Derivation of induced pluripotent stem cells from orangutan skin fibroblasts

Cell culture

Previously established primary orangutan skin fibroblast cultures were obtained from
the Zoological Society of San Diego (ZSSD) 33]. Cultured skin fibroblasts were maintained in fibroblast growth media containing
Dulbecco’s modified eagle medium (DMEM) containing 10 % fetal bovine serum (FBS),
2 mM L-glutamine, 100 µM nonessential amino acids and 0.5 % penicillin and streptomycin
(Invitrogen). 293T packaging cells were maintained in the same media except with 10 %
FBS. iPSCs were generated and maintained in human iPSC (hiPSC) growth medium containing
DMEM/F12 containing 20 % KOSR (vol/vol) (Invitrogen), 10 ng/ml bFGF, 1 mM L-glutamine, 100 µM nonessential amino acids, 100 µM ?-mercaptoethanol, 50 U/mL penicillin
and 50 mg/mL streptomycin.

Cellular reprogramming

Cellular reprogramming was conducted using the Yamanaka factors as previously described
37]. Retroviral pMX vectors for human OCT-3/4, SOX2, KLF4 and c-MYC were obtained from
Addgene (https://www.addgene.org/). Briefly, GP2-293 packaging cells (Clontech) were plated at 2 × 10
⁶
cells per 100-mm dish and incubated overnight. The cells were transfected with 5 µg
of pMX vectors and 5 µg of pVSV-G vectors in the presence of Lipofectamine Transfection
Reagent (Invitrogen) and the media was replaced the next day. Virus containing supernatant
was collected on days 2 and 3 post-transfection and filtered through a 0.4 micron
pore cellulose acetate filter (Corning).

Prior to transduction, primary orangutan cultures were plated at 8 × 10
⁵
cells per well of a 6-well plate and incubated overnight. The cells were transduced
with equal amounts of the four retroviruses in the presence of 5 ng/ml protamine sulfate.
The following day the virus containing media was replaced with Fibroblast Growth (FG)
medium. After another round of transduction, cells were trypsinized on day 6 and plated
on a murine embryonic fibroblast (MEF) feeder layer at 2 × 10
⁵
cells per well of a 6-well plate. The next day, the medium was replaced with hiPSC
growth medium containing 1 mM valproic acid and 10 ng/mL ?FGF. After daily media changing
for 1 week, hiPSC culture medium conditioned with mitomycin C (Sigma-Aldrich) inactivated
mouse embryonic fibroblasts (iMEFs) was used to support cell growth. Approximately
21 days after transduction, colonies exhibiting iPSC characteristics were picked mechanically
and plated on iMEFs for expansion.

Immunostaining

Cells were first fixed with 4 % paraformaldehyde, permeabilized with 1 % triton, and
incubated with the primary antibody overnight at 4 °C. After washing three times with
PBS, the cells were incubated with the secondary antibodies for 1 h at room temperature.
The following primary antibodies were used: TRA-1-81 (mouse monoclonal against human,
1:100 dilution, EMD Millipore, Catalog #MAB4381), TRA-1-60 (mouse monoclonal against
human, 1:100 dilution, EMD Millipore, Catalog #MAB4360), OCT4 (goat polyclonal against
human, 1:100 dilution, RD Systems, Catalog #AF1759), NANOG (goat polyclonal against
human, 1:50 dilution, RD systems, Catalog #1997), SOX2 (goat polyclonal against mouse,
rat and human, 1:100 dilution, Santa Cruz Biotechnology, Catalog #sc17320), SSEA4
(mouse monoclonal against human, 1:100, Developmental Studies Hybridoma Bank, Catalog
#MC-813-70), ?-fetoprotein (AFP) (mouse monoclonal against human, 1:250 dilution,
Sigma, Catalog #A8452), ?III-Tubulin (rabbit monoclonal against rat, 1:100 dilution,
Covance Catalog #MRB-435P) and alpha smooth muscle actin (?SMA) (1:500 dilution, Abcam,
Catalog #ab5694). Secondary antibodies used were rhodamine-labeled donkey anti-mouse
IgG (1:100 dilution, Santa Cruz Biotechnology, Catalog #sc-2300), FITC-labeled donkey
anti-rabbit IgG (1:100 dilution, Jackson ImmunoResearch, Catalog #711-095-152), FITC-labeled
donkey anti-goat IgG (1:100 dilution, Jackson ImmunoResearch, Catalog #705-095-003)
and Alexa Fluor 488 donkey anti-mouse IgG (1:500 dilution, ThermoFisher Scientific,
Catalog #R37114). The Alkaline Phosphatase Detection Kit (Sigma-Aldrich) was used
according to the manufacturer’s protocol.

Genotyping and copy number analysis

Human CytoSNP-12 Infinium HD BeadChips (Illumina) that interrogate the genotypes of
approximately 300,000 human single nucleotide polymorphisms (SNPs) were used to evaluate
copy number in total genomic DNA from orangutan fibroblasts and iPSCs. Data filtering
was performed using GenomeStudio (Illumina). Copy number analysis was performed using
CNVPartition version 2.4.4, as previously described 62].

Gene expression profiling

Biotin-labeled cRNA targets obtained from total RNA samples (Affymetrix GeneChip IVT
Labeling Kit) were processed and analyzed on Affymetrix Human Genome 133A 2.0 GeneChips,
as previously described 47]. All data normalization and analysis of differentially expressed genes (DEGSs) were
conducted using WebArray software 63], 64]. We applied the RMA algorithm to generate log2-transformed gene expression values
and used linear model statistical analysis (limma) to identify DEGSs with false discovery
rates (FDRs), based on the spacings LOESS histogram (SPLOSH) method (Additional file
1B). We used CIMminer software (http://discover.nci.nih.gov/cimminer/home.do) for hierarchical clustering analysis 65].

In vitro differentiation and teratoma assays

iPSCs were detached from culture dishes with collagenase IV, maintained in suspension
to induce embryoid body formation, and subjected to an in vitro differentiation procedure
as described 37]. For teratoma analysis, iPSCs from a confluent 10 cm
²
plate were harvested and subcutaneously injected to the dorsal flanks of immunodeficient
(SCID) mice (Jackson Laboratory). Teratomas were fixed in 10 % formalin, sectioned,
stained with hematoxylin and eosin, and subject to histological analysis as described
37]. All mice used in this study were maintained in accordance with the Guide for the
Care and Use of Animals (United States Department of Health and Human Services, Public
Health Service, Bethesda, MD, 2012).

Availability of supporting data

Gene expression scores and .cel files supporting the results of this article are available
in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus
(GEO) repository [Series Accession Number GSE69603 and http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE69603].