection followed the previously published protocol
(Mostoslavsky, 2006). Briefly, the STEMCAA-Red Light plasmid (24ug/100-mm dish)
together with four-plasmid pHelper (encoding human immunodeficiency virus (HIV)
Hpgm2, REV, TAT and VSV-G envelope, 1.2, 1.2, 1.2, and 2.4ug, respectively, 100-mm
dish) were transfected into 293T (HEK) cells using TransIT293 (3ul/ug of plasmid)
Mirus, Madison, WI) overnight, and media was replaced. After 48h, the virus-containing
supernatants were collected every 12h on two consecutive days and filtered through a
0.45-um polyethersulfone membrane (Waltman), and viral particles were concentrated by
ultracentrifugation at 24,000 rpm for 1.5h at 4C. Human fibroblast were seeded at
approximately 3 x 104 cells per well of a 6-well plate and incubated with 5ul of virus plus
5ug/ml of polybene in MEF media with a change in media 24h after infection. The media
is changed to IMDM with 10% FBS, 1x NEAA, 1x 1-Glutamine, 1x-Penn/Strep, 20ug/ml
of ascorbic acid, 4ng/ml bFGF. For three factor reprogramming, where indicated, GDK3beta (Bio:EMD Biosciences, San Diego, CA, http://www.emdchemicals.com, 361550;
5uM) was added to the culture media on days 2-6 of reprogramming. Six days after
infection, 4 x 10^5 cells were replated on two 100-mm plates prepared MEFs plates ((6)100-mm dishes are prepared), using 0.25% trypsin/EDTA, and replated in complete
IMDM media plus Thiazovivin (Stemgent, Boston, MA, http://www.stemgent.com;
0.5uM). The cells were maintained in IMDM with 10% FBS, 1x NEAA, 1x 1-Glutamine,
1x-Penn/Strep, 50ug/ml of ascorbic acid, 4ng/ml bFGF for 3-days post-splitting. Media
was changed to HES media on the 4th day after splitting, and media changes were
performed every 2-3 days using 15-ml of media/100-mm dish.
Initial iPSC expansion and Characterization
To establish iPSC lines, iPSC clones were picked about 1-month post-infection based on
cell morphology and size. One 100-mm dish was stained for alkaline phosphatase
according to manufacturers recommendation (Stemgent, Inc). Cells (12-24 clones) were
picked into 96-well plates and then transferred to a 12-well plate containing irMEFs,
matrigel (BD Bioscience, San Diego, CA, http://bdbiosciences.com) in HES media. The
picked clones were subsequently passaged and expanded in one well of a 6-well dishes
using 0.05% Trypsin/EDTA according to splitting protocol. When the cells reached 75%
confluence, they were subsequently split to three wells of a 6-well dish. Two of the three
wells were frozen into 4-freezing vials (freezing media (1ml): 50% HES, 40% FBS, and
10% DMSO). One well was further passaged to (1) 6-well dish. At 80% confluence,
three wells of the 6-well dish are taken for qDNA (Qiagen, DNeasy Blood & Tissue kit
for southern blot analysis), two wells are frozen in liquid nitrogen, one well was taken for
characterization of the clones including pluripotent ES cell markers; qPCR analysis:
DMNT3B, ABCG2, REX1, OCT4-endogenous, SOX2-endogenous, Nanong-endogenous
gene expression. Flow cytometry was utilized to identify pluripotency antigens:
SSEA3/4, TRA-1-81/TRA-1-60, and OCT4/SOX2. Cells were maintained in culture until
all clones reached 20 or greater passages.
Immunoflourescence
Cells were fixed in 3.7% paraformaldehyde for 10 min. Washed cells were treated with
0.1% Triton X (Sigma, St. Louis, MO, http://www.sigmaaldrich.com ) in PBS for 30 min.
Cells were blocked with 10% BSA (Invitrogen) in PBS for 30 min at RT. Cells were
stained in blocking buffer with primary antibodies at 4C overnight. Cells were washed
and stained with secondary antibodies in blocking buffer for 1 hr at RT, protected from
light. Primary antibodies were used, at 1:100 dilutions: TRA-1-60, TRA-1-81, or SSEA4 (Millipore, Billerica, MA, http://www.millipore.com), and an anti-goat IgG Alexa Flour
488 or anit-goat igM Alexa 649 (Invitrogen; 1:400 dilution). Primary OCT4 (Abcam,
Cambridge, MA, http://www.abcam.com) were used at 0.5 mg/mL, and an anti-rabbit
IgG Alexa Fluor 488 (Invitrogen, Carlsbad, CA) was used as the secondary (1:400
dilution). Images were acquired with a Leica 4000i B.
RNA isolation and real-time quantitative PCR analysis
RNA was isolated from cells using the RNeasy microkit (Qiagen, Inc), with the optional
on columm RNAse-free Dnase treatment, according the manufacturer’s instructions.
cDNA was produced using random hexamers with Superscript III Reverse Transcriptase
(Invitrogen), from 200ng – 1ug of starting RNA. Human gDNA was used for the qPCR
standard curve with gDNA ranging from 0.1 to 10ng per reaction to evaluate the
efficiency of the PCR and calculate copy number of each gene relative to the
housekeeping gene Cyclophilin. The expression level was expressed as number of
molecules of RNA for each indicated gene per number of molecules of cyclophilin,
following the protocol previously published (Somers et. al. 2011). Real-time quantitative
PCR reactions were set up in triplicates with the SYBR Green QPCR Master Mix
(Roche, Indianapolis, IN, http://www.roche.com) and run on a Light Cycler 480II qPCR
System (Roche, Indianapolis, IN, http://www.roche.com). Primer sequences are listed in
Supplemental Table ??.
Southern blot analysis
10ug (for hSTEMCAA vector Southern blot) of genomic DNA was digested with
BamHI, separated on a 0.8% agarose gel and blotted onto Hybond-N+ membrane
(Amersham Biosciences). Membrane was probed using WRPE element as previously
published (Somer, 2011), and labeling with performed using 32P--dCTP with the High
Prime Random Labeling (Roche, Indianapolis, IN, http://www.roche.com) following the
manufacturer’s instructions. iPSC clones that were shown to contain a single integration
of the lentivirus were expanded for Cre-excision protocols and teratoma formation.
Teratoma formation
For teratoma formation, cells were harvested from irMEF plates at ~75% confluence and
replated on Matrigel-coated 6-well dish ensuring a cells density of at least 6x10^5
cells/well. One million cells were resuspended in 300-ul of matrigel and subsequently
injected into the neck of a Fox Chase SCID Beige Mice. Mice were sacrificed 6-9 weeks
later, teratomas isolated and processed for histological analysis.
Cre-mediated hSTEMCAA Excision
The IPSC cells (>6 passages) were plated at 1x10^5 cells/well of a matrigel-coated 6-well
dish containing puromycin resistant irradiated MEFs. Cells were grown to roughly 40%
confluence and subsequently transfected with 2ug/well of pHAGE1-Cre-IRES-PuroR
plasmid DNA using Hela Monster transfection reagent (Mirus, Madison, Wi,
http://www.mirusbio.com) according to manufacturer’s instructions. Media was changed
to puromycin selection (1.2ug/ml) media 24 hours post-transfection, and lasted for 48
hours. The re-emergence of iPSCs was observed between 7-10 days post-puromycin
selection, and a total of 12 colonies were picked when colonies reached between 50-100
cell per cluster. Genomic DNA was collected using DNeasy Blood & Tissue kit (Qiagen,
Valencia, CA, http://www.qiagen.com) from each subclone after 2-3 weeks in culture and
screen for hSTEMCAA transgene using the following primers and conditions: OCT4-TgF: 5’-GGT GCG CCA GTA AAG CAG ACA TTA AAC 3’;KLF4-Tg-R: 5’-CAG ACG
CGA ACG TGG AGA AAG A-3’ and GAPDH-F: 5’-GTG GAC CTG ACC TGC CGT
CT-3’; GAPDH-R: 5’- GGA GGA GTG GGT GTC GCT GT-3’; 95C for 10 minutes;
followed by 30-cycles of 95C for 30 seconds, and 60C for 1 minute. Vector excision was
then confirmed by Southern blotting of BAMHI digested gDNA, using a probe
encompassing a 600bp WRPE segment of the hSTEMCAA backbone.